Anti-CD137 antigen-binding molecule and its use

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 using the same.

Background Art

[0002] Cancer is one of the lethal diseases that are difficult to cure except for some cases. The treatment results using chemotherapeutic agents, which are the main treatment method, are not very high. It has been suggested that factors making cancer treatment difficult include not only the heterogeneity of cancer cells themselves but also the tumor microenvironment playing a major role (Non-Patent Document 1). In recent years, the anti-CTLA-4 antibody that suppresses the function of CTLA-4, which suppresses the immune response, and promotes the activation of T cells has shown the possibility of cure for inoperable malignant melanoma and the like (Non-Patent Document 2). In 2011, an anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the US Food and Drug Administration (FDA) as the world's first immune activation antibody drug. Furthermore, the therapeutic effects of inhibitory antibodies against PD-1 and PD-L1, which are immune checkpoint molecules other than CTLA-4, have been reported (Non-Patent Document 3) and have been approved by the FDA. The activation of T cells, which plays an important role in tumor immunity, is understood to be achieved by two signals: 1) the binding and activation of the T cell receptor (TCR) to the antigen peptide presented by the major histocompatibility complex (MHC) class I molecule; and 2) the binding and activation of the co-stimulatory molecule on the T cell surface to its ligand on the antigen-presenting cell. Furthermore, it has also been stated that the activation of co-stimulatory 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, GITR, etc. 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 (DC), B cells, NK cells, macrophages, and neutrophils (Non-Patent Document 5). It has already been demonstrated in a mouse model that a CD137 agonist antibody exhibits an antitumor effect, and it has been experimentally shown in the mouse model that this is mainly due to the activation of CD8-positive T cells and NK cells (Non-Patent Document 6). However, side effects due to the non-specific hepatotoxicity of CD137 agonist antibodies have been a problem in both clinical and non-clinical settings, and the development of drugs has not progressed as expected (Non-Patent Documents 7 and 8). As the main cause of this side effect, the activation of immune cells in non-immune tissues other than tumors such as the liver, which is involved in the binding to Fcγ receptors via the antibody constant region, has been suggested (Non-Patent Document 9). On the other hand, it has been reported that for an agonist antibody of a receptor belonging to the TNF receptor superfamily to exhibit agonist activity in vivo, cross-linking of the antibody by Fcγ receptor-expressing cells (FcγRII-expressing cells) is necessary (Non-Patent Document 10). That is, since both the efficacy of the antitumor effect of the CD137 agonist antibody and side effects such as hepatotoxicity are involved in the binding of the antibody to the Fcγ receptor, if the binding of the antibody to the Fcγ receptor is increased, an improvement in efficacy is expected but the side effect of hepatotoxicity also increases, and if the binding of the antibody and the Fcγ receptor is reduced, although the side effect is reduced, the efficacy is also reduced. So far, a CD137 agonist antibody that separates efficacy and side effects has not been reported. Furthermore, in clinical settings, the antitumor effect itself of the CD137 agonist antibody is not very strong, and it is desired to increase the efficacy further while avoiding toxicity. Therefore, the development of a new drug that can induce an antitumor immune response while suppressing such side effects is desired.

[0004] When a therapeutic antibody is administered in vivo, it is desirable that the target antigen is specifically expressed only in the lesion site. However, in many cases, the same antigen is also expressed in normal tissues, which are non-lesion sites, and this can cause undesirable side effects from a therapeutic perspective. For example, an antibody against a tumor antigen can exhibit cytotoxic activity against tumor cells through ADCC or the like. On the other hand, if the same antigen is expressed in normal tissues, there is a possibility of damaging normal cells as well. To solve the above problems, a technique has been developed that focuses on the phenomenon in which a specific compound is present in a large amount in the target tissue (e.g., tumor tissue), and searches for an antigen-binding molecule whose binding activity to the antigen changes according to the concentration of such a compound (for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present disclosure relates to an anti-CD137 antigen-binding molecule and a method of using the same.

Means for Solving the Problems

[0008] The present disclosure provides an anti-CD137 antigen-binding molecule having an activating effect, cytotoxic activity, or antitumor activity on immune cells, but having a low effect on non-tumor tissues such as normal tissues and few side effects, and a method of using the same. Therefore, an anti-CD137 antigen-binding molecule having a characteristic that the binding activity to CD137 changes depending on various substances (for example, low molecular weight compounds) in a target tissue (for example, tumor tissue) is provided, and a method of using the same, a pharmaceutical preparation, etc. are provided. In one aspect, since the anti-CD137 antigen-binding molecule in the present disclosure has few side effects, the dosage can be increased without concern about side effects, and as a result, a stronger drug effect (cytotoxic activity or antitumor activity) can be exhibited. That is, the present disclosure specifically provides an anti-CD137 antigen-binding molecule, a method of using the same, a pharmaceutical preparation, etc., which are exemplified below. 〔1〕 An anti-CD137 antigen-binding molecule having CD137-binding activity that depends on a low molecular weight compound. [2] The binding activity to CD137 in the presence of a small molecule compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is at least 2-fold higher than the binding activity to CD137 in the absence of the small molecule compound, and the anti-CD137 antigen-binding molecule according to [1]. [2.1] The binding activity to CD137 in the presence of a small molecule compound at 10 μM or higher is at least 2-fold higher than the binding activity to CD137 in the absence of the small molecule compound, and the anti-CD137 antigen-binding molecule according to [1] or [2]. [2.2] The KD value for CD137 in the presence of a small molecule compound at 10 μM or higher is 5×10 -7 M or less, and the anti-CD137 antigen-binding molecule according to any one of [1] to [2.1]. [2.3] The KD value for CD137 in the absence of the small molecule compound is 1×10 -6 M or more, and the anti-CD137 antigen-binding molecule according to any one of [1] to [2.2]. [2.4] The KD value for CD137 in a solution prepared such that the concentration of the small molecule compound is 10 μM or higher is 5×10 -7 M or less, and the KD value for CD137 in a solution without adding the small molecule compound is 1×10 -6 M or more, and the anti-CD137 antigen-binding molecule according to [1]. [2.5] The KD value for CD137 in a solution prepared such that the concentration of the small molecule compound is 10 μM or higher, and the KD value for CD137 in a solution without adding the small molecule compound are each measured by a Biacore assay within 24 hours after contacting CD137 and the anti-CD137 antigen-binding molecule in the solution, and the anti-CD137 antigen-binding molecule according to [1]. [2.6] The anti-CD137 antigen-binding molecule according to any one of [1] to [2.5], which forms a ternary complex with the small molecule compound and CD137. [2.7] An anti-CD137 antigen-binding molecule according to any one of [1] to [2.6], which binds to CD137 derived from human and monkey. [2.8] An anti-CD137 antigen-binding molecule according to any one of [1] to [2.7], wherein the low molecular weight compound is an adenosine-containing compound. [2.9] An anti-CD137 antigen-binding molecule according to any one of [1] to [2.8], wherein the low molecular weight compound is ATP. [3] An anti-CD137 antigen-binding molecule according to any one 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] An anti-CD137 antigen-binding molecule according to any one 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) VH having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 43 to 53; or (b) An anti-CD137 antigen-binding molecule comprising a VL having at least 95% sequence identity with any one of the amino acid sequences 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) VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 43, and VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54; (b) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 44, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55; (c) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55; (d) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 46, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54; (e) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 47, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54; (f) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56; (g) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 49, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 57; (h) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 58; (i) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 51, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 59; (j) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 51, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 60; (k) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 52, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 60; (l) A VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50, and a VL having at least 95% sequence identity with 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 containing the amino acid sequence of SEQ ID NO: 53 and VL containing the amino acid sequence of SEQ ID NO: 54. [5.3] [Binding activity (binding amount) to CD137 in the presence of a low molecular weight compound of 10 μM or more] / [Binding activity (binding amount) to CD137 in the absence of the low molecular weight compound] is a value equal to or greater than that of the reference antigen-binding molecule, and is an anti-CD137 antigen-binding molecule, where the reference antigen-binding molecule is HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; an anti-CD137 antigen-binding molecule comprising a combination of [5.4] The anti-CD137 antigen-binding molecule according to [5.3], wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of VH containing the amino acid sequence of SEQ ID NO: 43 and VL containing the amino acid sequence of SEQ ID NO: 54. [5.5] [Binding activity (KD) to CD137 in the presence of a 1 μM low molecular weight compound] / [Binding activity (KD) to CD137 in the presence of the low molecular weight compound of 10 μM or more] is a value equal to or greater than that of the reference antigen-binding molecule, and is an anti-CD137 antigen-binding molecule, where the reference antigen-binding molecule is HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; an anti-CD137 antigen-binding molecule comprising a combination of [5.6] The anti-CD137 antigen-binding molecule according to [5.5], wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of VH containing the amino acid sequence of SEQ ID NO: 43 and VL containing the amino acid sequence of SEQ ID NO: 54. [5.7] An anti-CD137 antigen-binding molecule having a small molecule compound-dependent CD137 binding activity that competes with any of the antigen-binding molecules described in [3] to [5.2] with respect to binding to CD137 in the presence of a small molecule compound of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more. [5.8] An anti-CD137 antigen-binding molecule having a small molecule compound-dependent CD137 binding activity that binds to the same epitope of CD137 as that bound by any of the antigen-binding molecules described in [3] to [5.2] in the presence of a small molecule compound of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more. [5.8A] The anti-CD137 antigen-binding molecule according to any one of [5.3] to [5.8], wherein the small molecule compound is an adenosine-containing compound. [5.8B] The anti-CD137 antigen-binding molecule according to any one of [5.3] to [5.8A], wherein the small molecule compound is ATP. [5.9] The anti-CD137 antigen-binding molecule according to any one of [1] to [5.8B], which is a monoclonal antibody or an antigen-binding fragment thereof. [5.10] The anti-CD137 antigen-binding molecule according to any one 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 according to any one of [1] to [5.10], which is a full-length IgG1 antibody. [5.12] An anti-CD137 antigen-binding molecule according to any one of [1] to [5.11], comprising a modified Fc region in which at least one amino acid is modified, wherein the binding activity of the modified Fc region to FcγRIIb is increased as compared to the parental Fc region that does not contain the amino acid modification. [5.13] The anti-CD137 antigen-binding molecule according to [5.12], wherein the binding activity of the modified Fc to FcγRIIb is the same as or higher than that of the reference Fc region, where the reference Fc is a human IgG1 Fc region containing a combination of amino acid substitutions of G236N / H268D / A330K based on EU numbering. [5.14] The anti-CD137 antigen-binding molecule according to [5.12] or [5.13], wherein the reference Fc region contains the amino acid sequence of SEQ ID NO: 153. [5.15] The anti-CD137 antigen-binding molecule according to [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 based on EU numbering. [5.16] The anti-CD137 antigen-binding molecule according to [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 according to any one of [5.12] to [5.16], wherein the parental Fc region is derived from a human IgG1 Fc region. [5.18] An anti-CD137 antigen-binding molecule according to any one of [1] to [5.17], comprising a modified Fc region in which at least one amino acid is modified, and having an increased isoelectric point (pI) as compared to the parental anti-CD137 antigen-binding molecule containing the parental 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 parental Fc region. [5.20] wherein the at least one amino acid modification is (i) a modification that substitutes an amino acid residue having a negative charge in at least one side chain of the parental Fc region with an amino acid residue having no charge in the side chain, (ii) a modification that substitutes an amino acid residue having no charge in at least one side chain of the parental Fc region with an amino acid residue having a positive charge in the side chain, and / or (iii) a modification that substitutes an amino acid residue having a negative charge in at least one side chain of the parental Fc region with an amino acid residue having a positive charge in the side chain, and is the anti-CD137 antigen-binding molecule according to [5.18] or [5.19]. [5.21] The anti-CD137 antigen-binding molecule according to any one of [5.18] to [5.20], wherein the at least one amino acid modification is a combination of a plurality of amino acid substitutions, and the plurality of amino acid substitutions are present at positions that are sterically close to each other. [5.22] The anti-CD137 antigen-binding molecule according to any one of [5.18] to [5.21], wherein the binding activity of the modified Fc region to the Fcγ receptor (FcγR) is not substantially reduced as compared with the parental Fc region. [5.23] The anti-CD137 antigen-binding molecule according to [5.22], wherein the Fcγ receptor (FcγR) is FcγRIIb. [5.24] The anti-CD137 antigen-binding molecule according to any one 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 according to any one of [5.18] to [5.24], wherein the at least one amino acid modification is a combination of (i) the amino acid substitution of P343R, (ii) the amino acid substitution of Q311R / P343R, or (iii) the amino acid substitution of Q311R / D413K based on EU numbering. [6] An anti-CD137 antigen-binding molecule according to 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 according to any one of 〔1〕 to 〔6〕, wherein the modified Fc region is derived from the human IgG1 Fc region. 〔6.2〕 The anti-CD137 antigen-binding molecule according to any one of 〔1〕 to 〔6.1〕, wherein the modified Fc region further comprises a deletion at positions 446 and 447 based on EU numbering. 〔7〕 The anti-CD137 antigen-binding molecule according to any one of 〔1〕 to 〔6.2〕, comprising a heavy chain constant region containing any one amino acid sequence 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) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 64, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (ii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 66, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (iii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 67, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (iv) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 68, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (v) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 69, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (vi) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 70, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (vii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 71, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (viii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 73, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (ix) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (x) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xi) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xiii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xiv) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 85, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 65, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxiii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 81, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxiv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 83, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxiii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxiv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 81, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 83, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; and (xxxviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 85, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63. [8] An isolated nucleic acid encoding an anti-CD137 antigen-binding molecule according to any one of [1] to [7.1]. [9] A vector containing the nucleic acid according to [8].

[10] A host cell containing the nucleic acid according to [8], or the vector according to [9].

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

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

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

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

[12] ; and a pharmaceutical preparation comprising a pharmaceutically acceptable carrier.

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

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

[12] , or the pharmaceutical preparation according to

[13] for use in the treatment of tumors. [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] The anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], the immunoconjugate according to

[12] , or the pharmaceutical preparation according to

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

[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 according to any one of [1] to [7.1], or the pharmaceutical preparation according to

[13] for the activation of 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] The anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], the immunoconjugate according to

[12] , or the pharmaceutical preparation according to

[13] for use in cell injury.

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

[12] , or the pharmaceutical preparation according to

[13] , wherein the level of immune activation in non-tumor tissue is low as compared to an anti-CD137 antigen-binding molecule having no CD137-binding activity dependent on a low molecular weight compound. [16.1] The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation according to

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

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

[12] , which has an extended plasma 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 according to any one of [1] to [7.1], the immunoconjugate according to

[12] , or the pharmaceutical preparation according to

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

[17] , wherein the side effects are an increase in AST, an increase in ALT, fever, nausea, acute hepatitis, liver damage, splenomegaly, enteritis, suppurative inflammation of the skin, neutropenia, lymphopenia, thrombocytopenia, expression of transaminase, and / or hyperbilirubinemia.

[18] An anti-CD137 antigen-binding molecule having CD137 agonist activity dependent on a low molecular weight compound. [18.1] The anti-CD137 antigen-binding molecule according to

[18] , wherein the agonist activity against CD137 in the presence of a low molecular weight compound of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is at least 2-fold higher than the agonist activity against CD137 in the absence of the low molecular weight compound. [18.2] An anti-CD137 antigen-binding molecule described in

[18] or [18.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 10 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔18.3〕 An anti-CD137 antigen-binding molecule described in

[18] or [18.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 50 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔18.4〕 An anti-CD137 antigen-binding molecule described in

[18] or [18.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 250 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔18.5〕 The anti-CD137 antigen-binding molecule according to any one of

[18] to [18.4], wherein the agonist activity against CD137 is evaluated 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 (PBMC) or T cells derived from human peripheral blood mononuclear cells (PBMC). 〔18.7〕 The anti-CD137 antigen-binding molecule according to any one of

[18] to [18.4], wherein the agonist activity against CD137 is evaluated by a reporter gene assay. 〔18.8〕 An anti-CD137 antigen-binding molecule described in

[18] , which exhibits agonist activity against CD137 in a solution prepared such that the final concentration of the small molecule compound is 50 μM or higher, and substantially does not exhibit an agonist against CD137 in a solution without the addition of the small molecule compound. 〔18.9〕 The agonist activity against CD137 in a solution prepared such that the final concentration of the low-molecular-weight compound is 50 μM or higher, and the agonist activity against CD137 in a solution to which the low-molecular-weight compound is not added, are each evaluated by the amount of production of IL-2, IFN-γ, and / or IL-6 measured within 72 hours after contacting CD137-expressing cells with an anti-CD137 antigen-binding molecule in the solution, and the anti-CD137 antigen-binding molecule described in [18.8]. 〔18.10〕 The agonist activity against CD137 in a solution prepared such that the final concentration of the low-molecular-weight compound is 50 μM or higher, and the agonist activity against CD137 in a solution to which the low-molecular-weight compound is not added, are each evaluated by the luciferase luminescence signal measured within 6 hours after contacting a T cell expressing NF-kappaB-luciferase reporter construct and CD137 with an anti-CD137 antigen-binding molecule, and the anti-CD137 antigen-binding molecule described in [18.8]. 〔18.11〕 The anti-CD137 antigen-binding molecule according to any one of

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

[18] to [18.11], wherein the low-molecular-weight compound is ATP. 〔19〕 The anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], which has CD137 agonist activity dependent on a low-molecular-weight compound. 〔19.1〕 The agonist activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a low-molecular-weight compound is at least twice as high as the agonist activity against CD137 in the absence of the low-molecular-weight compound, and the anti-CD137 antigen-binding molecule described in

[19] . 〔19.2〕 An anti-CD137 antigen-binding molecule as described in

[19] or [19.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 10 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔19.3〕 An anti-CD137 antigen-binding molecule as described in

[19] or [19.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 50 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔19.4〕 An anti-CD137 antigen-binding molecule as described in

[19] or [19.1], wherein the agonist activity against CD137 in the presence of a small molecule compound at a concentration of 250 μM or higher is at least twice as high as the agonist activity against CD137 in the absence of the small molecule compound. 〔19.5〕 The anti-CD137 antigen-binding molecule according to any one of

[19] to [19.4], wherein the agonist activity against CD137 is evaluated 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 T cells derived from human peripheral blood mononuclear cells (PBMCs). 〔19.7〕 The anti-CD137 antigen-binding molecule according to any one of

[19] to [19.4], wherein the agonist activity against CD137 is evaluated by a reporter gene assay. 〔19.8〕 An anti-CD137 antigen-binding molecule as described in

[19] , which shows agonist activity against CD137 in a solution prepared such that the final concentration of the small molecule compound is 50 μM or higher and substantially does not show an agonist against CD137 in a solution without the addition of the small molecule compound. 〔19.9〕 The agonist activity against CD137 in a solution prepared such that the final concentration of the low molecular weight compound is 50 μM or more, and the agonist activity against CD137 in a solution to which the low molecular weight compound is not added, are each evaluated by the amount of production of IL-2, IFN-γ, and / or IL-6 measured within 72 hours after contacting CD137-expressing cells with an anti-CD137 antigen-binding molecule in the solution, the anti-CD137 antigen-binding molecule described in [19.8]. 〔19.10〕 The agonist activity against CD137 in a solution prepared such that the final concentration of the low molecular weight compound is 50 μM or more, and the agonist activity against CD137 in a solution to which the low molecular weight compound is not added, are each evaluated by the luciferase luminescence signal measured within 6 hours after contacting an NF-kappaB-luciferase reporter construct and T cells expressing CD137 with an anti-CD137 antigen-binding molecule, the anti-CD137 antigen-binding molecule described in [19.8]. 〔19.11〕 The anti-CD137 antigen-binding molecule according to any one of

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

[19] to [19.11], wherein the low molecular weight compound is ATP. 〔20〕 An agonist antigen-binding molecule comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification that results in an increase in the isoelectric point (pI) compared to a parental agonist antigen-binding molecule comprising a parental Fc region, and the agonist activity is increased compared to the parental agonist antigen-binding molecule. 〔20.1〕 The agonist antigen-binding molecule according to

[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 parental Fc region. 〔20.2〕 The at least one amino acid modification is (i) A modification in which an amino acid residue having a negative charge in at least one side chain of the parental Fc region is replaced with an amino acid residue having no charge in the side chain, (ii) A modification in which an amino acid residue having no charge in at least one side chain of the parental Fc region is replaced with an amino acid residue having a positive charge in the side chain, and / or, (iii) A modification in which an amino acid residue having a negative charge in at least one side chain of the parental Fc region is replaced with an amino acid residue having a positive charge in the side chain, which is the agonist antigen-binding molecule described in

[20] or [20.1]. [20.3] The agonist antigen-binding molecule according to any one of

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

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

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

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

[20] to [20.8], which is an anti-CD137 antibody.

[21] A method for producing an agonist antigen-binding molecule comprising a modified Fc region, comprising introducing at least one amino acid modification into the parental Fc region that results in an increase in the isoelectric point (pI) compared to the parental agonist antigen-binding molecule comprising the parental Fc region, wherein the agonist activity of the agonist antigen-binding molecule comprising the modified Fc region is increased compared to the parental agonist antigen-binding molecule. [21.1] The method according to

[21] , wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. [21.2] The method according to

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 10 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. [21.3] The method according to

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 50 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. [21.4] The method according to

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 250 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. [21.5] The method according to any one of

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

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

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

[21] to [21.7], comprising the above. [21.9] The method according to any one of

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

[21] to [21.9], wherein the agonistic 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.

[22] A method for increasing the agonist activity of an agonist antigen-binding molecule comprising an Fc region, the method 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 parental agonist antigen-binding molecule comprising a parental Fc region. 〔22.1〕 The method according to

[22] , wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. 〔22.2〕 The method according to

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 10 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. 〔22.3〕 The method according to

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 50 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. 〔22.4〕 The method according to

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of a low molecular weight compound at 250 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the low molecular weight compound. 〔22.5〕 The method according to any one of

[22] to [22.4], wherein the agonist activity against the antigen is evaluated by the amount of IL-2 and / or IFN-γ production by antigen-expressing cells. 〔22.6〕 The method according to [22.5], wherein the antigen-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or T cells derived from human peripheral blood mononuclear cells (PBMCs). 〔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 according to any one of

[22] to [22.7], wherein the agonist antigen-binding molecule is an anti-CD137 antigen-binding molecule. [22.9] The method according to any one 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 small molecule compound is an adenosine-containing compound. [22.11] The method according to any one of [22.1] to [22.10], wherein the small molecule compound is ATP.

[23] Use of at least one amino acid modification to increase the agonist activity of an agonist antigen-binding molecule comprising an Fc region, wherein the amino acid variation results in an increase in the isoelectric point (pI) compared to the parental agonist antigen-binding molecule comprising the parental Fc region. [23.1] The method according to

[23] , wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of a small molecule compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is at least 2-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. [23.2] The method according to

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of a small molecule compound at 10 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. [23.3] The method according to

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of a small molecule compound at 50 μM or higher is at least 2-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. [23.4] The method according to

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of a low-molecular-weight compound of 250 μM or more is at least twice as high as the agonist activity of the agonist antigen-binding molecule against the antigen in the absence of the low-molecular-weight compound. [23.5] The method according to any one of

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

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

[24] A screening method for an antigen-binding domain or an antigen-binding molecule having an antigen-binding activity dependent on a low-molecular-weight compound, comprising: (a) contacting a fusion molecule in which two or more units of an antigen are fused per unit of a fusion partner molecule with an antigen-binding domain or an antigen-binding molecule or a library thereof in the presence of a low-molecular-weight compound; (b) exposing 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 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 the above. [24.1] The method according to

[24] , wherein the fusion partner molecule is a dimeric Fc region. [24.2] The method according to [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 according to [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 according to any one of

[24] to [24.3], wherein the library of the antigen-binding domain or antigen-binding molecule is a phage library. [24.5] The method according to any one of

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

[24] to [24.5], wherein the phage contained in the phage library is a phage having a deletion in the helper phage-derived pIII gene.

[25] A screening method for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on two or more different small molecule 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) exposing the antigen-binding domain or antigen-binding molecule that has bound to the antigen in step (a) in the absence or low concentration of the first low molecular weight 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 low molecular weight compound; (e) exposing the antigen-binding domain or antigen-binding molecule that has bound to the antigen in step (d) in the absence or low concentration of the second low molecular weight compound; (f) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (e), comprising: wherein between (c) and (d), it does not include amplifying the gene encoding the antigen-binding domain or antigen-binding molecule isolated in (c), A screening method. [25.1] The method according to

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

[26] A screening method for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on a low molecular weight compound, (a) contacting an antigen with a naive library of antigen-binding domains or antigen-binding molecules in the presence of a low molecular weight compound; (b) exposing the antigen-binding domain or antigen-binding molecule that has bound to the antigen in step (a) in the absence or low concentration of the low molecular weight compound, and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b), comprising: wherein the naive library is a phage library containing phages presenting two or more antigen-binding domains or antigen-binding molecules on their surfaces. A screening method.

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

[28] A method for screening an antigen-binding domain or an antigen-binding molecule having antigen-binding activity dependent on a low molecular weight compound, comprising: (a) contacting an antigen with a library of antigen-binding domains or antigen-binding molecules in the presence of a low molecular weight compound; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) in the absence or low concentration presence of the low molecular weight compound; and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b). Including that, wherein the library is a library containing phages prepared by increasing the expression of an antigen-binding domain or an antigen-binding molecule with a low molecular weight additive that increases the expression level from a promoter that controls the expression of the antigen-binding domain or antigen-binding molecule. A screening method. [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 dependent on the concentration of a tumor tissue-specific compound, wherein the antigen-binding activity in the presence of 100 μM of the compound is 2-fold or more higher than the antigen-binding activity in the absence of the compound. [29.1] The KD value in the presence of 100 μM of the compound is 5×10 -7 M or less, the antigen-binding molecule according to

[29] . [29.2] The KD value in the absence of the compound is 1×10 -6 M or more, the antigen-binding molecule according to

[29] or [29.1]. [29.3] The antigen-binding molecule according to any one of

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

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

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

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

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

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

[30] A pharmaceutical preparation comprising an antigen-binding molecule according to any one of

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

[30] for use in the treatment of tumors. [30.2] A pharmaceutical preparation according to [30.1], which has lower cytotoxic activity in non-tumor tissues as compared to a pharmaceutical preparation containing a control antigen-binding molecule. [30.3] A pharmaceutical preparation according to [30.1] or [30.2], which has a lower level of side effects as compared to a pharmaceutical preparation containing a control antigen-binding molecule. [30.4] A pharmaceutical preparation 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 dependent on the concentration of a tumor tissue-specific compound.

[31] A method for producing an antigen-binding molecule for use in the treatment of tumors, the method comprising the step of selecting an antigen-binding molecule having an antigen-binding activity in the presence of 100 μM of a tumor tissue-specific compound that is at least 2-fold higher than the antigen-binding activity in the absence of the compound.

[32] A method for producing a pharmaceutical preparation for use in the treatment of tumors, the method comprising the step of mixing an antigen-binding molecule according to any one of

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

[33] An antigen-binding molecule having antigen-binding activity dependent 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 2-fold lower than the antigen-binding activity in the presence of a sufficient amount of the compound. [33.1] The KD value in the presence of 1 μM of the said compound is 2×10 -7 M or more, the antigen-binding molecule described in

[33] . [33.2] The KD value in the presence of a sufficient amount of the said compound is 1×10 -7 M or less, the antigen-binding molecule described in

[33] or [33.1]. [33.3] The said compound is a tumor tissue-specific compound, the antigen-binding molecule described in any one of

[33] to [33.2]. [33.4] The said compound is an adenosine-containing compound, the antigen-binding molecule described in [33.3]. [33.5] Compared with the control antigen-binding molecule, having high plasma retention and / or low plasma antigen accumulation ability, the antigen-binding molecule described in any one of

[33] to [33.4]. [33.6] The control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of the target tissue-specific compound, the antigen-binding molecule described in [33.5]. [33.7] The antigen-binding molecule is an antibody or antibody fragment, the antigen-binding molecule described in any one of

[33] to [33.6].

[34] A pharmaceutical preparation comprising the antigen-binding molecule described in any one of

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

[35] A method for producing an antigen-binding molecule having high plasma retention and / or low plasma antigen accumulation ability compared with a control antigen-binding molecule, comprising: (a) a step of producing an antigen-binding molecule whose antigen-binding activity increases as the concentration of the target tissue-specific compound increases; and (b) a step of measuring the plasma retention and / or plasma antigen accumulation ability of the antigen-binding molecule produced in (a). [35.1] A method according to

[35] , comprising the step of selecting an antigen-binding molecule having an antigen-binding activity that is at least twofold lower in the presence of 1 μM of a target tissue-specific compound than the antigen-binding activity in the presence of a sufficient amount of said compound. [35.2] A method according to

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

[36] A method for producing a pharmaceutical preparation, comprising the step of mixing an antigen-binding molecule according to any one of

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

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

[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 the interstitial fluid in in vivo tissue. [37.3] The method according to [37.2], wherein the tissue is a 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 in vivo tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

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Mode for Carrying Out the Invention

[0010] I. Definitions

[0011] The term "binding activity" refers to the total strength of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and a binding partner of the molecule (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 1:1 interaction in a monovalent manner, this binding activity is specifically referred to as the intrinsic binding affinity ("affinity"). When the 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 molecule X for its partner Y can generally be represented by the dissociation constant (KD) or the "amount of analyte bound per unit amount of ligand" (hereinafter sometimes referred to as the "binding amount"). Generally, those skilled in the art will understand that the lower the numerical value of the dissociation constant (KD), the higher the binding activity, and the higher the numerical value of the "amount of analyte bound per unit amount of ligand" or "binding amount", the higher the binding activity. The binding activity can be measured by conventional methods known in the art, including those described herein. Specific and exemplary embodiments for measuring the binding activity are described below.

[0012] An antigen-binding molecule or antibody with "matured binding activity" or an antigen-binding molecule or antibody with "enhanced binding activity" refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) that result in improved binding activity of the antigen-binding molecule or antibody to the antigen, as compared to the unmodified parental antigen-binding molecule or parental antibody.

[0013] The terms "anti-CD137 antigen-binding molecule", "anti-CD137 antibody" or "antigen-binding molecule that binds CD137", "antibody that binds CD137" refer to an antigen-binding molecule or antibody that can bind CD137 with sufficient binding activity such that the antigen-binding molecule or antibody is useful as a diagnostic agent and / or therapeutic agent when targeting CD137. In certain embodiments, the anti-CD137 antibody binds to an epitope of CD137 that is conserved among CD137s from different species.

[0014] The term "anti-CD137 antigen-binding molecule or anti-CD137 antibody having CD137-binding activity dependent on a low-molecular compound" refers to an antigen-binding molecule or antibody whose binding activity to CD137 in the presence of the low-molecular compound is higher than its binding activity to CD137 in the absence of the low-molecular compound. In one embodiment, "in the presence of the low-molecular compound" refers to a condition where the low-molecular 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 irrelevant non-CD137 protein in the presence of the low-molecular compound is less than about 10% of the binding of the antigen-binding molecule or antibody to CD137 when measured (e.g., by radioimmunoassay (RIA) or surface plasmon resonance (SPR)). In certain embodiments, the anti-CD137 antigen-binding molecule or antibody has a dissociation constant (KD) 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 to 10 -10 M, 10 -7 M to 10 -9 M, e.g., 10 -7 M to 10 -8 M).

[0015] As used herein, the term "antigen-binding molecule" is used in its broadest sense and refers 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). Thus, for example, when the antigen is CD137, an antigen-binding molecule or antibody having such agonistic activity is referred to as a "CD137 agonistic antigen-binding molecule" or a "CD137 agonistic antibody", respectively. Similarly, for example, when the antigen is CD3, an antigen-binding molecule or antibody having such agonistic activity is referred to as a "CD3 agonistic antigen-binding molecule" or a "CD3 agonistic antibody", respectively.

[0017] As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, encompassing various antibody structures as long as they exhibit the desired antigen-binding activity.

[0018] An "antibody fragment" refers to a molecule other than the intact antibody that comprises 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" 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 inhibits the binding of the reference antibody or reference antigen-binding molecule to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody inhibits the binding of the aforementioned antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein. In one aspect, 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 chain and / or light chain is derived from a particular source or species, while the remaining portion of the heavy chain 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 heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. And some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0022] "Effector function" refers to the biological activities that vary depending on the isotype of an antibody and are attributed to the Fc region of the antibody. Examples of the effector functions of antibodies include the following: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0023] "Cytotoxic activity" refers to the activity that inhibits or interferes with the function of cells and / or causes cell death or destruction. Cytotoxic activity may be, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and cytotoxic activity by T cells, etc., or it may be caused by cytotoxic agents (e.g., radioisotopes and chemotherapeutic agents) such as immunoconjugates.

[0024] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) 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 follows the EU numbering system (also referred to as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0025] As used herein, the term "variant Fc region" includes an amino acid sequence that differs from that of the 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 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide, as compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions herein preferably have at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0026] In this specification, amino acid modifications or substitutions in the Fc region or constant region can be represented by a combination of the EU numbering system and amino acids. For example, S424N represents the substitution of serine (Ser) at position 424 of the EU numbering to asparagine (Asn). Also, EU424N represents the substitution of the amino acid at position 424 (regardless of type) to asparagine (Asn).

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

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

[0029] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody clearly excludes humanized antibodies that contain non-human antigen-binding residues.

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

[0031] "Acceptor human framework" as used herein is a framework that includes the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that is 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 contain the same amino acid sequence as those, or may contain changes in the amino acid sequence. 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 to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0032] The "human consensus framework" is a framework that indicates the amino acid residues that most commonly occur in a selected group 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 the 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 by Kabat et al. as described above. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al. as described above.

[0033] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein in said variable regions, all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the 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. The "humanized form" of an antibody (e.g., a non-human antibody) refers to the antibody that has been humanized.

[0034] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain would be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may be isolated by screening a complementary library of VL or VH domains using the 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 the variable domain of an antibody that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs that occur at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts that occur at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732 - 745 (1996)); and (d) Combinations of (a), (b), and / or (c) that include 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 the amino acid. For example, N99 represents asparagine (Asn) at position 99 of the Kabat numbering, and N99A represents substitution of asparagine (Asn) at position 99 of the Kabat numbering with alanine (Ala).

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

[0037] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of cells and / or causes cell death or destruction. 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 radioisotopes of Pb and Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleolytic enzymes and fragments thereof; antibiotics; toxins such as, for example, low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor or anticancer agents disclosed below.

[0038] An "isolated" antibody is one that has been separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, 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, for example, 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 the components of its original environment. An isolated nucleic acid includes nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its native chromosomal location.

[0040] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell into which it has been introduced. A vector can effect the expression of a nucleic acid operably linked thereto. Such a vector is also referred to herein as an "expression vector".

[0041] "Encoded nucleic acid encoding an anti-CD137 antigen-binding molecule" refers to one or more nucleic acid molecules encoding a polypeptide constituting the antigen-binding molecule. "Isolated nucleic acid encoding an anti-CD137 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including nucleic acid molecules carried on one 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 a cell (including progeny of such a cell) into which foreign nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny derived from such cells regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell and may include mutations. Mutant progeny having the same function or biological activity as that used when 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 substantially homogeneous population of antibodies. That is, the individual antibodies that make up that population are identical and / or bind to the same epitope, except for variant antibodies that may arise (e.g., variant antibodies that include naturally occurring mutations, or variant antibodies that occur during the production of a monoclonal antibody preparation. Such variants typically occur in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, methods utilizing transgenic animals that include all or part of the human immunoglobulin locus, and 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 radioactive label. A naked antibody may be present in a pharmaceutical formulation.

[0045] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, natural IgG antibodies are approximately 150,000 Dalton heterotetrameric glycoproteins composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain 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 the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0046] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after the sequences are aligned to obtain the maximum percent sequence identity and gaps are introduced if necessary, and when any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics, Inc.). One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0047] The ALIGN-2 array comparison computer program is the work of Genentech, and its source code has been submitted to the U.S. Copyright Office (Washington D.C., 20559) together with user documents and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems including Digital UNIX V4.0D. All array 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, it can also be said as a given amino acid sequence A having or containing 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. Here, X is the number of amino acid residues scored as matches that are identical in the alignment of A and B by the alignment program ALIGN-2, 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 be equal to the % amino acid sequence identity of B to A. Unless otherwise specifically stated, all % amino acid sequence identity values used in this specification are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0048] The term "pharmaceutical preparation" refers to a preparation in a form such that the biological activity of the active ingredient contained therein can exert an effect, and the preparation does not contain additional elements that are toxic to an unacceptable degree to the subject to which the preparation is administered.

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

[0050] The "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to the amount that is effective and necessary in the required dosage and over the required period to achieve the desired therapeutic or prophylactic result.

[0051] "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 a human.

[0052] As used herein, the term "CD137" refers to any native CD137 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. This term encompasses CD137 that has not undergone "full-length" processing, as well as any form of CD137 resulting from processing in cells. This term also encompasses naturally occurring variants of CD137, such as splice variants and allelic variants. The exemplary full-length amino acid sequence of human CD137 is shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_001552.2), and the exemplary amino acid sequence of the extracellular region of human CD137 is shown in SEQ ID NO: 2. The exemplary full-length amino acid sequence of mouse CD137 is shown in SEQ ID NO: 3 (NCBI Reference Sequence: NP_035742.1), and the exemplary amino acid sequence of the extracellular region of mouse CD137 is shown in SEQ ID NO: 4. The exemplary full-length amino acid sequence of monkey CD137 is shown in SEQ ID NO: 5 (NCBI Reference Sequence: ABY47575.1), and the exemplary amino acid sequence of the extracellular region of 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 in 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 in cancer cells (Labiano et al., Oncoimmunology, Vol. 24: e1062967 (2015)). Its natural ligand, CD137L, has been shown on antigen-presenting cells such as B cells, monocytes / macrophages, and dendritic cells (Watts et al., Annu. Rev. Immunol., Vol. 23: pp. 23-68 (2005)). In its interaction with its ligand, CD137 results in 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, Vol. 5: pp. 357-363 (2005), Watts et al., Annu. Rev. Immunol., Vol. 23: pp. 23-68 (2005)).

[0053] The terms “cancer,” “carcinoma,” and “carcinogenic” refer to or describe a physiological state in a mammal typically characterized by unregulated cell growth / proliferation.

[0054] The term “tumor” refers to all neoplastic cell growth and proliferation and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. The terms “cancer,” “carcinoma,” “carcinogenic,” “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 a degree of abnormal cell proliferation. In one aspect, the cell proliferative disorder is cancer.

[0056] As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and can be carried out for prevention or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, reducing the rate of progression of the disease, restoring or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the onset of a disease or slow the progression of the disease.

[0057] II. Compositions and Methods (Anti-CD137 Agonist Antigen-Binding Molecules) In one aspect, the present disclosure is based on anti-CD137 agonist antigen-binding molecules and their use. In certain embodiments, antibodies that bind to CD137 are provided. The antibodies of the present disclosure can exhibit activating, cytotoxic, or anti-tumor activity of immune cells, and are thus 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 · has CD137-binding activity that depends on a small molecule compound; · binds to the extracellular region of CD137; · forms a ternary complex with a small molecule compound and CD137; · binds to CD137 derived from human and monkey; · is an agonist of CD137 activity; · exhibits agonist activity against CD137 in the presence of a small molecule compound; · has low agonist activity against CD137 in the absence of a small molecule compound; and / or · substantially does not exhibit agonist activity against CD137 in the absence of a small molecule compound.

[0059] [Binding Activity of Antigen-Binding Molecule or Antibody] In certain embodiments, the binding activity of the antigen-binding molecules or antibodies provided herein, in the presence of a small molecule compound, 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 to 10 -10 M, 10 -7 M to 10 -9 M, e.g., 10 -7 M to 10 -8 M) and has a dissociation constant (KD).

[0060] In one embodiment, the binding activity of the antigen-binding molecule or antibody is measured by a radiolabeled antigen binding assay (RIA) and is represented by KD. In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is the minimum concentration of ( 125I) It is measured by equilibrating the Fab with the labeled antigen and then capturing the bound antigen with a plate coated with an anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To construct the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 - 5 hours at room temperature (approximately 23°C). In non-adsorbing plates (Nunc #269620), 100 pM or 26 pM of 125 I]-antigen is mixed with serial dilutions of the Fab of interest (in the same manner as the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997), for example). The Fab of interest is then incubated overnight, although this incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is achieved. Thereafter, the mixture is transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 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 less than 20% of the maximum binding is selected for use in the competitive binding assay.

[0061] In one aspect, the binding activity of the antibody is measured by means of a ligand capture method using, for example, BIACORE® T200 or BIACORE® 4000 (GE Healthcare, Uppsala, Sweden) with surface plasmon resonance analysis as the measurement principle. BIACORE® Control Software is used for instrument operation. In one aspect, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and a ligand capture molecule such as an anti-tag antibody, an anti-IgG antibody, protein A, etc. is immobilized on a sensor chip (GE Healthcare, Uppsala, Sweden) coated with carboxymethyl dextran. The ligand capture molecule is diluted using a 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. For the measurement of binding activity, a buffer solution containing 0.05% polysorbate 20 (also known as Tween®-20) is used as the measurement buffer, the flow rate is 10 - 30 μL / min, and the measurement temperature is preferably 25°C or 37°C. When the measurement is carried out by capturing the antibody as a ligand on the ligand capture molecule, after injecting the antibody to capture the target amount, serial dilutions (analytes) of the antigen and / or Fc receptor prepared using the measurement buffer are injected. When the measurement is carried out by capturing the antigen and / or Fc receptor as a ligand on the ligand capture molecule, after injecting the antigen and / or Fc receptor to capture the target amount, serial dilutions (analytes) of the antibody prepared using the measurement buffer are injected.

[0062] In one aspect, the measurement results are analyzed using BIACORE (registered trademark) Evaluation Software. Calculation of the kinetics parameter is performed by simultaneously fitting the sensorgrams of binding and dissociation using a 1:1 Binding model, and the association rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, especially when dissociation is fast and it is difficult to calculate the kinetics parameter, the equilibrium dissociation constant (KD) may be calculated using the Steady state model. As another parameter of the binding activity, the amount of analyte bound per unit amount of ligand (resonance unit: RU) can also be calculated by dividing the amount of analyte bound (resonance unit: RU) at a specific concentration of the analyte by the amount of ligand captured (RU).

[0063] [Binding Activity Dependent on Small Molecule Compounds] In one aspect, the anti-CD137 antigen-binding molecule or antibody has a CD137-binding activity that depends on a small molecule compound. In one non-limiting aspect, the anti-CD137 antigen-binding molecule or antibody has a higher binding activity to CD137 in the presence of the small molecule compound than in the absence of the small molecule compound. In a different aspect, the anti-CD137 antigen-binding molecule or antibody has a higher binding activity to CD137 in the presence of a high concentration of the small molecule compound than in the presence of a low concentration of the small molecule compound. In a preferred aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity to CD137 in the presence of the small molecule 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 3 times or more, 2×10 3 times or more, up to 3×10 3 times or more, 5×10 3 times or more, 1×10 4 times or more, 2×10 4 times or more, 3×10 4 times or more, 5×104 times or more, or 1×10 5 times or more. In a different preferred embodiment, the anti-CD137 antigen-binding molecule or antibody has a binding activity to CD137 in the presence of a small molecule compound that is higher than 2-fold, higher than 3-fold, higher than 5-fold, higher than 10-fold, higher than 15-fold, higher than 20-fold, higher than 25-fold, higher than 30-fold, higher than 50-fold, higher than 100-fold, higher than 200-fold, higher than 300-fold, higher than 500-fold, higher than 1×10 3 times, higher than 2×10 3 times, higher than 3×10 3 times, higher than 5×10 3 times, higher than 1×10 4 times, higher than 2×10 4 times, higher than 3×10 4 times, higher than 5×10 4 times or more, or 1×10 5 times or more.

[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 the "presence of a high concentration of a small molecule compound" can be, for example, 100nM or more, 500nM or more, 1μM or more, 3μM or more, 5μM or more, 10μM or more, 50μM or more, 100μM or more, 150μM or more, 200μM or more, 250μM or more, 300μM or more, 400μM or more, 500μM or more, or 1mM or more. Alternatively, the concentration here can be a sufficient amount that each anti-CD137 antigen-binding molecule or antibody shows the maximum binding activity. In one embodiment, the concentration of the low molecular weight compound in the "presence of a low concentration of the low 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 less, or 1 nM or less. A case where the concentration of the low molecular weight compound is zero or substantially zero can also be selected as one embodiment of a low concentration. Here, "substantially zero concentration" refers to, for example, the presence of a low molecular weight compound, but at such an extremely small concentration that it cannot be detected using current technology.

[0065] In one aspect, the binding activity to CD137 in the presence of a small molecule compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM 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 of the binding activity to CD137 in the absence of the small molecule compound. In one aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity to CD137 in the presence of a small molecule compound at 10 μM or more that 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 of the binding activity to CD137 in the absence of the small molecule compound. In one aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity to CD137 in the presence of a small molecule compound at 100 μM or more that 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 of the binding activity to CD137 in the absence of the small molecule compound.

[0066] In one aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity (KD) to CD137 in the presence of a small molecule compound at 10 μM or more 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 less, or 4x10 -7 M or less dissociation constant (KD), preferably 5x10 -7 M or less dissociation constant (KD). In a further aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity (KD) to CD137 in the absence of the small molecule compound that is so large (weak binding activity) as to be impossible to calculate by Biacore, or 1x10 -7 M or more, 5x10 -7 M or more, 7x10 -7 M or more, 8x10 -7 M or more, 9x10 -7 M or more, 1x10 -6 M or more, 2x10 -6 M or more.-6 M or more, or 4x10 -6 M or more dissociation constant (KD), preferably 1x10 -6 M or more dissociation constant (KD). In another aspect, the anti-CD137 antigen-binding molecule or antibody has a binding activity (KD) to CD137 in the presence of a low molecular weight compound of 100 μM or more 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 less, 4x10 -7 M or less, 3x10 -7 M or less, 2x10 -7 M or less, or 1x10 -7 M or less dissociation constant (KD), preferably 2x10 -7 M or less dissociation constant (KD). In a further aspect, the anti-CD137 antigen-binding molecule or antibody further has a binding activity (KD) to CD137 in the absence of the low molecular weight compound that is so large (weak binding activity) that it is impossible to calculate with Biacore, or 1x10 -7 M or more, 5x10 -7 M or more, 7x10 -7 M or more, 8x10 -7 M or more, 9x10 -7 M or more, 1x10 -6 M or more, 2x10 -6 M or more, 3x10 -6 M or more, or 4x10 -6 M or more dissociation constant (KD), preferably 1x10 -6 M or more dissociation constant (KD).

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

[0068] In one aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody in which the value of [binding activity (binding amount) to CD137 in the presence of a small molecule compound of 10 μM or more] / [binding activity (binding amount) to CD137 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 in which the value of [binding activity (binding amount) to CD137 in the presence of a small molecule compound of 100 μM or more] / [binding activity (binding amount) to CD137 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 can be selected from anti-CD137 antibodies containing HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 having the same amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 as those included 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 described in Table 17.

[0069] In one aspect, the reference anti-CD137 antigen-binding molecule is an antibody comprising, as a heavy chain variable region / light chain variable region combination, 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. In a preferred aspect, the reference antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375 / B167. In a further aspect, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a heavy chain variable region / light chain variable region combination. In a different preferred aspect, 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 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379. In a further aspect, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as a heavy chain variable region / light chain variable region combination. In a preferred aspect, the reference antigen-binding molecule comprises a human-derived heavy chain constant region and light chain constant region (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human lambda 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 having a binding activity (binding amount) to CD137 in the absence of a small molecule compound that is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and having a binding activity (binding amount) to CD137 in the presence of 10 μM or more of the small molecule compound that is the same as or higher than the binding activity of the reference anti-CD137 antigen-binding molecule to CD137 under the same conditions. In another different aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody having a binding activity to CD137 in the absence of a small molecule compound that is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and having a binding activity (binding amount) to CD137 in the presence of 10 μM or more of the small molecule compound that is the same as or higher than the binding activity (binding amount) of the reference anti-CD137 antigen-binding molecule to CD137 under the same conditions. In any of the above aspects, the reference anti-CD137 antigen-binding molecule can 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 as described in Table 17.

[0071] In one aspect, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising, as a combination of a heavy chain variable region / light chain variable region, the amino acid sequences of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as set forth in Table 17. In a preferred aspect, 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 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375 / B167. In a further aspect, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a combination of a heavy chain variable region / light chain variable region. In a different preferred aspect, 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 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379. In a further aspect, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as a combination of a heavy chain variable region / light chain variable region. In a preferred aspect, the reference antigen-binding molecule comprises a human-derived heavy chain constant region and light chain constant region (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human λ chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

[0072] In one aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (KD) to CD137 in the presence of a low-molecular-weight compound at 1 μM] / [binding activity (KD) to CD137 in the presence of the low-molecular-weight compound at 10 μM or higher] is the same as or greater than that of a reference antigen-binding molecule. In another aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (KD) to CD137 in the presence of a low-molecular-weight compound at 1 μM] / [binding activity (KD) to CD137 in the presence of the low-molecular-weight compound at 100 μM or higher] 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 can 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 as described in Table 17.

[0073] In one aspect, the reference antigen-binding molecule is an antibody that includes, as a combination of heavy chain variable region / light chain variable region, the amino acid sequences of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as set forth in Table 17. In a preferred aspect, the reference antigen-binding molecule is an antibody that includes HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 included in A375 / B167. In a further aspect, the reference antigen-binding molecule is an antibody that includes A375 / B167 as a combination of heavy chain variable region / light chain variable region. In a different preferred aspect, the reference antigen-binding molecule is an antibody that includes HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 that are the same as the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 included in A551 / B379. In a further aspect, the reference antigen-binding molecule is an antibody that includes A551 / B379 as a combination of heavy chain variable region / light chain variable region. In a preferred aspect, the reference antigen-binding molecule includes a human-derived heavy chain constant region and light chain constant region (for example, 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).

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

[0075] Details of an exemplary method for measuring the binding activity of an anti-CD137 antibody to CD137 are described below. In one aspect, the binding activity of the anti-CD137 antibody to CD137 is evaluated with a BIACORE™ T200. In a preferred aspect, this measurement is carried out at 37° C. using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 0.05% Tween 20 as the running buffer. In one aspect, in this measurement, the antibody is captured as a ligand by a ligand-capturing molecule for measurement. Specifically, first, an antibody solution prepared with the running buffer is allowed to interact with a chip obtained by immobilizing Sure Protein A (GE Healthcare) on a Series S Sensor Chip CM3 (GE Healthcare), whereby an appropriate amount (e.g., about 100 RU, 200 RU, 300 RU, 400 RU, or 500 RU) of the antibody is captured.

[0076] In a preferred embodiment, antibodies of about 100 to 500 RU, preferably about 250 to 400 RU are captured. Next, the binding activity of CD137 in the presence and absence of a small molecule compound is evaluated by interacting a CD137 solution prepared with a running buffer to which a small molecule compound has been added to reach a target concentration (for example, 1 μM, 10 μM, 50 μM or 100 μM), or a CD137 solution prepared with a running buffer not containing the small molecule compound. The concentration of CD137 in the CD137 solution can be determined as appropriate. For example, when hCD137-HisBAP (see Example 1-1) is used as an antigen, the antigen concentration is measured at 0 nM, 15.625 nM, 62.5 nM, 250 nM, and 1000 nM, respectively. In one embodiment, the dissociation constant (KD) of the anti-CD137 antibody for human CD137 is calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s) are calculated by globally fitting the sensorgram obtained by measurement with a 1:1 Langmuir binding model, and the dissociation constant KD (mol / L) is calculated from these values.

[0077] Details of a further exemplary method for measuring the binding activity of an anti-CD137 antibody to CD137 are described below. The binding of the anti-CD137 antibody to human CD137 is evaluated using a Biacore T200. The binding measurement to human CD137 was carried out at 37 °C using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 0.05 % Tween20 as the running buffer. First, an antibody solution prepared in the running buffer was allowed to interact with a chip in which Sure Protein A (GE Healthcare) was immobilized on a Series S Sensor Chip CM3 (GE Healthcare), and about 250 to 400 RU of the antibody was captured. Next, a human CD137 solution prepared in a running buffer supplemented with ATP to a target concentration (for example, 1 μM, 10 μM, 50 μM or 100 μM), or a human CD137 solution prepared in a running buffer without ATP was allowed to interact, and the binding activity with human CD137 in the presence and absence of ATP was evaluated. The antigen human CD137 was measured at antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM, and 1000 nM using hCD137-HisBAP prepared by the method of Example (1-1). The chip was regenerated using 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and the antibody was repeatedly captured and measured. The dissociation constant of each antibody for human CD137 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the sensorgram obtained by the measurement was globally fitted with a 1:1 Langmuir binding model to calculate the association rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) was calculated from these values.

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

[0079] As used herein, the terms "small molecule" and "small molecule compound" refer to natural or non-natural chemical substances other than "biopolymers" present in a living body. Preferably, they are target tissue-specific compounds or non-natural compounds, but are not limited thereto. In one aspect, the "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" as used in the present disclosure refers to a compound that is differentially present in cancer tissue as compared to non-cancer tissue. As used herein, the term "cancer" is generally used to represent a malignant neoplasm, which may be metastatic or non-metastatic. The term "metabolism" refers to chemical changes occurring within a biological tissue and includes "assimilation" and "dissimilation". Assimilation refers to the biosynthesis or accumulation of molecules, and dissimilation refers to the decomposition of molecules. A "metabolite" is an intermediate or product resulting from substance metabolism.

[0080] The term "target tissue" means 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 distinguishable tissue such as various organs, or a pathologically distinguishable tissue such as a healthy tissue and a tissue in a diseased state. In a specific aspect, the target tissue is a tumor tissue. On the other hand, "non-target tissue" means a tissue other than the target tissue in a living body.

[0081] The term "tumor tissue" means a tissue containing at least one tumor cell. Tumor tissue usually consists of a population of tumor cells (parenchyma) that forms the main body of the tumor and connective tissue and blood vessels (stroma) that exist between them and support the tumor. Some are clearly distinguishable between the two, while others are a mixture of both. There may also be infiltration of immune cells, etc. in the tumor tissue. On the other hand, "non-tumor tissue" means tissue in a living body other than tumor tissue. Healthy tissue / normal tissue without a disease state is a typical example of non-tumor tissue.

[0082] As a non-limiting aspect of the cancer tissue-specific compound or cancer tissue-specific metabolite used in the present disclosure, at least one compound selected from the compounds detailed below is preferably mentioned. The at least one compound means that the binding activity to an antigen by the same antigen-binding domain described later depends on a kind of cancer tissue-specific compound or cancer tissue-specific metabolite, and also includes the case where it depends on a plurality of kinds 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 a target tissue as compared to a 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 the target tissue but not in the non-target tissue, or being present in the non-target tissue but not in the target tissue. In another embodiment, a target tissue-specific compound can be a compound defined by quantitative target tissue specificity, such as being present in the target tissue at a different concentration (e.g., a higher or lower concentration) as compared to the non-target tissue. In certain embodiments, 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, 100-fold or more, 10 3 -fold or more, 10 4 -fold or more, 10 5 -fold or more, 10 6 -fold or more, or at a concentration even higher than that, in the target tissue. In another embodiment, the target tissue-specific compound is present in the non-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, 100-fold or more, 10 3 -fold or more, 10 4 -fold or more, 10 5 -fold or more, 10 6at a concentration that is several-fold or more higher in non-target tissues. In certain embodiments, the target tissue-specific compound is present in target tissue at a statistically significantly higher or lower concentration (i.e., p-value < 0.05 and / or q-value < 0.10 as determined using either Welch's t-test or Wilcoxon's rank sum test) compared to non-target tissues. In certain embodiments, the target tissue-specific compound is a tumor tissue-specific compound.

[0084] In certain embodiments, a 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 life activities (primary metabolite), or a product produced by metabolism not necessarily required for life activities (secondary metabolite). Examples of primary metabolites include sugars, proteins, lipids, nucleic acids, etc. Examples of secondary metabolites include antibiotics and pigments, etc. The metabolite may be a biopolymer or a low molecular weight molecule. In certain embodiments, a biopolymer is a molecule with a molecular weight of approximately 5000 or more consisting of one or more repeating units, and includes, for example, polysaccharides, polypeptides, and polynucleotides. In certain embodiments, a low molecular weight molecule is a molecule with a molecular weight of approximately 500 or less and is a chemical substance present in the living body. In a further embodiment, the tumor tissue-specific compound is a low molecular weight metabolite specifically produced 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 the tumor tissue (e.g., immune cells, etc.) or stromal cells present in the tumor tissue (such as cancer-associated fibroblast (CAF), etc.). Examples of immune cells infiltrating the tumor tissue include dendritic cells, inhibitory dendritic cells, regulatory T cells, exhausted T cells, myeloid-derived suppressor cells (MDSC), etc. In a further embodiment, metabolites produced by cells present in the tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.) and released extracellularly when those cells undergo cell death by apoptosis, necrosis, etc. may also be included in the tumor tissue-specific compounds of the present disclosure.

[0085] To identify tumor tissue-specific compounds, in addition to transcriptome-level analysis (e.g., as exemplified by Dhanasekaran et al. (Nature (2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. U.S.A. (2004) 101, 811-816), or Perou et al. (Nature (2000) 406, 747-752)) or proteome-level analysis (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15), Hood et al. (Mol. Cell. Proteomics (2005) 4, 1741-1753)), metabolomics (metabolomics) analysis centered on metabolic profiling is appropriately used. That is, for identifying 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, etc. alone and / or in combination can be appropriately used.

[0086] In certain embodiments, 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 glucose 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), inosine, (2) Amino acids such as alanine, glutamic acid, aspartic acid, (3) Metabolites of amino acids such as kynurenine, anthranilic acid, 3-hydroxykynurenine, kynurenic acid, (4) Metabolites of arachidonic acid such as prostaglandin E2, (5) Primary metabolites of the glycolysis or Krebs cycle such as lactic acid, succinic acid, and citric acid, 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), inosine, etc. When tumor cells undergo cell death, it is known that a large amount of ATP within the cells leaks out extracellularly. Therefore, the ATP concentration in tumor tissue is significantly higher compared to normal tissue (PLoS One. (2008) 3, e2599). AMP is metabolized by cell surface enzymes such as extracellular - 5'-nucleotidase (ecto - 5'-nucleotidase) (CD73) (Resta and Thompson (Immunol. Rev. (1998) 161, 95 - 109) as well as Sadej et al. (Melanoma Res. (2006) 16, 213 - 222)). Adenosine is a purine nucleoside that constitutively exists in the extracellular environment at low concentrations, but a significant increase in extracellular adenosine concentration 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 tumors and immune cells (Kobie et al. (J. Immunol. (2006) 177, 6780 - 6786)), and increased activity has been found 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 tissue 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 tissue also express ATP - degrading enzymes and produce adenosine (Proc. Natl. Acad. Sci. USA (2006) 103(35), 13132 - 13137, Curr. Med. Chem. (2011) 18, 5217 - 5223).The adenosine produced is thought to create an immunosuppressive environment in tumor tissues via adenosine receptors such as the A2A receptor (Curr. Med. Chem. (2011) 18, 5217-5223). From the above, ATP, ADP, AMP, or adenosine, etc., which are thought to accumulate at high concentrations in tumor tissues due to the metabolism of purine nucleotides, are cited as examples of the tumor tissue-specific compounds used in the present disclosure. Furthermore, since adenosine is decomposed into inosine by adenosine deaminase, inosine accumulates at high concentrations.

[0088] In certain embodiments, the nucleosides having a purine ring structure include adenosine-containing compounds. In certain embodiments, examples of adenosine-containing compounds 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 γ-thiotriphosphate (ATPγS), and the like. In another embodiment, the nucleosides having a purine ring structure include inosine, which is a metabolite of adenosine. Furthermore, in certain embodiments, the 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, aspartic acid, etc. In tumor cells, the rate of intracellular uptake of glutamine, which acts as a nitrogen carrier in vivo, is increased, and such glutamine uptake and the resulting conversion to glutamate and lactate (glutaminolysis) are considered to be characteristics 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, while plasma glutamine levels are decreased, glutamate concentrations are increased (Droge et al. (Immunobiology (1987) 174, 473-479)), and 13 By metabolic studies of 13 C-radiolabeled glucose, 13 C-labeled succinic acid, 13 C-labeled alanine, 13 C-labeled glutamate, and

[0090] (3) Metabolites of amino acids such as kynurenine, anthranilic acid, 3-hydroxykynurenine, kynurenic acid, etc. a correlation was observed among the concentrations of C-labeled citrate. From the above, alanine, glutamate, aspartic acid, etc., which are considered to accumulate at high concentrations in tumor tissues by glutaminolysis and the like, are cited as examples of the tumor tissue-specific compounds used in the present disclosure.Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzyme that is highly expressed in many cancers such as melanoma, colon cancer, and renal cancer (Uyttenhove et al. (Nat. Med. (2003) 9, 1269-1274)), and IDO catalyzes the conversion of tryptophan to kynurenine. In gliomas that do not express IDO, kynurenine is produced from tryptophan by liver tryptophan 2,3-dioxygenase (TDO) (Opitz et al. (Nature (2011) 478(7368), 197-203)). IDO is also expressed in dendritic cells infiltrating tumor tissues, and dendritic cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). In addition, IDO is expressed in myeloid-derived suppressor cells (MDSC) in tumor tissues, and MDSC 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, which is a precursor of NAD. Kynurenine is converted to kynurenic acid by kynurenine aminotransferase. From the above, kynurenine, as well as its metabolites, such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, are exemplified as tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in the present disclosure.

[0091] (4) Metabolites of arachidonic acid such as prostaglandin E2 Prostaglandin E2 (PGE2) promotes the growth of colon cancer cells and suppresses 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 has been mainly 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 of breast cancer (Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)) and rapid disease progression of ovarian cancer (Denkert et al. (Mod. Pathol. (2006) 19, 1261-1269)). Regulatory T cells infiltrating tumor tissues also produce PGE2 (Curr. Med. Chem. (2011) 18, 5217-5223). From the above, metabolic products of arachidonic acid such as PGE2 can be cited as examples of tumor tissue-specific compounds used in the present disclosure, particularly tumor cell-specific metabolic products and immune cell-specific metabolic products infiltrating tumor tissues. In addition to PGE2, thromboxane A2 (TXA2) is produced in increased amounts in tumor tissues such as colorectal cancer (J. Lab. Clin. Med. (1993) 122, 518-523).

[0092] (5) Primary metabolites of glycolysis or the Krebs cycle such as lactic acid, succinic acid, citric acid, etc. The glycolytic phenotype characterized by the upregulation of glycolytic enzymes such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH) (Embden-Myerhof pathway) is conventionally known to be a characteristic of solid tumors as the Warburg effect. It is known that lactate, the end product of glycolysis, and succinic acid and citric acid produced by the Krebs cycle accumulate in tumor tissues (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). From the above, primary metabolites produced by glycolysis, such as lactate, succinic acid, and citric acid, are exemplified as tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in the present disclosure. Also, it is known that succinic acid, which is present intracellularly at a high concentration due to cell death, leaks extracellularly (Nature Immunology, (2008) 9, 1261-1269). Therefore, in tumor tissues where cell death occurs frequently, it is considered that the concentration of succinic acid is increasing.

[0093] (6) Metabolites of nicotinamide such as 1-methylnicotinamide, etc. In multiple human tumor tissues, it is known that nicotinamide N-methyltransferase is highly expressed. 1-Methylnicotinamide, a stable metabolite of nicotinamide by this enzyme, is known to be secreted extracellularly by tumor cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). From the above, 1-methylnicotinamide and the like, which are considered to accumulate at a high concentration in tumor tissues by the metabolism of nicotinamide, are exemplified as tumor tissue-specific compounds used in the present disclosure.

[0094] The "antigen-binding molecule" of the present disclosure includes an "antigen-binding domain". As long as it binds to the target antigen, any structurally configured domain can be used as the "antigen-binding domain". In one aspect, examples of the antigen-binding domain of the present disclosure include, for example, the variable regions of the heavy and / or light chains of an antibody, Avimer (International Publication WO2004 / 044011, WO2005 / 040229) containing a module of about 35 amino acids (A domain) included in various cell membrane proteins in vivo, Adnectin (International Publication WO2002 / 032925) containing a 10Fn3 domain in fibronectin, which is a glycoprotein expressed on the cell membrane, Affibody (International Publication WO1995 / 001937) with the IgG-binding domain consisting of 58 amino acids of Protein A as a scaffold, DARPins (Designed Ankyrin Repeat proteins) (International Publication WO2002 / 020565) containing an ankyrin repeat (AR), which is a 33-amino acid repeat sequence, as a backbone, Anticalin (International Publication WO2003 / 029462) containing a lipocalin such as neutrophil gelatinase-associated lipocalin (NGAL) as a backbone, variable lymphocyte receptor (VLR) (International Publication WO2008 / 016854) which is a protein functioning in the acquired immune system of agnathans such as lampreys and hagfishes and contains a leucine-rich-repeat (LRR) module, and the like. In a specific aspect, the antigen-binding domain of the present disclosure includes the variable regions of the heavy and light chains of an antibody. In a further aspect, examples of the antigen-binding domain of the present disclosure include, for example, scFv (single chain Fv), single chain antibody, Fv, scFv2 (single chain Fv 2), Fab, or F(ab')2, and the like.

[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 NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NO: 17, 18, 19, or 20. 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 any one amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NO: 17, 18, 19, or 20.

[0096] In one aspect, the anti-CD137 antigen-binding molecule is an antibody comprising, as a heavy chain variable region / light chain variable region combination, 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. In a preferred aspect, 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 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375 / B167. In a further aspect, the anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a heavy chain variable region / light chain variable region combination. In a different preferred aspect, 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 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A551 / B379.

[0097] In one aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 NO: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 NO:27, 28, and 29. In one aspect, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1 comprising any one amino acid sequence selected from SEQ ID NO: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 NO:27, 28, and 29.

[0109] In one aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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, an anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences 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, one or more amino acids of any of the above 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): positions 3, and / or 6 -HVR-L1 (SEQ ID NO: 32): positions 4, 5, 9, and / or 11 -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: -HVR-H2 (SEQ ID NO: 8): K5H or S; S6G; T7S; E10Y; D13E; S14Q; V17G or L -HVR-H3 (SEQ ID NO: 17): A3P, K or I; F6E -HVR-L1 (SEQ ID NO: 21): R4S; Y5T; Y9F; E11N -HVR-L3 (SEQ ID NO: 27): E6P; H7A; Q8I

[0132] All possible combinations of the above substitutions are included 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 aspects, the anti-CD137 antigen-binding molecule or antibody is humanized. In one aspect, the anti-CD137 antigen-binding molecule or antibody comprises the HVRs in any of the above aspects and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another aspect, the anti-CD137 antigen-binding molecule or antibody comprises the HVRs in any of the above aspects and further comprises a heavy chain variable region (VH) or a light chain variable region (VL) that comprises a framework (FR) sequence. In one aspect, 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 aspect, 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, the anti-CD137 antigen-binding molecule or antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CD137 antigen-binding molecule or antibody comprising such sequence retains the ability to bind 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 of the HVRs (i.e., within the FRs). Optionally, the anti-CD137 antibody comprises the VH sequence in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53, including those with post-translational modifications of the sequence. In certain embodiments, 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 of the amino acid sequences selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3 comprising any one of the amino acid sequences selected from SEQ ID NO: 17, 18, 19, or 20. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0135] In another aspect, there is provided an anti-CD137 antigen-binding molecule or antibody 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 to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-CD137 antigen-binding molecule or antibody comprising such a 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 of the HVRs (i.e., in the FRs). Optionally, the anti-CD137 antigen-binding molecule or antibody includes the VL sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60, including those with post-translational modifications of the sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising any one of the amino acid sequences selected from SEQ ID NO: 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 of the amino acid sequences selected from SEQ ID NO: 27, 28, and 29. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

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

[0137] The correspondence between the heavy and light chain variable regions of the preferred anti-CD137 antigen-binding molecules or antibodies of the present disclosure, and the amino acid sequences of their HVR1, 2, and 3, and the SEQ ID NOs, is shown in the following list.

[0138]

Table 1

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

[0140] In a preferred aspect, any of the anti-CD137 antigen-binding molecules or antibodies including the above-described HVR, heavy chain variable region, and / or light chain variable region have a binding activity to CD137 that depends on the above-described small molecule compound.

[0141] [Constant 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 the 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 wild-type sequence constant region. Exemplary heavy chain constant regions derived from wild-type antibodies include, for example, the heavy chain constant regions of human IgG1 (SEQ ID NOs: 61, 62), human IgG2, human IgG3, human IgG4, etc. Exemplary light chain constant regions derived from wild-type antibodies include, for example, human κ chain, human λ chain (e.g., SEQ ID NO: 63).

[0142] As used herein, "parent constant region" or "parent Fc region" refers to the constant region or Fc region prior to the introduction of the amino acid modifications described herein. Also, "parent antigen-binding molecule" refers to an antigen-binding molecule that includes a parent constant region or a parent Fc region. In some embodiments, the parent Fc region is a wild-type sequence Fc region (or the Fc region of a wild-type antibody). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, etc. Antibodies can be derived from humans or monkeys (e.g., cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon). Wild-type 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 can 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 that includes a native sequence Fc region or a parental Fc region. In some embodiments, the mutant Fc region includes at least one amino acid modification. In a further embodiment, the amino acid modification increases the isoelectric point (pI) of the mutant Fc region compared to the parental Fc region. Without being bound by a particular theory, the pH of a biological fluid (e.g., plasma) is thought to be within a neutral pH range. In a biological fluid, 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 surface of endothelial cells having a net negative charge by physicochemical Coulombic interactions compared to an antigen-binding molecule or antibody that does not have an increased pI. Thereby, the agonist antigen-binding molecule (or antibody), or the agonist antigen-binding molecule (or antibody) bound to an antigen, approaches the surface of Fcγ receptor-expressing cells, and the binding of the antigen-binding molecule or antibody to Fcγ receptor-expressing cells can be increased. In an anti-CD137 agonist antigen-binding molecule or antibody in which the binding activity to the Fcγ receptor contributes to the CD137 agonist activity, an anti-CD137 agonist antigen-binding molecule or antibody in which the binding to Fcγ receptor-expressing cells is increased by an amino acid modification that increases the pI can exhibit higher CD137 agonist activity compared to an anti-CD137 agonist antigen-binding molecule or antibody that does not include the amino acid modification that increases the pI.

[0144] In the present disclosure, the pI may be either the theoretical pI or the measured pI. The value of pI can be measured, for example, by an isoelectric focusing method known to those skilled in the art. The value of the theoretical pI can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx). In this case, the characteristics of the antibody may be reflected in the calculation formula. For example, (i) Usually, Cys conserved in the antibody forms a disulfide bond and has no charge on the side chain. Such Cys may be excluded from the calculation, and only free-type Cys that does not form a disulfide bond may be added to the calculation. Also, (ii) Regarding antibodies, the charged state, that is, the isoelectric point, may change due to post-translational modification. Considering such post-translational modification, the calculation formula may be modified as follows: (a) When the N-terminal of the heavy chain is Q (glutamine), assuming that pyroglutamylation occurs, the amino group at the N-terminal is excluded from the calculation, (b) When the C-terminal of the heavy chain is K (lysine), assuming that cleavage occurs, K (for 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 (i) and (ii) above may be reflected in the calculation formula.

[0145] In one embodiment, the pI value can increase, for example, by 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 the modification.

[0146] In one aspect, amino acid modifications related to pI increase, and methods for increasing the pI of an antigen-binding molecule or antibody are detailed in Section III. Compositions and Methods (agonist antigen-binding molecules comprising isoelectric point (pI)-elevated mutant Fc regions) herein. It will be understood by those skilled in the art that any amino acid modification and method for increasing pI described in Section III. Compositions and Methods (agonist antigen-binding molecules comprising isoelectric point (pI)-elevated mutant Fc regions) are applicable to an anti-CD137 antigen-binding molecule or antibody.

[0147] In one aspect, the anti-CD137 antigen-binding molecule or antibody has a mutant Fc region with an increased pI, and the mutant 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 as represented by EU numbering. In a further aspect, the mutant Fc region with an increased pI comprises Arg or Lys at each of the selected positions.

[0148] In a further aspect, the anti-CD137 antigen-binding molecule or antibody has a mutant Fc region with an increased pI, and the mutant 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 represented by EU numbering. In a further aspect, the mutant Fc region with an increased pI comprises an amino acid modification at position 311, 343, or 413 as represented by EU numbering. In a further aspect, the mutant Fc region with an 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 an increased pI, comprising 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, represented by EU numbering. In a further aspect, the mutant Fc region with an increased pI comprises Arg or Lys at each of the selected positions.

[0150] In one aspect, the anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a mutant Fc region comprising the amino acid modifications described in Table 2 below.

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

[0152] In one aspect, the anti-CD137 antigen-binding molecule or antibody comprises a mutated Fc region created by introducing amino acid modifications into the Fc region of the native sequence. In one aspect, the mutated Fc region has increased binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb as compared to the Fc region of the native sequence or the parental Fc region. Preferably, the mutated Fc region has increased binding activity to FcγRIIb as compared to the Fc region of the native sequence or the parental Fc region. It has been reported that an anti-CD137 antibody comprising a mutated Fc region with increased binding activity to FcγRIIb has increased agonist activity as compared to an anti-CD137 antibody comprising an Fc region of the native sequence. In one aspect, the amino acid modifications that increase the binding activity to FcγRIIb may be, for example, the amino acid modifications described in WO2012 / 115241, WO2014 / 030728, WO2014 / 163101, and / or WO2017 / 104783. In a preferred aspect, the modification that increases the binding activity to FcγRIIb 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 represented by EU numbering.

[0153] "Fcγ receptor" (referred to herein 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 virtually means any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) containing isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) containing isoforms FcγRIIa (including allotypes H131 (H-type) and R131 (R-type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) containing isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and furthermore, includes any and all human FcγR, FcγR isoforms or allotypes that have not been discovered, but are not limited thereto. FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb. Furthermore, a splicing variant called FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes AAI46679.1 registered in NCBI, and all splicing variants registered in NCBI, 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 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)) and 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 replaced by arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25 (1990)).

[0155] FcγR includes, but is not limited to, FcγR derived from human, mouse, rat, rabbit, and monkey, and may be derived from any organism. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0156] In another aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising a mutant Fc region with increased binding activity to FcγRIIb, which comprises 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; represented by EU numbering.

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

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

[0159] In one aspect, the present disclosure provides a modified Fc region in which at least one amino acid is modified, and the binding activity of the modified Fc region to FcgammaRIIb is the same as or higher than that of a reference Fc region. In one aspect, the reference Fc region is an Fc region containing any combination of amino acid modifications described in Table 3 above. In a preferred aspect, the reference Fc region is an Fc region contained in TT14 (SEQ ID NO: 149), TT16 (SEQ ID NO: 150), MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154) which are heavy chain constant regions. In a preferred aspect, the reference Fc region is an Fc region contained in MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154) which are heavy chain constant regions.

[0160] In another aspect, the present disclosure provides an isolated agonist antigen-binding molecule or antibody comprising a mutant Fc region associated with an increase in Fcγ receptor (preferably FcγRIIb) binding activity and an increase in pI. In certain aspects, the mutant Fc regions described herein include at least two amino acid modifications in the parental Fc region. As described above, an antigen-binding molecule or antibody with an increased pI is more strongly attracted to the surface of endothelial cells having a net negative charge by physicochemical Coulomb interactions as compared to an antigen-binding molecule or antibody with an unchanged pI. Thus, in an agonist antigen-binding molecule or antibody in which the binding activity to the Fcγ receptor (preferably FcγRIIb) contributes to agonist activity, it is possible to increase the agonist activity of the antigen-binding molecule or antibody by combining an amino acid modification that increases the Fcγ receptor (preferably FcγRIIb) and an amino acid modification that increases the pI.

[0161] In one aspect, the anti-CD137 antigen-binding molecule or antibody comprises a mutated Fc region that includes both of the above-described 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, an antigen-binding molecule or antibody with an increased pI is more strongly attracted to the surface of endothelial cells having a net negative charge by physicochemical Coulomb interactions compared to an antigen-binding molecule or antibody with an unchanged pI. Thus, in an anti-CD137 agonist antigen-binding molecule or antibody in which binding activity to an Fcγ receptor (preferably FcγRIIb) contributes to CD137 agonist activity, by combining an amino acid modification that increases the Fcγ receptor (preferably FcγRIIb) and an amino acid modification that increases the pI, it is possible to increase the agonist activity of the anti-CD137 antigen-binding molecule or antibody.

[0162] In one aspect, the present disclosure provides a polypeptide comprising a mutated 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 represented by 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 represented by EU numbering.

[0163] In another aspect, the present disclosure provides a polypeptide comprising a mutated Fc region with increased FcγRIIb binding activity and increased pI, comprising any one of the following amino acid modifications (1) to (26): represented by EU numbering, (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 aspect, the mutant Fc region of the present disclosure includes any combination of amino acid modifications described in Table 4 below.

[0165] [Table 4]

[0166] In one aspect, the mutant Fc region including any combination of amino acid modifications described in Table 4 above lacks the amino acid at position 447 based on EU numbering. In a preferred aspect, the mutant Fc region including any combination of amino acid modifications described in Table 4 above lacks the amino acids at positions 446 and 447 based on EU numbering.

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

[0168] Furthermore, amino acid modifications carried out for other purposes can be combined in the mutant Fc region 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 for improving antibody heterogeneity or stability (WO2009 / 041613) may be added. Alternatively, a polypeptide having a property of promoting antigen clearance described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, a polypeptide having specific binding properties to a target tissue described in WO2013 / 180200, or a polypeptide having a property of repeatedly binding to multiple antigen molecules described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752 can be combined with the mutant Fc region described herein. Alternatively, for the purpose of conferring binding activity to other antigens, the amino acid modifications disclosed in EP1752471 and EP1772465 may be combined in the CH3 of the mutant Fc region described herein.

[0169] In one aspect, the 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, the 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 mutated Fc region described above has binding activity to CD137 that is dependent on the small molecule compound described above.

[0171] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises the following variable region and constant region: a variable region comprising the above-described HVR, heavy chain variable region, and / or light chain variable region; and the above-described mutated Fc region. 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 described in Table 52.

[0172] In a further aspect, the present disclosure provides an antigen-binding molecule or antibody that binds to the same epitope of CD137 as the anti-CD137 antigen-binding molecule or antibody 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 the small molecule compound). For example, in a particular embodiment, as a combination of heavy chain variable region / light chain variable region, an antigen-binding molecule or antibody that binds to the same epitope as the anti-CD137 antigen-binding molecule or antibody comprising A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, and / or A549 / B167 described in Table 17 is provided. In one embodiment, the anti-CD137 antigen-binding molecule or antibody having CD137 binding activity that depends on the antigen-binding activity that depends on the 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).

[0173] In a further aspect, the present disclosure provides an antigen-binding molecule or antibody that competes with the anti-CD137 antigen-binding molecule or antibody provided herein with respect to 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, with respect to the binding site to CD137, as a combination of heavy chain variable region / light chain variable region, A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, competes with an anti-CD137 antigen-binding molecule or antibody comprising 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 embodiments is a monoclonal antibody, including chimeric, humanized, or human antibodies. 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 defined herein.

[0175] In a further aspect, 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. Agonist Activity of Anti-CD137 Antigen-Binding Molecule or Antibody In certain embodiments, the anti-CD137 antigen-binding molecule or antibody in the present disclosure has CD137 agonist activity. CD137 signaling not only stimulates IFN-γ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), but is also known to promote DC activation as shown 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 sensitivity of T lymphocytes to activation-induced cell death (reviewed in Snell et al., 2011). Among these phenomena, the physiological phenomena observed after CD137 signaling on T cells are mediated by downstream signals activated by the CD137 signal, such as TRAF2, TRAF1, particularly NF-kappaB, JNK, Erk, Akt, survivin, Bcl-XL, and / or Bcl-2 (Ward-Kavanagh et al., Immunity, 44:1005 (2016)).

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

[0178] In certain aspects, the anti-CD137 antigen-binding molecules or antibodies in the present disclosure have CD137 agonist activity that is dependent on a small molecule compound. In one non-limiting aspect, the anti-CD137 antigen-binding molecule or antibody has a higher CD137 agonist activity against CD137 in the presence of the small molecule compound than the CD137 agonist activity of CD137 in the absence of the small molecule compound. In a different aspect, the anti-CD137 antigen-binding molecule or antibody has a higher CD137 agonist activity in the presence of a high concentration of the small molecule compound than 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 the 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 3 times or more, 2×10 3 times or more, up to 3×10 3 times or more, 5×10 3times or more, 1 × 10 4 times or more, 2 × 10 4 times or more, 3 × 10 4 times or more, 5 × 10 4 times or more, or 1 × 10 5 times or more.

[0179] As the concentration of the low molecular weight compound, any concentration can be selected 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 transmits a CD137 signal 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 having CD137 binding activity dependent on the low molecular weight compound has CD137 agonist activity dependent on the low molecular weight compound. However, on the other hand, since the measurement methods for binding activity and agonist activity are different, it will be understood by those skilled in the art that the concentration of the low molecular weight compound at which a difference in binding activity is detected and the concentration of the low molecular weight compound at which a difference in agonist activity is detected may be different (for example, in an anti-CD137 antigen-binding molecule or antibody in which the CD137 binding activity in the presence of 10 μM of the low molecular weight compound is 2 times or more the CD137 binding activity in the absence of the low molecular weight compound, the CD137 agonist activity (measured value) in the presence of 10 μM of the low molecular weight compound may be less than 2 times the CD137 agonist activity (measured value) in the absence of the low molecular weight compound). Also, it will be understood by those skilled in the art 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 exhibits agonist activity against CD137 (i) in the presence of a low molecular weight compound of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM, and (ii) does not substantially exhibit agonist activity against CD137 in the absence of the low molecular weight compound, or the agonist activity against CD137 in the absence of the low molecular weight compound is low (compared to the presence of the low molecular weight compound).

[0181] In one aspect, when the agonistic activity of an anti-CD137 antigen-binding molecule or antibody is evaluated in a) the agonistic activity assay (PBMC) detailed in C. Measurement method (assay), the anti-CD137 antigen-binding molecule or antibody shows (i) agonistic activity against CD137 in the presence of a small molecule compound at 250 μM, and (ii) the agonistic activity against CD137 in the absence of the small molecule compound is lower (compared to the presence of the small molecule compound). In a further aspect, the anti-CD137 antigen-binding molecule or antibody shows (i) agonistic activity against CD137 in the presence of a small molecule compound at 250 μM, and (ii) substantially no agonistic activity against CD137 in the absence of the small molecule compound.

[0182] In one aspect, when the agonistic activity of an anti-CD137 antigen-binding molecule or antibody is evaluated in b) the agonistic activity assay (reporter gene assay) detailed in C. Measurement method (assay), the anti-CD137 antigen-binding molecule or antibody shows (i) agonistic activity against CD137 in the presence of a small molecule compound at 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM, and (ii) substantially no agonistic activity against CD137 in the absence of the small molecule compound or the agonistic activity is lower (compared to the presence of the small molecule compound). The antibody concentration in the reporter gene assay can be arbitrarily selected. For example, the final concentration of the antibody is 0, 0.001, 0.01, 0.1, 1, or 10 μg / mL. In a preferred aspect, the final concentration of the antibody is 0.1 μg / mL or 1 μg / mL.

[0183] In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in the C. measurement method (assay), when the final concentration of the antibody is 0.1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of a 10 μM 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in the C. measurement method (assay), when the final concentration of the antibody is 0.1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of a 100 μM 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in the C. measurement method (assay), when the final concentration of the antibody is 0.1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of a 250 μM 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In any of the above aspects, further, the anti-CD137 antigen-binding molecule or antibody at 0.1 μg / mL substantially does not exhibit CD137 agonist activity in the absence of the small molecule compound.

[0184] In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement method (assay), when the final concentration of the antibody is 1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of 10 μM of the 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement method (assay), when the final concentration of the antibody is 0.1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM of the 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In one aspect, in the b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement method (assay), when the final concentration of the antibody is 0.1 μg / mL, (i) the CD137 agonist activity (relative luminescence) of the anti-CD137 antigen-binding molecule or antibody in the presence of 250 μM of the 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 (ii) the CD137 agonist activity (relative luminescence) in the absence of the small molecule compound. In any of the above aspects, further, the anti-CD137 antigen-binding molecule or antibody at 1 μg / mL substantially does not exhibit CD137 agonist activity in the absence of the small molecule compound.

[0185] 2. Antibody Fragments 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, and other fragments described hereinbelow. For a review of certain 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); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab’)2 fragments that include salvage receptor binding epitope residues and that have extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0186] A diabody is an antibody fragment with two antigen-binding sites that can 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 tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0187] A single domain antibody is an antibody fragment that includes 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, the 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 made by various techniques including, but not limited to, proteolytic digestion of a full antibody as described herein and production by recombinant host cells (e.g., E. coli or phage).

[0189] 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Specific 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 a further example, a chimeric antibody is a “class switch” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include their antigen-binding fragments.

[0190] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody 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 the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) to, for example, 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 are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. 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); 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 the consensus sequences of specific subgroups of human antibodies of the 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 (somatic mutated) framework regions or human germline framework regions (see, for example, 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 a variety of methods 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 a transgenic animal modified to produce a fully human antibody or a complete antibody with human variable regions in response to an antigen challenge (loading). Such animals typically contain all or a portion of the human immunoglobulin locus, which may replace the endogenous immunoglobulin locus or be present extrachromosomally or randomly integrated within the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. See, for example, Lonberg, Nat. Biotech. 23:1117-1125 (2005) for a review of methods for obtaining human antibodies from transgenic animals. Also see, 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 K-M MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0195] Human antibodies can also be made by methods based on hybridomas. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already 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, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing 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. Such variable domain sequences can then be combined with the desired human constant domains. Methods for selecting human antibodies from antibody libraries are described below.

[0197] 5. Library-Derived Antibodies The antibodies of the present disclosure may be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies with desired binding properties are known in the art. 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 certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and the phage library can be screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages display antibody fragments, typically 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 to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), 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. Finally, naive libraries can also be made synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning V-gene segments from stem cells prior to rearrangement and using PCR primers that encode the hypervariable CDR3 region and contain random sequences to achieve recombination in vitro. Patent documents describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, as well as 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 molecule or antibody having antigen-binding activity dependent on a low-molecular compound of the present disclosure may be selected by screening from a library of antigen-binding molecules. As the library, the above-described combinatorial library may be used. Further, the library of antigen-binding molecules may be a library (naïve library) in which the repertoire of antigen-binding molecules is not biased, or a biased library. Examples of the latter library include a library of antigen-binding molecules to which binding activity to a predetermined compound is previously imparted. In a specific embodiment, the library of antigen-binding molecules is a library of antigen-binding molecules into which amino acid modifications for imparting binding activity to a predetermined compound have been previously introduced. As an example of such a library, for example, the library described in International Publication WO2015 / 083764 can be referred to.

[0201] 6. Bispecific Antibodies In a specific embodiment, the antibody provided herein is a multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is a monoclonal antibody having binding specificities at at least two different sites. In a specific embodiment, one of the binding specificities is for CD137 and the other is for any other antigen. In a specific embodiment, the bispecific antibody may bind to two different epitopes of CD137. The bispecific antibody may be used to localize a cytotoxic agent to cells expressing CD137. The bispecific antibody can be prepared as a full-length antibody or as an antibody fragment.

[0202] In one aspect, the anti-CD137 antigen-binding molecule or antibody of the present disclosure is a bispecific antibody in which one arm has a small molecule-dependent CD137 binding activity and the other arm binds to an antigen different from CD137. The "antigen" different from CD137 is not limited to a specific structure in terms of its structure. In another sense, the antigen can be either an inorganic substance or an organic substance. Exemplary antigens are disclosed herein (e.g., "IV. Compositions and Methods (Antigen-Binding Molecules Whose Binding Activity to Antigens Varies Depending on the Concentration of Compounds), B. Antigens"). In one aspect, as the antigen, antigens expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues or inflammatory tissues are preferred.

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

[0204] Also included herein are modified antibodies with three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576 A1).

[0205] As used herein, antibodies or fragments also include "dual acting Fabs" or "DAFs" (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820) that include one antigen binding site that binds to CD137 and a different antigen.

[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 variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for introducing substitutional mutations include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1 and are detailed below with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest and the products screened for the 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 grouped 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 affecting chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the replacement of one member of these classes with one from another class.

[0210] One type of substitution variant involves substitution of 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 has a modification (e.g., improvement) (e.g., increased affinity, decreased immunogenicity) in a particular biological property compared to the parent antibody and / or will substantially retain a particular biological property of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be made as appropriate 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 antibody is displayed on a phage and screened for a particular biological activity (e.g., binding affinity).

[0211] Modifications (e.g., substitutions) can be made in the HVRs, for example, to improve the affinity of an antibody. Such modifications can be made at the “hot spots” of the HVRs, i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or at residues that contact the antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and rescreening from a secondary library 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 created. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves an HVR-directed approach that randomizes some HVR residues (e.g., 4-6 residues at a time). 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 can be made within one or more HVRs so long as such modifications do not substantially decrease the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially decrease binding affinity can be made in the HVRs. Such modifications can be, for example, outside of the antigen contact residues of the HVRs. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

[0213] A method useful for identifying residues or regions of an antibody that can be targeted for mutagenesis is what is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one residue or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified, replaced with a neutral or negatively charged amino acid (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 showed functional sensitivity to this initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex may be analyzed to identify the contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the substitution candidates. Variants can be screened to determine whether they possess the desired properties.

[0214] Insertions of amino acid sequences include fusions in the range of the length of a polypeptide from 1 residue to more than 100 residues at the amino terminus and / or carboxyl terminus, as well as insertions of single or multiple amino acid residues into the interior of the sequence. Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus. Other insertion mutants of the antibody molecule include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0215] b) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or removal of glycosylation sites to the antibody can be readily accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0216] If the antibody includes an Fc region, the carbohydrates added thereto may be modified. Naturally-occurring antibodies produced by mammalian cells typically include branched, biantennary oligosaccharides that are usually added by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and also include fucose added to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present disclosure may be performed to create antibody variants with certain improved properties.

[0217] In one aspect, there is provided an antibody variant having a carbohydrate structure lacking fucose (directly or indirectly) added to the Fc region. For example, the amount of fucose in such an antibody can be 1% - 80%, 1% - 65%, 5% - 65% or 20% - 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry as described, for example, in WO2008 / 077546. Asn297 represents the asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to minor sequence diversity between 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 can have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related 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. J. Mol. Biol. 336:1239 - 1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., particularly Example 11) and knockout cell lines such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0218] Also provided are antibody variants having bisected oligosaccharides, for example, where the bisected oligosaccharide added to the Fc region of the antibody is 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.); US 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 added 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 variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein, thereby generating an Fc region variant (which may also be referred to as a "modified Fc region"). The Fc region variant may comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0220] In certain embodiments, antibody variants with some, but not all, effector functions are also contemplated within the present disclosure, and such effector functions make the antibody a desirable candidate for applications where its in vivo half-life is important, but certain effector functions, such as complement and ADCC, are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm a decrease / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements can be performed to confirm that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity) while maintaining FcRn binding activity. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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. Patent 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)).Effector cells useful for such measurement methods include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model as described, for example, in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Also, to confirm that the antibody cannot bind to C1q and thus lacks CDC activity, C1q binding measurement may be performed. See, for example, the C1q and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402. Also, CDC measurement may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Furthermore, FcRn binding and determination of clearance / half-life in vivo can also be performed using methods known in the art (see, for example, Petkova, S.B. 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 so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581), and Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327.

[0222] Certain antibody variants with increased or decreased binding to FcRs are described. (See U.S. Patent 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 (residues in 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), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0225] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn: which is responsible for transferring maternal IgG 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 that increase binding to FcRn in the Fc region. Such Fc variants include those with substitutions at one or more of the 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 WO94 / 29351.

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

[0228] Details of an exemplary method for measuring the binding activity of the antibody Fc region to each human Fcγ receptor (FcγR) are described below. In one aspect, the binding activity of the antibody Fc region to FcγR is evaluated with BIACORE® T200. In a preferred aspect, this measurement is carried out at 25°C using 50 mM phosphate, 150 mM NaCl, 0.05 w / v%-P20, pH 7.4 as the measurement buffer. Specifically, first, a sensor chip immobilized with CaptureSelect® Human Fab-lambda Kinetics Biotin Conjugate (ThermoFisher scientific) as a molecule for ligand capture is used, and an antibody containing a modified Fc region is captured at about 1000 RU. Human FcγR is diluted to 8 nM for FcγRIa and 1000 nM for other FcγR in the measurement buffer and bound to the captured antibody. The binding activity of each antibody to each FcγR is evaluated by calculating the amount of FcγR bound per unit amount of antibody (RU) using Biacore T200 Evaluation Software 2.0. In one aspect, the binding activity of the 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 measurement method may be the extracellular domain of FcγR prepared by the following method. First, the gene of the extracellular domain of FcγR is synthesized by a method known to those skilled in the art. At this time, the sequence of each FcγR is prepared based on the information registered in NCBI. Specifically, for FcγRI, it is prepared based on the sequence of NCBI accession # NM_000566.3, for FcγRIIa, it is based on the sequence of NCBI accession # NM_001136219.1, for FcγRIIb, it is based on the sequence of NCBI accession # NM_004001.3, and for FcγRIIIa, it is based on the sequence of NCBI accession # NM_001127593.1, and a His tag is added to the C-terminus. For the polymorphic site of FcγRIIa, J. Exp. Med., 1990, 172, 19-25 is referred to, and for the polymorphic site of FcγRIIIa, J. Clin. Invest., 1997, 100, 1059-1070 is referred to for preparation. The obtained gene fragment is inserted into an animal cell expression vector to prepare an expression vector. The prepared expression vector is transiently introduced into FreeStyle293 cells (Invitrogen) derived from human fetal renal carcinoma cells, and the target protein is expressed. After the culture supernatant is recovered, it is passed through a 0.22 μm filter and purified in principle by the following 4 steps. The first step is cation exchange column chromatography (SP Sepharose FF), the second step is affinity column chromatography for the His tag (HisTrap HP), the third step is gel filtration column chromatography (Superdex200), and the fourth step is sterile filtration. However, for FcγRI, anion exchange column chromatography using Q sepharose FF is performed in 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 by methods such as PACE from the obtained value (Protein Science, 1995, 4, 2411-2423).

[0230] In one aspect, the binding activity of the antibody Fc region to human FcRn is measured by a ligand capture method using, for example, BIACORE® T200 with surface plasmon resonance analysis as the measurement principle.

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

[0232] d) Cysteine-modified antibody variant In certain embodiments, it may be desirable to generate cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residues to be replaced occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, and the reactive thiol groups may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as further detailed herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). The cysteine-modified antibodies may be generated, for example, as described in U.S. Patent No. 7,521,541.

[0233] e) Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known in the art and readily available. Moieties suitable 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 / prolylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in manufacture due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary widely and if more than one polymer is attached they may be the same or different molecules. In general, the number and / or type of polymer 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 is to be used in therapy under defined conditions, and the like.

[0234] In another aspect, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one aspect, 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 and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature that kills cells in proximity to the antibody-non-protein moiety.

[0235] B. Recombinant Methods and Constructs For example, as described in U.S. Patent No. 4,816,567, antibodies can be produced using recombinant methods and constructs. In one aspect, 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 of the antibody and / or an amino acid sequence comprising the VH (e.g., the light and / or heavy chains of the antibody). In a further aspect, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further aspect, a host cell comprising such a nucleic acid is provided. In one such aspect, the host cell comprises (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 (e.g., is transformed). In one aspect, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells)). In one aspect, a method of making an anti-CD137 antigen-binding molecule or antibody is provided that includes culturing a host cell comprising a 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 the recombinant production of an anti-CD137 antigen-binding molecule or antibody, a nucleic acid encoding an antibody (such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0237] Host cells suitable for cloning or expressing vectors encoding antibodies include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (In addition, see Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. 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 from the bacterial cell paste into the soluble fraction and further purified.

[0238] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast, including strains of fungi and yeast in which the glycosylation pathway has been "humanized" to result in the production of antibodies with a partial or complete human glycosylation pattern, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0239] Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that are used for conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0240] Plant cell cultures can also be utilized as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants.

[0241] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension would be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human fetal kidney lines (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; and FS4 cells, etc. 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, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

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

[0243] 1. Binding Assays and Other Assays In one aspect, the 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., 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), a competition assay in the presence of the small molecule compound can be used to identify an antigen-binding molecule or antibody that competes with an anti-CD137 antigen-binding molecule or antibody comprising, as a combination of a heavy chain variable region / light chain variable region, A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, and / or A549 / B167 as set forth in Table 17. 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, as a combination of a heavy chain variable region / light chain variable region, A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, and / or A549 / B167 as set forth in Table 17. Detailed exemplary methods for mapping the epitope to which an antigen-binding molecule or antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In one aspect, an anti-CD137 antigen-binding molecule or antibody having CD137-binding activity that depends on the antigen-binding activity that depends on the 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 competitive assay using an antibody, immobilized CD137 is incubated in a solution containing a first labeled antibody that binds to CD137 (e.g., an anti-CD137 antibody comprising a combination of a heavy chain variable region / light chain variable region such as A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, and / or A549 / B167 as described in Table 17) and a second unlabeled antibody that is tested for its ability to compete with the first antibody 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 greater, 50 μM or greater, 100 μM or greater, 150 μM or greater, 200 μM or greater, or 250 μM or greater). The second antibody can be present in the hybridoma supernatant. As a control, immobilized CD137 is incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that permit 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 the immobilized CD137 is substantially decreased in the test sample compared to the control sample, this indicates that the second antibody is competing 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). It will be understood by those skilled in the art that the assay can be similarly performed for antigen-binding molecules other than antibodies.

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

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

[0248] a) Agonist activity assay (PBMC) In one aspect, the agonist 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 a small molecule compound added. In one aspect, the agonist activity against CD137 in a solution with a small molecule compound added and the agonist activity against CD137 in a solution without a small molecule compound added are each evaluated by the amount of cytokine production (e.g., IL-2, IFN-γ, and / or IL-6 production) measured 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 respective solution. In one aspect, the solution with a small molecule compound added is adjusted such that the adjusted concentration of the small molecule compound is 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM. In a further aspect, the CD137-expressing cells used are isolated human peripheral blood mononuclear cells (PBMCs) or T cells expanded from isolated human PBMCs.

[0249] In one embodiment, human PBMCs isolated from the blood collection of healthy subjects by centrifugation at 400 x g for 30 minutes at room temperature are used. Preferably, human PBMCs isolated in the following two steps are used. In the first step, blood diluted with PBS is added after centrifugation of Leucosep (greiner bio-one) added with Ficoll-Paque PLUS (GE Healthcare) at 1000 x g for 1 minute at room temperature, and centrifugation is performed at 400 x g for 30 minutes at room temperature. In the second step, after the buffy coat is collected from the tube after centrifugation, it is washed with 60 mL of PBS (Wako). Details of an exemplary method for measuring CD137 agonist activity using human PBMCs are described below. In the following examples, ATP is exemplarily used as a small molecule compound, but other small molecule compounds are not excluded. In one embodiment, the isolated human PBMCs are diluted to a cell density of 5 x 10 6 / mL in a medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific)). Thereafter, the isolated human PBMCs are contacted with an anti-human CD3ε antibody and / or an anti-human CD28 antibody. Thereby, CD137 expression is induced in human PBMCs. Preferably, to the isolated human PBMCs (100 μL at a cell density of 5 x 10 6 / mL), 50 μL of an anti-human CD3ε antibody (clone SP34, manufactured by BD) diluted in a medium at 0.04 μg / mL and an anti-human CD28 antibody (BD, clone: CD28.2) at 20 μg / mL are added.

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

[0251] In another embodiment, the amount of IFN-γ production is measured within about 48 hours after contacting the human PBMCs with the anti-human CD3ε antibody and / or anti-human CD28 antibody. Preferably, th...

Claims

Claim 1. An anti-CD137 antibody comprising any combination of VH, VL, CH, and CL selected from the following (i) to (xxxviii): (i) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 64, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (ii) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 66, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (iii) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 67, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (iv) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 68, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (v) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 69, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (vi) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 70, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (vii) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 71, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (viii) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 73, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (ix) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 75, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (x) VH comprising the amino acid sequence of SEQ ID NO: 43, CH comprising the amino acid sequence of SEQ ID NO: 78, VL comprising the amino acid sequence of SEQ ID NO: 54, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xi) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xiii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xiv) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 85, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63; (xv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 65, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxiii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 81, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxiv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 83, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; (xxxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing 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 amino acid sequence of SEQ ID NO: 82, 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 amino acid sequence of SEQ ID NO: 83, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxvii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 84, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; and (xxxviii) VH 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.

2. An isolated nucleic acid encoding the anti-CD137 antibody according to claim 1.

3. A vector into which the nucleic acid according to claim 2 has been introduced.

4. A host cell comprising the nucleic acid according to claim 2, or the vector according to claim 3.

5. A method for producing an anti-CD137 antibody, the method comprising culturing the host cell according to claim 4 so that the anti-CD137 antibody is produced.

6. An immunoconjugate comprising the anti-CD137 antibody according to claim 1 and a cytotoxic agent.

7. A pharmaceutical preparation comprising the anti-CD137 antibody according to claim 1 or the immunoconjugate according to claim 6; and a pharmaceutically acceptable carrier.

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