HK40076755A

HK40076755A - Anti-cd137 antigen-binding molecule for use in cancer treatment

Info

Publication number: HK40076755A
Application number: HK62022066286.5A
Authority: HK
Inventors: 樱井实香; 成田义规; 谷口健治; 三上纮史; 堀川小百合; 内川亮; 小野夏生; 滨田孝树
Original assignee: 中外制药株式会社
Priority date: 2020-02-12
Filing date: 2021-02-10
Publication date: 2023-02-17

Description

anti-CD 137 antigen binding molecules for cancer therapy

Technical Field

The present disclosure relates to anti-cancer agents comprising anti-CD 137 antigen-binding molecules, and therapeutic methods that use such agents in combination with another anti-cancer agent.

Background

Cancer is a fatal disease that, except for some cases, is difficult to completely cure. The therapeutic results of chemotherapeutic agents as the primary treatment method are not very good. It has been proposed that not only heterogeneity of cancer cells themselves plays an important role, but also the tumor microenvironment as a factor that makes cancer treatment difficult (non-patent document 1). Recently, it has been shown that unresectable malignant melanoma and the like may be cured with an anti-CTLA-4 antibody that inhibits the immunosuppressive function of CTLA-4 to promote T cell activation (non-patent document 2). In 2011, anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the Food and Drug Administration (FDA) as the first immune-activating antibody drug in the world. Furthermore, inhibitory antibodies against PD-1 and PD-L1 (other immune checkpoint molecules than CTLA-4) have been reported to have therapeutic effects (non-patent document 3) and have been approved by the FDA.

It is understood that T cells, which play an important role in tumor immunity, are activated by two signals: 1) Binding of a T Cell Receptor (TCR) to an antigenic peptide presented by a Major Histocompatibility Complex (MHC) class I molecule, and activation of the TCR; 2) The binding of a costimulatory molecule on the surface of a T cell to its ligand on an antigen presenting cell and the activation of the costimulatory molecule. In addition, it has been described that activation of costimulatory molecules belonging to the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) including CD137 (4-1 BB) on the surface of T cells is important for T cell activation (non-patent document 4).

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

CD137 agonist antibodies have been shown to have anti-tumor effects in mouse models, and this is mainly caused by the experimental activation of CD 8-positive T cells and NK cells in mouse models (NPL 6). However, side effects due to non-specific hepatotoxicity of CD137 agonist antibodies have become clinical and non-clinical problems, hindering the anticipated progress of drug development (non-patent document 7, non-patent document 8). It is suggested that the side effects are mainly caused by activation of immune cells in non-tumor, non-immune tissues (e.g., liver), which involves binding of an antibody to an Fc γ receptor through an antibody constant region (non-patent document 9). On the other hand, it has been reported that in order for an agonistic anti-TNF receptor superfamily member antibody to exhibit agonistic activity in vivo, it is necessary to crosslink the antibody with cells expressing Fc γ receptor (cells expressing Fc γ RII) (non-patent document 10). That is, binding of a CD137 agonist antibody to an Fc γ receptor is involved in both the pharmacodynamic effects of the anti-tumor effect of the antibody and its side effects such as hepatotoxicity. Thus, increasing binding between the antibody and the Fc γ receptor is expected to enhance drug efficacy, but may also increase hepatotoxic side effects, while decreasing binding between the antibody and the Fc γ receptor may decrease side effects but also decrease drug efficacy. To date, there has been no report on the separation of the pharmacological effects and side effects of CD137 agonist antibodies. Furthermore, the antitumor effect of the CD137 agonist antibody itself is not all clinically effective, and further improvement in the drug efficacy while avoiding toxicity is desired. Therefore, it is desired to develop a novel drug capable of inducing an anti-tumor immune response while reducing those side effects.

When a therapeutic antibody is administered to a living body, its target antigen is desirably specifically expressed only at a lesion site. However, in many cases the same antigen is also expressed in non-diseased sites, i.e. normal tissues, which may be responsible for the generation of undesirable side effects from a therapeutic point of view. For example, although antibodies against tumor antigens may exhibit cytotoxic activity against tumor cells through ADCC or the like, they may also damage normal cells if the same antigens are expressed in the normal cells. In order to solve the above problems, a technique of searching for an antigen-binding molecule that changes the antigen-binding activity depending on the concentration of a certain compound has been developed, focusing on the phenomenon that the compound is present in a large amount in a target tissue (e.g., tumor tissue) (e.g., patent document 1).

In recent years, the efficacy of immunotherapeutic agents represented by inhibitors targeting immune checkpoint molecules such as CTLA-4, PD-1, PD-L1 has been clinically proven. However, these drugs are not effective for all patients, and further improvement in drug efficacy is required. Regarding the combination of immunotherapy, it has been confirmed that the combined use of nivolumab and ipilimumab improves the drug efficacy in melanoma, compared to the use of ipilimumab alone (non-patent document 11).

Reference list

Patent literature

[PTL 1]WO2013/180200

Non-patent document

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

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

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

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

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

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

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

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

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

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

[NPL 11]N Eng J Med(2015)vol.373,p.23-34

Disclosure of Invention

Technical problem

The present disclosure relates to anti-cancer agents comprising anti-CD 137 antigen binding molecules, and combination therapies with another anti-cancer agent.

Means for solving the problems

In order to provide an anti-CD 137 antigen-binding molecule having an immune cell activating effect, a cytotoxic activity, or an antitumor activity while having a reduced effect on non-tumor tissues such as normal tissues and less side effects, and to provide a method of using the anti-CD 137 antigen-binding molecule, the present invention provides an anticancer agent comprising the anti-CD 137 antigen-binding molecule as an active ingredient, characterized in that its binding activity to CD137 varies depending on various compounds (e.g., small molecule compounds) in a target tissue (e.g., tumor tissue). The disclosure also provides combination therapies using such anti-cancer agents comprising an anti-CD 137 antigen binding molecule and another agent.

In particular, the present disclosure provides anti-cancer agents comprising anti-CD 137 antigen binding molecules, methods of use thereof, combination therapies using such an anti-cancer agent and another anti-cancer agent, kits, and the like, as exemplarily described below.

[1]

An anticancer agent comprising, as an active ingredient, an anti-CD 137 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.

[1.1]

An anticancer agent comprising, as an active ingredient, an anti-CD 137 antigen-binding molecule comprising:

(a) VH having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs 43 to 53; or alternatively

(b) A VL having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NO 54 to 60.

[1.2]

An anti-cancer agent comprising an anti-CD 137 antigen binding molecule comprising any combination of a VH and a VL selected from the following (a) to (m):

(a) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 43; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54;

(b) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55;

(c) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 45; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55;

(d) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 46; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54;

(e) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54;

(f) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48; and VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56;

(g) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 49; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 57;

(h) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 50; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 58;

(i) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 59;

(j) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51; and VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 60;

(k) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 52; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 60;

(l) VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 50; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 59; and

(m) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 53; and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54.

[2]

(a) VH comprising the amino acid sequence of SEQ ID NO 43; and VL comprising the amino acid sequence of SEQ ID NO: 54;

(b) VH comprising the amino acid sequence of SEQ ID NO: 44; and VL comprising the amino acid sequence of SEQ ID NO: 55;

(c) VH comprising the amino acid sequence of SEQ ID NO 45; and VL comprising the amino acid sequence of SEQ ID NO: 55;

(d) VH comprising the amino acid sequence of SEQ ID NO 46; and VL comprising the amino acid sequence of SEQ ID NO: 54;

(e) VH comprising the amino acid sequence of SEQ ID NO: 47; and VL comprising the amino acid sequence of SEQ ID NO: 54;

(f) VH comprising the amino acid sequence of SEQ ID NO 48; and VL comprising the amino acid sequence of SEQ ID NO 56;

(g) VH comprising the amino acid sequence of SEQ ID NO: 49; and VL comprising the amino acid sequence of SEQ ID NO: 57;

(h) VH comprising the amino acid sequence of SEQ ID NO 50; and VL comprising the amino acid sequence of SEQ ID NO: 58;

(i) VH comprising the amino acid sequence of SEQ ID NO: 51; and VL comprising the amino acid sequence of SEQ ID NO 59;

(j) VH comprising the amino acid sequence of SEQ ID NO: 51; and VL comprising the amino acid sequence of SEQ ID NO 60;

(k) VH comprising the amino acid sequence of SEQ ID NO 52; and VL comprising the amino acid sequence of SEQ ID NO: 60;

(l) VH comprising the amino acid sequence of SEQ ID NO 50; and VL comprising the amino acid sequence of SEQ ID NO 59; and

(m) a VH comprising the amino acid sequence of SEQ ID NO: 53; and VL comprising the amino acid sequence of SEQ ID NO 54.

[2.1]

[1] The anticancer agent according to any one of [1] to [2], wherein the anti-CD 137 antigen-binding molecule has a CD137 binding activity depending on a small molecule compound.

[2.2]

An anti-cancer agent comprising an anti-CD 137 antigen-binding molecule having a CD137 binding activity that is dependent on a small molecule compound, wherein the anti-CD 137 antigen-binding molecule competes for binding to CD137 with the antigen-binding molecule according to any one of [1] to [2.1] in the presence of 10 μ Μ or more, 50 μ Μ or more, 100 μ Μ or more, 150 μ Μ or more, 200 μ Μ or more, or 250 μ Μ or more of the small molecule compound.

[2.3]

An anti-cancer agent comprising an anti-CD 137 antigen binding molecule having a small molecule compound dependent CD137 binding activity, wherein the anti-CD 137 antigen binding molecule binds to the same epitope of CD137 as the antigen binding molecule of any one of [1] to [2.1] in the presence of 10 μ Μ or more, 50 μ Μ or more, 100 μ Μ or more, 150 μ Μ or more, 200 μ Μ or more, or 250 μ Μ or more of the small molecule compound.

[2.4]

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

[2.5]

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

[2.6]

[1] The anticancer agent of any one of [3.4], wherein the anti-CD 137 antigen-binding molecule is a monoclonal antibody or an antigen-binding fragment thereof.

[3]

[1] The anti-cancer agent of any one of [2.6], wherein the anti-CD 137 antigen-binding molecule is a human antibody, a humanized antibody, or a chimeric antibody, or an antigen-binding fragment of any one thereof.

[3.1]

[1] The anticancer agent according to any one of [1] to [3], wherein the anti-CD 137 antigen-binding molecule is a full-length IgG1 antibody.

[3.2]

[1] The anti-cancer agent of any one of [3.1], wherein the anti-CD 137 antigen binding molecule comprises an altered Fc region in which at least one amino acid is altered, wherein the altered Fc region has increased binding activity to fcyriib compared to a parent Fc region that does not comprise the amino acid alteration.

[3.3]

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

[3.4]

The anti-cancer agent of [3.2] or [3.3], wherein the at least one amino acid change is a combination of amino acid substitutions G236N/H268D/A330K according to EU numbering.

[3.5]

[1] The anti-cancer agent of any one of [3.4], wherein the anti-CD 137 antigen binding molecule comprises an altered Fc region in which at least one amino acid is altered, wherein the anti-CD 137 antigen binding molecule has an increased isoelectric point (pI) as compared to a parent anti-CD 137 antigen binding molecule comprising a parent Fc region that does not comprise the amino acid alteration.

[3.6]

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

[3.7]

The anti-cancer agent of [3.5] or [3.6], wherein the at least one amino acid change is (i) an amino acid substitution of P343R, (ii) a combination of amino acid substitutions Q311R/P343R, or (iii) a combination of amino acid substitutions Q311R/D413K, according to EU numbering.

[4]

[1] The anti-cancer agent of any one of [3.7], wherein the anti-CD 137 antigen-binding molecule comprises an altered Fc region, and the altered Fc region comprises any one combination of amino acid alterations according to EU numbering selected from the group consisting of:

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。

[4.1]

the anticancer agent of any one of [3.2] to [4], wherein the altered Fc region is derived from a human IgG1Fc region.

[4.2]

The anti-cancer agent of any one of [3.2] to [4.1], wherein the altered Fc region further comprises deletions at positions 446 and 447 according to EU numbering.

[4.3]

[1] The anti-cancer agent of any one of [4.2], wherein the anti-CD 137 antigen-binding molecule comprises a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOS 64 to 85.

[5]

An anti-cancer agent comprising an anti-CD 137 antigen binding molecule comprising any combination of VH, VL, CH, and CL selected from the group consisting of (i) to (xxxviii) below:

(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 comprising the amino acid sequence of SEQ ID NO 43; CH comprising the amino acid sequence of SEQ ID NO 80; VL comprising the amino acid sequence of SEQ ID NO: 54; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xii) VH comprising the amino acid sequence of SEQ ID NO 43; CH, comprising the amino acid sequence of SEQ ID NO: 82; VL comprising the amino acid sequence of SEQ ID NO: 54; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xiii) VH comprising the amino acid sequence of SEQ ID NO 43; CH comprising the amino acid sequence of SEQ ID NO: 84; VL comprising the amino acid sequence of SEQ ID NO: 54; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xiv) VH comprising the amino acid sequence of SEQ ID NO 43; CH comprising the amino acid sequence of SEQ ID NO 85; VL comprising the amino acid sequence of SEQ ID NO: 54; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xv) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 65; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xvi) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 72; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xvii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO 74; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xviii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 75; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xix) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 77; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xx) VH comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO: 78; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxi) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO: 79; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 80; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxiii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO: 81; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxiv) VH comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO: 82; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxv) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 83; VL comprising the amino acid sequence of SEQ ID NO 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxvi) 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 59; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxvii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 72; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxviii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO 74; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxix) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 75; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxx) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO: 77; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxxi) VH comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO: 78; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxxii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO: 79; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxxiii) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO 80; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxxiv) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the amino acid sequence of SEQ ID NO: 81; VL comprising the amino acid sequence of SEQ ID NO 60; and CL comprising the amino acid sequence of SEQ ID NO 63;

(xxxv) VH comprising the amino acid sequence of SEQ ID NO: 51; CH comprising the 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.

[6]

[1]To [5 ]]The anti-cancer agent according to any one of the above,it is administered to a cancer patient with: (i) Selected from the group consisting of B cells, dendritic cells, natural killer cells, macrophages and CD8 ⁺ A solid cancer infiltrated by one or more of T cells, and/or (ii) regulatory T (Treg) cells or CD4 ⁺ T cell infiltrated solid cancer.

[6.1]

[1]To [6 ]]The anti-cancer agent of any one of, which is administered to a patient suffering from CD8 ⁺ A cancer patient with a T cell infiltrated solid cancer.

[7]

[1] The anti-cancer agent of any one of [6.1], which is administered to a patient having a cancer refractory to treatment with an immune checkpoint inhibitor.

[8]

[1] The anti-cancer agent of any one of [1] to [7], which is administered to a cancer patient having one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[8.1]

[1] The anti-cancer agent of any one of [1] to [8], which is administered to a cancer patient having one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[9]

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

[9.1]

[9] The anti-cancer agent of (a), wherein the anti-cancer agent is characterized by being administered concurrently with other anti-cancer agents.

[9.2]

[9] The anti-cancer agent of (a), wherein the anti-cancer agent is characterized by being administered before or after the administration of the other anti-cancer agent.

[10]

[9] The anticancer agent of any one of [9.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[10.1]

[10] The anti-cancer agent of (a), wherein the other anti-cancer agent is a chemotherapeutic agent.

[10.2]

[10] The anticancer agent of [ 1] or [10.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from the group consisting of antimetabolites, plant alkaloids, and platinum compounds.

[10.3]

[10] The anti-cancer agent of (a), wherein the additional anti-cancer agent is a T cell activation agonist.

[10.4]

[10] The anticancer agent of [10.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[11]

[10] The anti-cancer agent of (a), wherein the other anti-cancer agent is an immune checkpoint inhibitor.

[12]

[10] The anti-cancer agent of [ 1] or [11], wherein the other anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[12.1]

[10] The anticancer agent of any one of [11] and [12], wherein the other anticancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[12.2]

[9] The anti-cancer agent of any one of [12.1] administered to a patient having a cancer refractory to treatment with an immune checkpoint inhibitor.

[12.3]

[12.2] the anticancer agent, wherein the cancer refractory to treatment by the immune checkpoint inhibitor is a cancer having a gene mutation in JAK1, JAK2 and/or B2M.

[13]

[10] The anti-cancer agent of (a), wherein the additional anti-cancer agent is a T cell redirecting antigen binding molecule.

[13.1]

[10] The anticancer agent according to [ 5 ] or [13], wherein the other anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[13.2]

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

[14]

[9]To [9.2]The anti-cancer agent of any one of, wherein said other anti-cancer agent is one or more of depleting and/or inactivating regulatory T cells, CD4 ⁺ An agent for one or more cells of T cells, B cells, NK cells and macrophages.

[15]

[9] The anti-cancer agent of any one of [ 1] to [14], which is administered to a cancer patient having one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[15.1]

[9] The anti-cancer agent of any one of [1] to [15], which is administered to a cancer patient suffering from one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[16]

[1] The anti-cancer agent of any one of [1] to [5], for use in combination therapy with at least one other anti-cancer agent to enhance activation of T cells in tumor tissue of an individual.

[16.1]

[16] The anticancer agent of (a), wherein when the expression level of at least one gene selected from the group consisting of CD8b1, gzmb, prf1 and Ifng in the tumor tissue is increased, it is judged that the activation of T cells in the tumor tissue has been enhanced.

[17]

[1]To [5]]The anti-cancer agent of any one of the above for use in combination therapy with at least one other anti-cancer agent to promote CD8 in tumor tissue of an individual ⁺ Proliferation of T cells.

[17.1]

[16] The anticancer agent according to any one of [1] to [17], which is administered simultaneously with other anticancer agents.

[17.2]

[16] The anticancer agent according to any one of [1] to [17], which is administered before or after the administration of the other anticancer agent.

[17.3]

[16] The anticancer agent of any one of [17.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[17.4]

[16]To [17.2 ]]The anti-cancer agent of any one of, wherein said other anti-cancer agent is one or more of depleting and/or inactivating regulatory T cells, CD4 ⁺ An agent for one or more cells of T cells, B cells, NK cells and macrophages.

[18]

A pharmaceutical composition for the treatment of cancer comprising the anti-CD 137 antigen-binding molecule of any one of [1] to [5] in combination with at least one other anti-cancer agent.

[18.1]

[18] The pharmaceutical composition of (a), wherein the anti-CD 137 antigen binding molecule is used for simultaneous administration to a patient with other anti-cancer agents.

[18.2]

[18] The pharmaceutical composition of (a), wherein the anti-CD 137 antigen-binding molecule is used for administration to a patient before or after administration of other anti-cancer agents.

[19]

[18] The pharmaceutical composition of any one of [18.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[19.1]

[19] The pharmaceutical composition of (a), wherein the other anti-cancer agent is a chemotherapeutic agent.

[19.2]

[19] The pharmaceutical composition of [19.1] or [19.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from the group consisting of antimetabolites, plant alkaloids, and platinum compounds.

[19.3]

[19] The pharmaceutical composition of (a), wherein the other anticancer agent is a T cell activation agonist.

[19.4]

[19] The pharmaceutical composition of [19.3] or [19.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[20]

[19] The pharmaceutical composition of (a), wherein the other anti-cancer agent is an immune checkpoint inhibitor.

[21]

[19] The pharmaceutical composition of [ 1] or [20], wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[21.1]

[19] The pharmaceutical composition of any one of [20] and [21], wherein the additional anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[21.2]

[18] The pharmaceutical composition of any one of [21.1], for use in treating a cancer refractory to treatment with an immune checkpoint inhibitor.

[21.3]

[21.2] the pharmaceutical composition of, wherein the immune checkpoint inhibitor treatment-refractory cancer is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[22]

[19] The pharmaceutical composition of (a), wherein the additional anti-cancer agent is a T cell redirecting antigen binding molecule.

[22.1]

[19] The pharmaceutical composition of [ 5 ] or [22], wherein the additional anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[22.2]

[22.1] the pharmaceutical composition of, wherein the multispecific antibody is a bispecific antibody.

[23]

[18]To [18.2]The pharmaceutical composition of any one of the above, wherein the other anticancer agent is one or both of depleting and inactivating regulatory T cells and CD4 ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[24]

[18] The pharmaceutical composition of any one of [ 1] to [23], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[24.1]

[18] The pharmaceutical composition of any one of [ 1] to [24], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[25]

[1] The anti-CD 137 antigen-binding molecule of any one of [1] to [5] in combination with at least one other anti-cancer agent for use in the treatment of cancer.

[25.1]

[25] The combination of (a) wherein the anti-CD 137 antigen binding molecule is used so as to be administered to a patient simultaneously with other anti-cancer agents.

[25.2]

[25] The combination of (a) wherein the anti-CD 137 antigen binding molecule is used for administration to a patient before or after administration of other anti-cancer agents.

[26]

[25] The combination of any one of [25.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[26.1]

[26] The combination of (a), wherein the additional anti-cancer agent is a chemotherapeutic agent.

[26.2]

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

[26.3]

[26] The combination of (a), wherein the additional anti-cancer agent is a T cell activation agonist.

[26.4]

[26] The combination of [26.3] or [26.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[27]

[26] The combination of (a), wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

[28]

[26] The combination of [ or [27], wherein the additional anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[28.1]

[26] The combination of any one of [27] and [28], wherein the other anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[28.2]

[25] The combination of any one of [28.1] to [ 2] for use in the treatment of a cancer refractory to treatment with an immune checkpoint inhibitor.

[28.3]

[28.2] the combination of (1) wherein the cancer refractory to treatment by the immune checkpoint inhibitor is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[29]

[26] The combination of (a), wherein the additional anti-cancer agent is a T-cell redirecting antigen binding molecule.

[29.1]

[26] The combination of [ 5] or [29], wherein the other anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[29.2]

[29.1] the combination of, wherein the multispecific antibody is a bispecific antibody.

[30]

[25]To [25.2]The combination of any one of the above, wherein the other anticancer agent is one or both of depleting and inactivating regulatory T cells and CD4 ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[31]

[25] The combination of any one of [1] to [30], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[31.1]

[25] The combination of any one of [1] to [31], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[32]

[1] Use of the anti-CD 137 antigen-binding molecule of any one of [1] to [5] in combination with at least one other anticancer agent as an anticancer agent.

[32.1]

[32] The use of (a), wherein the anti-CD 137 antigen-binding molecule is used so as to be administered to a patient simultaneously with other anti-cancer agents.

[32.2]

[32] The use of (a), wherein the anti-CD 137 antigen binding molecule is used for administration to a patient before or after administration of a further anti-cancer agent.

[33]

[32] The use of any one of [32.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[33.1]

[33] The use of (a), wherein the other anti-cancer agent is a chemotherapeutic agent.

[33.2]

[33] The use of [ 4.1 ] or [33.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from the group consisting of antimetabolites, plant alkaloids, and platinum compounds.

[33.3]

[33] The use of (a), wherein the other anti-cancer agent is a T cell activation agonist.

[33.4]

[33] The use of [ or [33.3], wherein the other anticancer agent is an agonistic antibody to TNFRSF.

[34]

[33] The use of (a), wherein the other anti-cancer agent is an immune checkpoint inhibitor.

[35]

[33] The use of [ 5 ] or [34], wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[35.1]

[33] The combination of any one of [34] and [35], wherein the additional anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[35.2]

[32] The use of any of [35.1] for the treatment of cancer refractory to treatment with an immune checkpoint inhibitor.

[35.3]

[35.2] the use of wherein the immune checkpoint inhibitor-refractory cancer is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[36]

[33] The use of (a), wherein the other anti-cancer agent is a T cell redirecting antigen binding molecule.

[36.1]

[33] The use of [ 9 ] or [36], wherein the other anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[36.2]

[36.1] the use, wherein the multispecific antibody is a bispecific antibody.

[37]

[32]To [32.2]The use of any one of the above, wherein the other anticancer agent is one or more of depleting and inactivating regulatory T cells, CD4 ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[38]

[32] The use of any one of [ 1] to [37], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[38.1]

[32] The use of any one of [1] to [38], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[39]

A kit for treating cancer, comprising:

(1) A pharmaceutical composition comprising the anti-CD 137 antigen-binding molecule of any one of [1] to [5] as an active ingredient; and

(2) A package insert or label indicating that at least one other anti-cancer agent is administered prior to, concurrently with, or after administration of the pharmaceutical composition.

[39.1]

[39] The kit of (a), wherein the pharmaceutical composition is filled in a container.

[39.2]

[39] The kit of any one of [39.2], wherein a pharmaceutical composition comprising an anti-CD 137 antigen-binding molecule as an active ingredient is used so as to be administered to a patient simultaneously with other anticancer agents.

[39.3]

[39] Or [39.1], wherein a pharmaceutical composition comprising the anti-CD 137 antigen-binding molecule as an active ingredient is used for administration to a patient before or after administration of other anticancer agents.

[40]

[39] The kit of any one of [39.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[40.1]

[40] The kit of (a), wherein the other anti-cancer agent is a chemotherapeutic agent.

[40.2]

[40] The kit of [40.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from the group consisting of antimetabolites, plant alkaloids, and platinum compounds.

[40.3]

[40] The kit of (a), wherein the other anti-cancer agent is a T cell activation agonist.

[40.4]

[40] The kit of [40.3] or [40.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[41]

[40] The kit of (a), wherein the anti-cancer agent is an immune checkpoint inhibitor.

[42]

[40] The kit of [4 ] or [41], wherein the anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[42.1]

[40] The kit of any one of [41] and [42], wherein the anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[42.2]

[39] The kit of any one of [42.1], for use in treating a cancer refractory to treatment with an immune checkpoint inhibitor.

[42.3]

The kit of [42.2], wherein the cancer refractory to treatment with the immune checkpoint inhibitor is a cancer having a gene mutation in JAK1, JAK2 and/or B2M.

[43]

[40] The kit of (a), wherein the anti-cancer agent is a T-cell redirecting antigen binding molecule.

[43.1]

[40] The kit of [ 5 ] or [43], wherein the anticancer agent is a multispecific antibody having a binding activity to CD3 and a cancer antigen.

[43.2]

The kit of [43.1], wherein the multispecific antibody is a bispecific antibody.

[44]

[39]To [39.2 ]]The kit of any one of the above, wherein the other anticancer agent is one selected from the group consisting of regulatory T cells, CD4, and the like, and the other anticancer agent is one selected from the group consisting of a cytotoxic T cell inhibitor and a cytotoxic T cell inhibitor ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[45]

[39] The kit of any one of [ 6 ] to [44], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[45.1]

[39] The kit of any one of [ 6 ] to [44], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[46]

A method for treating cancer, which comprises administering [ 1]]To [5 ]]The anti-CD 137 antigen binding molecule of any one of claims is administered to a patient having: (i) Selected from the group consisting of B cells, dendritic cells, natural killer cells, macrophages and CD8 ⁺ In T cellsAnd/or (ii) regulatory T (Treg) cells or CD4 ⁺ T cell infiltrated solid cancer.

[46.1]

[46]The method of (a), wherein the patient is suffering from CD8 ⁺ Patients with T cell-infiltrated solid cancers.

[47]

[46] The method of [5 ] or [46.1], wherein the patient has a cancer refractory to treatment with an immune checkpoint inhibitor.

[48]

[46] The method of any one of [ 1] to [47], wherein the patient has one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[48.1]

[46] The method of any one of [ 6] to [48], wherein the patient is a patient suffering from one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[49]

A method for treating cancer comprising administering the anti-CD 137 antigen-binding molecule of any one of [1] to [5] to a patient in combination with at least one additional anti-cancer agent.

[49.1]

[49] The method of (a), wherein the anti-CD 137 antigen binding molecule is administered to the patient concurrently with other anti-cancer agents.

[49.2]

[49] The method of (a), wherein the anti-CD 137 antigen binding molecule is administered to the patient before or after the administration of the additional anti-cancer agent.

[50]

[49] The method of any one of [49.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[50.1]

[50] The method of (a), wherein the additional anti-cancer agent is a chemotherapeutic agent.

[50.2]

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

[50.3]

[50] The method of (a), wherein the additional anti-cancer agent is a T cell activation agonist.

[50.4]

[50] The method of [50.3] or [50.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[51]

[50] The method of (a), wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

[52]

[50] The method of [5 ] or [51], wherein the additional anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[52.1]

[50] The method of any one of [51] and [52], wherein the additional anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[52.2]

[49] The method of any one of [52.1], for treating a cancer refractory to treatment with an immune checkpoint inhibitor.

[52.3]

The method of [52.2], wherein the cancer refractory to treatment with the immune checkpoint inhibitor is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[53]

[50] The method of (a), wherein the additional anti-cancer agent is a T cell redirecting antigen binding molecule.

[53.1]

[50] The method of [5 ] or [53], wherein the additional anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[53.2]

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

[54]

[49]To [49.2 ]]The method of any one of the above, wherein the additional anti-cancer agent is one or more of depleting regulatory T cells, CD4, and/or inactivating ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[55]

[49] The method of any one of [ 1 ] to [54], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[55.1]

[49] The method of any one of [ 5] to [55], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[56]

[1] The anti-CD 137 antigen-binding molecule of any one of [ 6] to [5], for use in treating:

(i) Selected from B cell and dendritic cellCells, natural killer cells, macrophages and CD8 ⁺ A solid cancer infiltrated by one or more of the T cells, and/or

(ii) Regulatory T (Treg) cells or CD4 ⁺ T cell infiltrated solid cancer.

[56.1]

[56]The anti-CD 137 antigen binding molecule of wherein the cancer is CD8 ⁺ T cell infiltrated solid cancer.

[57]

[56] The anti-CD 137 antigen binding molecule of [56.1], wherein the cancer is a cancer refractory to treatment with an immune checkpoint inhibitor.

[58]

[56] The anti-CD 137 antigen binding molecule of any one of [1] to [57], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[58.1]

[56] The anti-CD 137 antigen binding molecule of any one of [1] to [58], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[59]

[1] The anti-CD 137 antigen-binding molecule of any one of [ 6] to [5], for use in combination with at least one other anti-cancer agent for treating cancer.

[59.1]

[59] The anti-CD 137 antigen binding molecule, wherein the treatment is characterized by administering the anti-CD 137 antigen binding molecule concurrently with other anti-cancer agents.

[59.2]

[59] The anti-CD 137 antigen binding molecule, wherein the treatment is characterized by administering the anti-CD 137 antigen binding molecule before or after administering the additional anti-cancer agent.

[60]

[59] The anti-CD 137 antigen-binding molecule of any one of [59.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[60.1]

[60] The anti-CD 137 antigen-binding molecule, wherein the additional anti-cancer agent is a chemotherapeutic agent.

[60.2]

[60] The anti-CD 137 antigen-binding molecule of [60.1], wherein the additional anti-cancer agent is at least one chemotherapeutic agent selected from the group consisting of an antimetabolite, a plant alkaloid, and a platinum compound.

[60.3]

[60] The anti-CD 137 antigen binding molecule, wherein the additional anti-cancer agent is a T cell activation agonist.

[60.4]

[60] The anti-CD 137 antigen-binding molecule of [60.3], wherein the additional anti-cancer agent is an agonistic antibody of TNFRSF.

[61]

[60] The anti-CD 137 antigen binding molecule, wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

[62]

[60] The anti-CD 137 antigen-binding molecule of [61], wherein the additional anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[62.1]

[60] The anti-CD 137 antigen-binding molecule of any one of [61] and [62], wherein the additional anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[62.2]

[59] The anti-CD 137 antigen-binding molecule of any one of [62.1], for use in treating a cancer refractory to treatment with an immune checkpoint inhibitor.

[62.3]

[62.2] the anti-CD 137 antigen-binding molecule of, wherein the immune checkpoint inhibitor treatment-refractory cancer is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[63]

[60] The anti-CD 137 antigen-binding molecule, wherein the additional anti-cancer agent is a T-cell redirecting antigen-binding molecule.

[63.1]

[60] The anti-CD 137 antigen-binding molecule according to [63], wherein the additional anti-cancer agent is a multispecific antibody having a binding activity to CD3 and a cancer antigen.

[63.2]

[63.1] the anti-CD 137 antigen-binding molecule of, wherein the multispecific antibody is a bispecific antibody.

[64]

[59]To [59.2 ]]The anti-CD 137 antigen binding molecule of any one of, wherein the additional anti-cancer agent is one or both of depleting and inactivating a regulatory T cell, CD4 ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[65]

[59] The anti-CD 137 antigen-binding molecule of any one of [ 1] to [64], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[65.1]

[59] The anti-CD 137 antigen binding molecule of any one of [1] to [65], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[66]

[1] Use of the anti-CD 137 antigen-binding molecule of any one of [5] for the preparation of a medicament for the treatment of cancer, wherein the cancer is

(i) Selected from the group consisting of B cells, dendritic cells, natural killer cells, macrophages and CD8 ⁺ One or more of T cells infiltrating solid cancer, and/or

(ii) Regulatory T (Treg) cells or CD4 ⁺ T cell infiltrated solid cancer.

[66.1]

[66]The use of (a), wherein the cancer is CD8 ⁺ T cell infiltrated solid cancer.

[67]

[66] The use of [66.1] or [66.1], wherein the cancer is a cancer refractory to treatment with an immune checkpoint inhibitor.

[68]

[66] The use of any one of [ 6] to [67], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[68.1]

[66] The use of any one of [5] to [68], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[69]

[1] Use of the anti-CD 137 antigen binding molecule of any one of [1] to [5] in the manufacture of a medicament for the treatment of cancer, wherein the medicament is characterized by being used in combination with at least one additional anti-cancer agent.

[69.1]

[69] The use of (a), wherein the medicament is characterized by being administered concurrently with other anti-cancer agents.

[69.2]

[69] The use of (a), wherein the medicament is characterized by being administered before or after the administration of the other anti-cancer agent.

[70]

[69] The use of any one of [69.2], wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

[70.1]

[70] The use of (a), wherein the other anti-cancer agent is a chemotherapeutic agent.

[70.2]

[70] The use of [70.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from the group consisting of antimetabolites, plant alkaloids, and platinum compounds.

[70.3]

[70] The use of (b), wherein the other anti-cancer agent is a T cell activation agonist.

[70.4]

[70] The use of [70.3] or [70.3], wherein the other anticancer agent is an agonistic antibody of TNFRSF.

[71]

[70] The use of (a), wherein the other anti-cancer agent is an immune checkpoint inhibitor.

[72]

[70] The use of [ a ] or [71], wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

[72.1]

[70] The use of any one of [71] and [72], wherein the other anti-cancer agent is an anti-PDL 1 antibody and/or an anti-TIGIT antibody.

[72.2]

[69] The use of any of [72.1] for the treatment of a cancer refractory to treatment with an immune checkpoint inhibitor.

[72.3]

[72.2] the use of wherein the immune checkpoint inhibitor is for treating a refractory cancer is a cancer having a genetic mutation in JAK1, JAK2 and/or B2M.

[73]

[70] The use of (a), wherein the other anti-cancer agent is a T cell redirecting antigen binding molecule.

[73.1]

[70] The use according to [ 9] or [73], wherein the other anticancer agent is a multispecific antibody having binding activity to CD3 and a cancer antigen.

[73.2]

[73.1] the use, wherein the multispecific antibody is a bispecific antibody.

[74]

[69]To [69.2]The use of any one of the above, wherein the additional anti-cancer agent is one or more of depleting and/or inactivating regulatory T cells, CD4 ⁺ An agent for one or more of T cells, B cells, NK cells and macrophages.

[75]

[69] The use of any of [ 9] to [74], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannomas, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

[75.1]

[69] The use of any one of [ 5] to [75], wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

Drawings

FIG. 1 shows the antitumor effect of A551-MB110/B379-ml0r in a mouse model transplanted with LLC1/OVA/GPC3 clone C5 cells.

Each dot shows the average of a set (n = 7) of tumor volumes.

FIG. 2 shows the gene expression levels in tumor samples administered A551-MB110/B379-ml0r in a mouse model transplanted with LLC1/OVA/GPC3 clone C5 cells.

Sub-graph (a) shows the expression level of CD8B1, sub-graph (B) shows the expression level of Gzmb, sub-graph (C) shows the expression level of Prf1, and sub-graph (D) shows the expression level of Ifng.

FIG. 3 shows CD8 in tumor tissue by administration of A551-MB110/B379-ml0r in a mouse model transplanted with LLC1/OVA/GPC3 clone C5 cells ⁺ Degree of activation of T cells.

Scheme (A) shows OVA tetramer ⁺ T cells in CD8 ⁺ Proportion in T cells. Panel (B) shows granzyme B ⁺ T cells in CD8 ⁺ Proportion in T cells. Panel (C) shows PD-1 ⁺ T cells in CD8 ⁺ Proportion in T cells. Subfigure (D) shows KLRG-1 ⁺ T cells in CD8 ⁺ Proportion in T cells. Subfigure (E) shows ICOS ⁺ T cells in CD8 ⁺ Proportion in T cells.

FIG. 4 shows the antitumor effect of A551-MB110/B379-ml0r in a mouse model transplanted with E.G7-OVA cells.

Panel (a) shows the change in tumor volume of each mouse in the vehicle-administered group. Panel (B) shows the change in tumor volume per mouse in the A551-MB110/B379-ml0r (2.5 mg/kg) administration group.

FIG. 5 shows the antitumor effect of A375-mIgG1/B167-ml0r in a mouse model transplanted with E.G7-OVA cells.

Panel (a) shows the change in tumor volume of each mouse in the vehicle-administered group. Panel (B) shows the change in tumor volume per mouse in the A375-mIgG1/B167-ml0r (2.5 mg/kg) administration group.

FIG. 6 shows the anti-tumor effect of A551-MB110/B379-ml0r in a mouse model transplanted with C1498 cells.

FIG. 7 shows the antitumor effect of A551-MB110/B379-ml0r in a mouse model transplanted with Hepa1-6/hGPC3 cells.

FIG. 8 shows the antitumor effect of A375-mIgG1/B167-ml0r in a mouse model transplanted with Hepa1-6/hGPC3 cells.

Panel (a) shows the change in tumor volume of each mouse in the vehicle-administered group. Panel (B) shows the change in tumor volume per mouse in the A375-mIgG1/B167-ml0r (7.5 mg/kg) administration group.

FIG. 9 shows various immune cells (CD 4) ⁺ T cell, CD8 ⁺ T cells, B cells, NK cells, granulocytes, macrophages) on the antitumor effect of a551-MB110/B379-ml0r in a mouse model transplanted with LLC1/OVA/GPC3 clone C5 cells.

FIG. 10 shows the anti-tumor effect of A551-MB110/B379-ml0r, anti-mouse PD-L1 antibodies, and combinations thereof in a mouse model transplanted with MC38 cells.

Each point shows the mean of a set (n = 6) of tumor volumes.

FIG. 11 shows the anti-tumor effect (body weight change) of A551-MB110/B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof in a mouse model transplanted with MC38 cells.

Panel (a) shows the body weight change of each mouse in the vehicle administration group. Panel (B) shows the change in body weight of each mouse in the A551-MB110/B379-ml0r administration group. Panel (C) shows the change in body weight of each mouse in the anti-mouse PD-L1 antibody administration group. Panel (D) shows the body weight change of each mouse in the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination administration group.

FIG. 12 shows the change in expression of various immune-related genes before or after administration of various antibodies or combinations thereof by administering A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or combinations thereof to a mouse model transplanted with MC38 cells.

FIG. 13 shows the extent of CD8 expression in tumor tissues of a mouse model implanted with MC38 cells by administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof.

Panel (a) shows the degree of expression of CD8 in the tumor tissues of the vehicle-administered group, panel (B) shows the degree of expression of CD8 in the tumor tissues of the a551-MB110/B379-ml0 r-administered group, panel (C) shows the degree of expression of CD8 in the tumor tissues of the anti-mouse PD-L1 antibody-administered group, and panel (D) shows the degree of expression of CD8 in the tumor tissues of the a551-MB110/B379-ml0 r-administered group in combination with the anti-mouse PD-L1 antibody.

FIG. 14 shows the degree of expression of PD-L1 in tumor tissue by administering A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

Panel (A) shows the degree of expression of PD-L1 in the tumor tissues of the vehicle-administered group, panel (B) shows the degree of expression of PD-L1 in the tumor tissues of the A551-MB110/B379-ml0 r-administered group, panel (C) shows the degree of expression of PD-L1 in the tumor tissues of the anti-mouse PD-L1 antibody-administered group, and panel (D) shows the degree of expression of PD-L1 in the tumor tissues of the A551-MB110/B379-ml0 r-administered group in combination with the anti-mouse PD-L1 antibody.

FIG. 15 shows the extent of liver function markers detected by blood tests as a result of administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

The top panel shows ALT (U/L) and the bottom panel shows AST (U/L).

FIG. 16 shows CD8 in tumor tissue due to administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells ⁺ The degree of increase in the number of T cells.

FIG. 17 shows the anti-tumor effect of A551-MB110/B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof in a mouse model transplanted with LLC1/OVA/GPC3 clone C5 cells.

Each point shows the mean of a set (n = 5) of tumor volumes.

FIG. 18 shows the anti-tumor effect of A375-mIgG1/B167-ml0r, anti-mouse PD-L1 antibody, or a combination thereof in a mouse model transplanted with C1498 cells.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 19 shows the anti-tumor effect of A375-mIgG1/B167-ml0r, anti-mouse PD-L1 antibody, or a combination thereof in a mouse model transplanted with AE17 cells.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 20 shows the anti-tumor effect of A551-MB110/B379-ml0r, anti-hGPC 3-mCD3 antibodies, or a combination thereof in a mouse model transplanted with LLC1/hGPC 3.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 21 shows the gene expression levels in tumor samples resulting from administration of A551-MB110/B379-ml0r, anti-hGPC 3-mCD3 antibody, or a combination thereof, in a mouse model transplanted with LLC1/hGPC 3.

Respectively, panel (a) shows the expression level of CD3E, panel (B) shows the expression level of CD8B1, panel (C) shows the expression level of Gzmb, panel (D) shows the expression level of Prf1, and panel (E) shows the expression level of Ifng.

Figure 22 is a graph showing agonist activity of various anti-CD 137 antibodies tested using Jurkat cells in the presence or absence of ATP.

The X-axis shows antibody concentration (. Mu.g/mL) and the Y-axis shows relative light units.

Figure 23 is a graph showing agonist activity of various anti-CD 137 antibodies tested using Jurkat cells in the presence or absence of ADP.

Fig. 24 is a graph showing the agonist activity of various anti-CD 137 antibodies tested using human T cells in the presence or absence of ADP β S.

FIG. 25 is a graph showing agonist activity of dBAT 119-P253/dBAT 119L-LamLib (small molecule switch anti-CD 137 antibody) or NS1-P253 (non-switch anti-CD 137 antibody) tested using human T cells in the presence or absence of ADPss S.

The X-axis shows the antibody concentration (. Mu.g/mL) and the Y-axis shows the IFN-. Gamma.production (ng/mL).

Fig. 26 is a graph showing ATP-dependent antigen binding activity of various anti-CD 137 antibodies (conversion anti-CD 137 antibodies with enhanced binding activity) tested by phage ELISA.

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

FIG. 27 is a graph showing the binding activity of various variants of anti-CD 137 antibodies (dBAT 119H-P253/dBAT 119L-LamLib) to human CD137 in the presence or absence of ATP.

The upper row shows the binding activity to human CD137 in the absence of ATP and the lower row shows the binding activity to human CD137 in the presence of ATP.

FIG. 28 is a graph showing agonist activity of dBAT 119H-P253/dBAT 119L-LamLib, dBATk 119H 024-P253/dBATk 119L020-LamLib, IC17HdK-hIgG1/IC17L-k0 (control), or NS1-P253 (non-transducing anti-CD 137 antibody) tested using human T cells in the presence or absence of ADP β S.

Panel (a) shows the test results in the absence of ADP β S, and panel (B) shows the test results in the presence of ADP β S.

FIG. 29 is a graph showing agonist activity of various switch anti-CD 137 antibodies tested using the 4-1BB Jurkat reporter gene assay in the presence or absence of ATP.

Panel (a) shows the test results without ATP and panel (B) shows the test results with ATP present.

Figure 30 is a graph showing enhanced agonist activity of various switch anti-CD 137 antibodies in the presence of ATP due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-. Gamma.production as an index.

Fig. 31 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence of ATP due to increased binding activity of the heavy chain constant region to the Fc γ receptor or increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 32 is a graph showing the enhanced agonist activity of various switch anti-CD 137 antibodies, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Figure 33 is a graph showing enhanced agonist activity of various switch anti-CD 137 antibodies, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to Fc γ receptors, tested using human peripheral blood mononuclear cells.

Panel (A) shows the agonist activity measured using the IL-2 production amount as an index, and Panel (B) shows the agonist activity measured using the IFN- γ production amount as an index.

Figure 34 is a graph showing enhanced agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Figure 35 is a graph showing enhanced agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Figure 36 is a graph showing enhanced agonist activity of various switch anti-CD 137 antibodies, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Figure 37 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 38 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 39 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 40 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 41 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to increased pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 42 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to an increase in pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 43 is a graph showing the enhancement of agonist activity of various switch anti-CD 137 antibodies in the presence or absence of ATP due to an increase in pI of the heavy chain constant region, tested using human peripheral blood mononuclear cells.

Figure 44 is a graph showing the enhanced agonist activity of various switch anti-CD 137 antibodies, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fc γ receptor, tested using human peripheral blood mononuclear cells.

Fig. 45 is a graph showing plasma concentrations of various converted and non-converted anti-CD 137 antibodies tested using human CD137 knock-in mice.

Fc is mIgG1.

Fig. 46 is a graph showing plasma concentrations of various converted and non-converted anti-CD 137 antibodies tested using human CD137 knock-in mice.

Fc is MB110.

Fig. 47 is a graph showing plasma concentrations of various converted and non-converted anti-CD 137 antibodies tested using human CD137 knock-in mice.

Fc is MB492.

FIG. 48 is a graph showing the antitumor effect of A375-mIgG1/B167-ml0r in a mouse model in which MC38 cells were transplanted.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 49 is a graph showing the weight of organs in a mouse model in which MC38 cells were transplanted after administration of an antibody (NO 1-mIgG1 or A375-mIgG1/B167-ml0 r).

Panel (a) shows lymph node weight, and panel (B) shows spleen weight.

FIG. 50 is a graph showing the degree of T cell activation in lymph nodes in a mouse model in which MC38 cells were transplanted after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0 r.

Panel (a) shows the percentage of PD-1 positive T cells in CD8 positive T cells, panel (B) shows the percentage of ICOS positive T cells in CD8 positive T cells, and panel (C) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

FIG. 51 is a graph showing the degree of T cell activation in spleen in a mouse model in which an MC38 cell line was transplanted after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0 r.

FIG. 52 is a graph showing the degree of T cell activation in the liver in a mouse model in which an MC38 cell line was transplanted after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0 r.

Panel (a) shows the percentage of PD-1 positive T cells in CD8 positive T cells, and panel (B) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

FIG. 53 is a graph showing the antitumor effect of A356-MB110/B040-ml0r in a mouse model in which an MC38 cell line was transplanted.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 54 is a graph showing organ weight in a mouse model in which an MC38 cell line was transplanted after administration of NS2-MB110 or A356-MB110/B040-ml0 r.

Panel (a) shows lymph node weight and panel (B) shows spleen weight.

FIG. 55 is a graph showing the degree of T cell activation in the liver of a mouse model in which an MC38 cell line was transplanted after administration of NS2-MB110 or A356-MB110/B040-ml0 r.

Panel (a) shows the percentage of PD-1 positive T cells among CD8 positive T cells, and panel (B) shows the percentage of ICOS positive T cells among CD8 positive T cells.

FIG. 56 is a graph showing the antitumor effect of A372-mIgG1/B040-ml0r in a mouse model into which an MC38 cell line was transplanted.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 57 shows lymph node cell number (panel (A)) and spleen weight (panel (B)) in a mouse model transplanted with the MC38 cell line after administration of A372-mIgG1/B040-ml0 r.

FIG. 58 is a graph showing the extent of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of A372-mIgG1/B040-ml0r (percentage of granzyme B-positive T cells in CD 8-positive T cells).

FIG. 59 is a graph showing the antitumor effect of A372-MB110/B040-ml0r in a mouse model into which an MC38 cell line was transplanted.

Each point shows the mean of a set (n = 5) of tumor volumes.

FIG. 60 is a graph showing organ weight in a mouse model transplanted with the MC38 cell line following administration of NS2-MB110 or A372-MB110/B040-ml0 r.

Panel (a) shows lymph node weight, and panel (B) shows spleen weight.

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

FIG. 62 is a graph showing the antitumor effect of A372-MB492/B040-ml0r in a mouse model in which an MC38 cell line was transplanted.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 63 is a graph showing lymph node cell number and spleen organ weight in a mouse model transplanted with an MC38 cell line after administration of NS1-MB492 or A372-MB492/B040-ml0 r.

Panel (a) shows the cell number of lymph nodes and panel (B) shows the organ weight of spleen.

FIG. 64 is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with an MC38 cell line following administration of NS1-MB492 or A372-MB492/B040-ml0r (percentage of granzyme B-positive T cells in CD 8-positive T cells).

FIG. 65 is a graph showing the antitumor effect of A486-MB492/B167-ml0r or A488-MB492/B226-ml0r in a mouse model in which an MC38 cell line was transplanted.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 66 is a graph showing the cell number and spleen weight of each lymph node in a mouse model in which an MC38 cell line was transplanted after administration of NS1-MB492, A486-MB492/B167-ml0r, or A488-MB492/B226-ml0 r.

Panel (a) shows the number of cells per lymph node, and panel (B) shows the weight of the spleen.

FIG. 67 is a graph showing the level of infiltration of effector cells in the liver (percentage of CD 3-positive and CD 8-positive T cells among CD 45-positive T cells) of a mouse model transplanted with the MC38 cell line following administration of NS1-MB492, A486-MB492/B167-ml0r, or A488-MB492/B226-ml0 r.

FIG. 68 is a graph showing the antitumor effect of A489-MB492/B223-ml0r in a mouse model in which an MC38 cell line was transplanted.

Each point shows the mean of a set (n = 5) of tumor volumes.

FIG. 69 is a graph showing the number of lymph node cells and the number of cells in the spleen lymphocyte fraction in a mouse model transplanted with an MC38 cell line after administration of NS1-MB492 or A489-MB492/B223-ml0 r.

Panel (a) shows the number of cells in the lymph node, and panel (B) shows the number of cells in the lymphocyte fraction of the spleen.

FIG. 70 is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A489-MB492/B223-ml0r (percentage of CD 8-positive T cells among CD 45-positive T cells).

FIG. 71 is a graph showing the antitumor effects of A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r in a mouse model in which an MC38 cell line was transplanted.

Panel (A) shows the anti-tumor effect of A548-mIgG1/B256-ml0r, and Panel (B) shows the anti-tumor effect of A551-mIgG1/B256-ml0 r.

FIG. 72 is a graph showing the weight of organs in a mouse model transplanted with an MC38 cell line after administration of NS1-mIgG1, A548-mIgG1/B256-ml0r, or A551-mIgG1/B256-ml0 r.

Panel (a) shows lymph node weight and panel (B) shows spleen weight.

FIG. 73 is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with an MC38 cell line following administration of NS1-mIgG1, A548-mIgG1/B256-ml0r, or A551-mIgG1/B256-ml0 r.

FIG. 74 is a graph showing the antitumor effect of A551-MB110/B379-ml0r in a mouse model into which an MC38 cell line was transplanted.

FIG. 75 is a graph showing the weight of organs in a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1 or A551-MB110/B379-ml0 r.

Panel (a) shows lymph node weight and panel (B) shows spleen weight.

FIG. 76 is a graph showing the degree of T cell activation in spleens of a mouse model in which an MC38 cell line was transplanted after administration of NS1-mIgG1 or A551-MB110/B379-ml0 r.

FIG. 77 is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1 or A551-MB110/B379-ml0 r.

Panel (a) shows the percentage of PD-1 positive T cells in CD8 positive T cells, panel (C) shows the percentage of ICOS positive T cells in CD8 positive T cells, and panel (B) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

FIG. 78 is a graph showing agonist activity of various anti-CD 137 antibodies tested using 4-1BB Jurkat cells in the presence or absence of small molecule compounds (ATP or ADP).

FIG. 79 is a graph showing agonist activity of various switch anti-CD 137 antibodies tested using the 4-1BB Jurkat reporter gene assay in the presence of ATP.

FIG. 80 is a graph showing a comparison of the plasma kinetics of each of the anti-CD 137 transition antibodies A375-SCF041aPh/B167-Lamlib and A375-MY201 aPh/B167-Lamlib. The vertical axis of the graph shows the plasma concentration of each antibody.

FIG. 81 is a graph showing the anti-tumor effect of each of A375/B167-SCF041aPh and A375/B167-MY201aPh in a mouse model prepared by transplanting an LLC1/OVA/GPC3 cell line into hCD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mice.

Each dot shows the average of a set (n = 5) of tumor volumes.

FIG. 82 shows CD8 in tumor tissue after administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells ⁺ The number of cells.

FIG. 83 shows PD-L1 in tumor tissue after administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells ⁺ The number of cells.

FIG. 84 shows the number of cells in tumor tissue that are in or highly expressed in PD-L1 after administration of A551-MB110/B379-ml0r, an anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

FIG. 85 shows tumor growth inhibition effects in a mouse model transplanted with MC38 cells by administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or combined administration of A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or combined administration of UreH-MB110/UreL-mk1 ("Uree-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab").

FIG. 86 shows the degree of fluctuation of blood parameters (leukocyte concentration, platelet concentration, and lymphocyte concentration) in a mouse model transplanted with MC38 cells due to administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Ure-MB"), or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or combined administration of A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or combined administration of UreH-MB110/UreL-mk1 ("Ure-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab").

FIG. 87 shows organ weights of spleen and tumor Draining Lymph Nodes (DLN) in a mouse model transplanted with MC38 cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or A551-MB110/B379-ml0r ("Sta-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab"), or UreH-MB110/UreL-mk1 ("Uree-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab").

FIG. 88 shows spleen KLRG-1, ICOS, PD-1, LAG-3 in CD8 cells in a mouse model transplanted with MC38 cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or A551-MB110/B379-ml0r ("Sta-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab"), or UreH-MB110/UreL-mk1 ("Uree-L1-Ab") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab ⁺ Expression ratio in T cells and Foxp3 ⁺ Regulatory T cells in CD4 ⁺ Ratio in T cells.

FIG. 89 shows CD45 in spleen of mouse model transplanted with MC38 cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or in combination with A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or in combination with UreH-MB110/UreL-mk1 ("Uree-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab ⁺ Ratio of individual activation marker positive CD8+ T cells in leukocytes and CD45 ⁺ Foxp3 in leukocytes ⁺ Ratio of regulatory T cells.

FIG. 90 shows KLRG-1, ICOS, PD-1 and LAG-3 in CD8 in tumor Draining Lymph Nodes (DLNs) of a mouse model transplanted with MC38 cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Ure-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or A551-MB110/B379-ml0r ("Sta-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab"), or UreH-MB110/UreL-mk1 ("Ure-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab ″) ⁺ Expression ratio in T cells and Foxp3 ⁺ Regulatory T cells in CD4 ⁺ Ratio in T cells.

FIG. 91 shows that each activation marker positive CD8 in tumor Draining Lymph Nodes (DLNs) of a MC38 cell transplanted mouse model after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Ure-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or in combination with A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or in combination with UreH-MB110/UreL-mk1 ("Ure-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab") ⁺ Absolute number of T cells and CD4 ⁺ Foxp3 in T cells ⁺ Absolute number of regulatory T cells.

FIG. 92 shows KLRG-1, ICOS, PD-1 and LAG-3 in the liver of a mouse model transplanted with MC38 cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Ure-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or A551-MB110/B379-ml0r ("Sta-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab"), or UreH-MB110/UreL-mk1 ("Ure-MB") in combination with anti-mouse PD-L1 antibody ("PD-L1-Ab"), or KLRG-1, ICOS, PD-1 and LAG-3 in the liver of a mouse model transplanted with MC38 cells ⁺ Expression ratio in T cells and Foxp3 ⁺ Regulatory T cells in CD4 ⁺ Ratio in T cells.

FIG. 93 shows CD45 in the liver of a mouse model transplanted with MC38 cells following administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or in combination with A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or in combination with UreH-MB110/UreL-mk1 ("Uree-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab ⁺ Activation marker positive CD8 in leukocytes ⁺ Ratio of T cells and CD45 ⁺ Foxp3 in leukocytes ⁺ Ratio of regulatory T cells.

FIG. 94 shows the extent of binding of Alexa488 labeled A551-MB110/B379-ml0r, alexa488 labeled IC17HdK-MB110/IC17L-mk (negative control substance), and non-switching anti-CD 137 antibody Alexa488 labeled UreH-MB110/UreL-mk1 (positive control substance) to lymphocyte fractions in tumors, spleen, and liver in a mouse model transplanted with MC38 cells, as analyzed by Flow Cytometry (FCM).

FIG. 95 shows a heat map of the gene set showing the changes in expression associated with extracellular ATP in various normal organ tissues and various tumor tissues.

FIG. 96 shows the degree of expression of CD73 in various cancer cell lines, as analyzed by Flow Cytometry (FCM).

FIG. 97 shows tumor cells, CD4, from mice transplanted with LLC1/OVA/GPC3 clone C5 cell line ⁺ T cell, CD8 ⁺ Expression rates of CD39 and CD73 in T cells and non-T cells as analyzed by Flow Cytometry (FCM).

Tumor cells, CD4, for expression of CD39 and CD73 ⁺ T cell, CD8 ⁺ T cells and non-T cells were examined 5 times with different samples, respectively, and the average value was calculated.

FIG. 98 is a representative graph showing tumor cells, CD4, from mice transplanted with LLC1/OVA/GPC3 clone C5 cell line ⁺ T cell, CD8 ⁺ Expression rates of CD39 and CD73 in T cells and non-T cells as analyzed by Flow Cytometry (FCM).

FIG. 99 shows tumor cells, CD4, from mice transplanted with the MC38 cell line ⁺ T cell, CD8 ⁺ Expression rates of CD39 and CD73 in T cells and non-T cells as analyzed by Flow Cytometry (FCM).

Tumor cells, CD4, for expression of CD39 and CD73 ⁺ T cell, CD8 ⁺ T cells and non-T cells were tested 5 times with different samples, respectively, and the average was calculated.

FIG. 100 is a representative diagram showing tumor cells, CD4, from mice transplanted with the MC38 cell line ⁺ T cell, CD8 ⁺ Expression rates of CD39 and CD73 in T cells and non-T cells as analyzed by Flow Cytometry (FCM).

FIG. 101 shows the anti-tumor effect in a mouse model transplanted with LLC1/OVA cells by administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Urea-MB"), or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or in combination with A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or in combination with UreH-MB110/UreL-mk1 ("Urea-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab").

FIG. 102 shows CD8 in tumor Draining Lymph Nodes (DLNs) of a mouse model transplanted with LLC1/OVA cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Uree-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or in combination with A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or in combination with UreH-MB110/UreL-mk1 ("Uree-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab ⁺ Expression rates of KLRG-1, ICOS and PD-1 in T cells.

FIG. 103 shows respective activation marker positive CD8 in tumor Draining Lymph Nodes (DLNs) of a mouse model transplanted with LLC1/OVA cells after administration of A551-MB110/B379-ml0r ("Sta-MB"), ureH-MB110/UreL-mk1 ("Ure-MB") or anti-mouse PD-L1 antibody ("PD-L1-Ab") alone, or combined administration of A551-MB110/B379-ml0r ("Sta-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab"), or combined administration of UreH-MB110/UreL-mk1 ("Ure-MB") and anti-mouse PD-L1 antibody ("PD-L1-Ab") ⁺ Absolute number of T cells.

FIG. 104 shows the tumor growth inhibition effect in a mouse model transplanted with LLC1/OVA cells by administration of A551-MB110/B379-ml0r ("Sta-MB") alone.

FIG. 105 shows tumor growth inhibition effects in a mouse model engrafted with a mouse AML C1498 cell line by administering either the switch anti-CD 137 antibody A551-MB110/B379-ml0r or anti-TIGIT antibody alone, or a combination of switch anti-CD 137 antibody A551-MB110/B379-ml0r and anti-TIGIT antibody.

FIG. 106 shows the anti-tumor effect of the switch anti-CD 137 antibody A551-MB110/B379-ml0r in a mouse model transplanted with MC38-hGPC3# G64B2M KO clone 5 cell line.

[ detailed description of the invention ]

I. Definition of

The term "binding activity" refers to the strength of the sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Herein, "binding activity" is not strictly limited to 1:1 interaction. For example, when the member of the binding pair reflects a monovalent 1:1 interaction, the binding activity is particularly referred to as intrinsic binding affinity (avidity). When the members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of the strengths of each binding. The binding activity of a molecule X to its partner Y can generally be expressed in terms of the dissociation constant (KD) or the "amount of analyte bound per unit amount of ligand" (hereinafter referred to as "bound amount"). It will be understood by those skilled in the art that, in general, a lower dissociation constant (KD) means a higher binding activity, a higher value of "analyte binding amount per unit amount of ligand" or "binding amount" means a higher binding activity. Binding activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

An "antigen binding molecule or antibody with mature binding activity, or an" antigen binding molecule or antibody with increased (enhanced) binding activity, refers to an antibody with one or more hypervariable regions (HVRs) having one or more alterations which result in an increase in the binding activity of the antigen binding molecule or antibody to an antigen, as compared to the parent antigen binding molecule or parent antibody without the alterations.

The terms "anti-CD 137 antigen-binding molecule" or "anti-CD 137 antibody" and "antigen-binding molecule that binds to CD 137" or "antibody that binds to CD 137" refer to an antigen-binding molecule or antibody that is capable of binding to CD137 with sufficient binding activity such that the antigen-binding molecule or antibody is useful as a diagnostic and/or therapeutic agent for targeting CD 137. In certain embodiments, the anti-CD 137 antibody binds to a CD137 epitope that is conserved between CD137 of different species.

The term "anti-CD 137 antigen-binding molecule or anti-CD 137 antibody having CD137 binding activity dependent on a small molecule compound" refers to binding activity to CD137 in the presence of a small molecule compound compared to binding activity to CD137 in the absence of a small molecule compoundCD137 has an antigen binding molecule or antibody with higher binding activity. In one embodiment, "the presence of a small molecule compound" refers to a condition wherein the small molecule compound is present at a concentration of 10 micromolar or more, 50 micromolar or more, 100 micromolar or more, 150 micromolar or more, 200 micromolar or more, or 250 micromolar or more. In one embodiment, the extent of binding activity of the anti-CD 137 antigen-binding molecule or antibody to an unrelated, non-CD 137 protein in the presence of the small molecule compound is less than about 10% of the binding of the antigen-binding molecule or antibody to CD137, e.g., as measured by Radioimmunoassay (RIA) or Surface Plasmon Resonance (SPR). In certain embodiments, the anti-CD 137 antigen-binding molecule or antibody has a dissociation constant (KD) of 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10nM or less) in the presence of the low molecular weight compound ^-6 M is less than or equal to 10 ^-7 M < 10 ^-8 M is less than or equal to 10 ^-9 M is less than or equal to 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)。

As used herein, the term "antigen binding molecule" is used in its broadest sense to refer to a molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding molecule is an antibody, an antibody fragment or an antibody derivative.

As used herein, an "agonistic antigen binding molecule" or "agonistic antibody" is an antigen binding molecule or antibody that significantly induces or enhances the biological activity of the antigen (e.g., CD137 and CD 3) to which it binds.

Thus, if the antigen is, for example, CD137, such an antigen binding molecule or antibody that has agonistic effects is referred to as a "CD137 agonistic antigen binding molecule" or "CD137 agonistic antibody," respectively. In the same way, if the antigen is, for example, CD3, such an antigen binding molecule or antibody having agonistic effects is referred to as a "CD3 agonistic antigen binding molecule" or "CD3 agonistic antibody", respectively.

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

An "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, fv, fab '-SH, F (ab') ₂ (ii) a Diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibodies formed from antibody fragments.

An antigen binding molecule that binds to "the same epitope" or an "antibody that binds to the same epitope" of a reference antigen binding molecule or a reference antibody refers to an antibody or antigen binding molecule that: in a competition assay, the binding of a reference antibody or reference antigen binding molecule to its antigen is blocked by 50% or more, and conversely, in a competition assay, the reference antibody blocks the binding of an antibody to its antigen by 50% or more. Exemplary competition assays are provided herein. In one embodiment, where the reference antigen binding molecule or reference antibody shows antigen binding activity in a manner dependent on the low molecular weight compound, the competition assay is performed in the presence of the low molecular weight compound.

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

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. Antibodies are mainly classified into five classes: igA, igD, igE, igG and IgM, some of which may be further divided into subclasses (subclasses), e.g. IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

"effector functions" refer to those biological activities attributed to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"cytotoxicity" refers to the activity of inhibiting or preventing cellular function and/or causing cell death or destruction. The cytotoxicity may be, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T-cell cytotoxicity; possibly caused by cytotoxic agents (e.g., radioisotopes and chemotherapeutic agents), such as immunoconjugates.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys 447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), as described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, national Institutes of Health, bethesda, MD, 1991.

The term "variant Fc region" herein encompasses an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

Herein, amino acid changes or substitutions within the Fc region or constant region may be represented by the EU numbering system and combinations of amino acids. For example, S424N represents a substitution at position 424 from serine (Ser) to asparagine (Asn) in EU numbering. EU424N represents a substitution at position 424 from amino acid (of any type) to asparagine (Asn) in EU numbering.

The term "antibody comprising an Fc region" herein refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine lysine (residues 446-447) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region according to the present disclosure may comprise an antibody having G446-K447, an antibody having G446 and not having K447, an antibody having all G446-K447 removed, or a mixture of the three types of antibodies described above.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region or a variant Fc region as defined herein.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen binding residues.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4,

for the purposes herein, an "acceptor human framework" is a framework defined below comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or may comprise amino acid sequence variations. 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 sequence is identical to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

A "human consensus framework" is a framework representing the most common amino acid residues in the selection of human immunoglobulin VL framework sequences or VH framework sequences. Typically, the selection of human immunoglobulin VL sequences or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, NIH publication 91-3242, bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is the kappa I subgroup as in Kabat et al, supra. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al, supra.

"humanized" antibodies are chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which 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. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies, e.g., non-human antibodies, refer to antibodies that have undergone humanization.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., kindt et al, kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated from antigen-binding antibodies using VH or VL domains to screen libraries of complementary VL or VH domains, respectively. See, e.g., portolano et al, j.immunol. (journal of immunology) 150; clarkson et al, nature 352, 624-628 (1991).

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which comprises antigen-contacting residues ("antigen contacts"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) The hypervariable loops 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. (J.Mol.Biol.) 196;

(b) CDRs at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al, protein sequence of immunological interest, fifth edition, national institutes of health, besserda, mass. (1991));

(c) Antigen contacts at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al, J.mol.biol. (J.Mol.Biol.) 262-732-745 (1996)); and

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

Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in variable domains are numbered herein according to Kabat et al, supra. Herein, HVR residues or other residues (e.g., FR residues) within the variable domain, as well as amino acid changes or substitutions at these residues, may be represented by the Kabat numbering system and combinations of amino acids. For example, N99 represents asparagine (Asn) at position 99 in the Kabat numbering, and N99A represents a substitution from asparagine (Asn) to alanine (Ala) at position 99 in the Kabat numbering.

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

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function 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 drugs (e.g., methotrexate, doxorubicin (adriamycin), vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin (doxorubicin), melphalan, mitomycin C, chlorambucil, daunomycin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor agents or anticancer agents disclosed below.

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

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures, as well as vectors which integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

By "encoding nucleic acid that encodes an anti-CD 137 antigen-binding molecule" is meant one or more nucleic acid molecules that encode the polypeptides that make up the antigen-binding molecule. An "isolated nucleic acid encoding an anti-CD 137 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not have the same nucleic acid content as the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, such as those containing naturally occurring mutations or those that occur during the production of monoclonal antibody preparations, which variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the present disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies are described herein.

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

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy or heavy chain variable domain, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, each light chain has, from N-terminus to C-terminus, a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light chain structure (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after the sequences are aligned and gaps, if necessary, introduced to achieve the maximum percent sequence identity and no conservative substitutions are considered as part of the sequence identity. Alignment can be achieved by various means within the skill in the art to determine the percentage of amino acid sequence identity, for example, using public computer software such as BLAST, BLAST-2, ALIGN, megalign (DNASTAR) software or GENETYX (registered trademark) (GENETYX ltd). One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

The ALIGN-2 sequence comparison computer program was written by Genettech, inc., and the source code was submitted with the user document to the U.S. copyright office of 20559, washington, D.C., and registered with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from GeneTak corporation, san Francisco, calif., and may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including Digital UNIX V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and were unchanged. In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity (which may be expressed in terms of phrases as the% amino acid sequence identity a has or comprises for a given amino acid sequence B) for a given amino acid sequence a with, or with, a given amino acid sequence B is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of amino acid residues that sequence alignment program ALIGN-2 is scored as an identical match in an alignment of the program to A and B, wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is different from the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the% amino acid sequence identity of B to A. Unless otherwise specifically stated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

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

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation that is not toxic to the subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, adjuvants, stabilizers or preservatives.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, is an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage and for the desired period of time.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs and 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.

As used herein, unless otherwise indicated, 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). The term encompasses "full-length" untreated CD137 as well as any form of CD137 that results from processing in a cell. The term also encompasses naturally occurring variants of CD137, such as splice variants or allelic variants.

The amino acid sequence of an exemplary full-length human CD137 is shown in SEQ ID NO 1 (NCBI reference sequence: NP-001552.2) and the amino acid sequence of an exemplary extracellular region of human CD137 is shown in SEQ ID NO 2. The amino acid sequence of an exemplary full-length mouse CD137 is shown in SEQ ID NO 3 (NCBI reference: NP-035742.1) and the amino acid sequence of an exemplary extracellular region of mouse CD137 is shown in SEQ ID NO 4. The amino acid sequence of the exemplary monkey's full-length CD137 is shown in SEQ ID NO:5 (NCBI reference sequence: ABY 47575.1) and the amino acid sequence of the exemplary monkey's CD137 extracellular region 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 (TNFRSF 9), 4-1BB, and ILA. Except in activated CD4 ⁺ T cells and CD8 ⁺ In addition to expression on T cells, CD137 may also be expressed on B cells, dendritic cells, natural Killer (NK) and NK-T cells, macrophages, monocytes, neutrophils, CD4 ⁺ CD25 ⁺ Regulatory T cells and vascular endothelial cells. Expression in cancer cells has also been reported (Labiano et al, tumor immunology, vol.24: e1062967 (2015)). The natural CD137 ligand, CD137L, is presented by antigen presenting cells (e.g., B cells, monocytes/macrophages and dendritic cells) (Watts et al, annual review in immunology, vol. 23: pages 23-68 (2005)). CD137 leads to TCR-induced increases in T cell proliferation through interaction with ligands Cytokine production, functional maturation, apoptosis inhibition and CD8 ⁺ Long-term survival of T cells (Nam et al, recent advances in cancer Drug target research (curr. Cancer Drug Targets), vol.5: pp.357-363 (2005); watts et al, annual review in immunology (Annu. Rev. Immunol.), vol.23: pp.23-68 (2005)).

The term "regulatory T cells" or "Treg cells" are T cells responsible for the suppressive regulation of the immune response (immune tolerance). In one embodiment, the regulatory T cell is CD4 ⁺ And/or CD25 ⁺ T cells.

The terms "cancer," "cancer," and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled growth/proliferation of cells.

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

The terms "cell proliferative disease" and "proliferative disease" refer to a disease associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disease is cancer.

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treating") refers to a clinical intervention that attempts to alter the natural course of a treated individual, and may be used prophylactically or during the course of a clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, alleviating or lessening the state of a disease, alleviating or improving prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the onset of or slow the progression of a disease.

Compositions and methods (anti-CD 137 agonistic antigen binding molecules)

In one aspect, the invention is based, in part, on anti-CD 137 agonistic antigen binding molecules and uses thereof. In certain embodiments, antibodies that bind CD137 are provided. The antibodies of the present disclosure may exhibit activation of immune cells, cytotoxicity, or anti-tumor activity, and thus may be useful, for example, in diagnosing or treating cancer.

A. Exemplary anti-CD 137 antigen binding molecules or antibodies

In one aspect, the present disclosure provides an isolated antigen binding molecule or antibody that binds CD 137. In certain embodiments, an anti-CD 137 antigen binding molecule or antibody

-has a small molecule compound dependent CD137 binding activity;

-an extracellular region bound to CD137;

-forming a ternary complex with a low molecular weight compound and CD137;

-binding to human-derived CD137 and monkey-derived CD137;

-agonism on CD137 activity;

-shows agonistic activity on CD137 in the presence of low molecular weight compounds;

-has low agonist activity on CD137 in the absence of low molecular weight compounds; and/or

Substantially no agonist activity on CD137 in the absence of low molecular weight compounds.

[ binding Activity of antigen-binding molecule or antibody]

In certain embodiments, the binding activity of an antigen-binding molecule or antibody provided herein has a dissociation constant (KD) of 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10nM or less) in the presence of a low molecular weight compound ^-6 M is less than or equal to 10 ^-7 M is less than or equal to 10 ^-8 M is less than or equal to 10 ^-9 M is less than or equal to 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)。

In one embodiment, the binding activity of the antigen binding molecule or antibody is mediated byRadiolabeled antigen binding assay (RIA) and is represented by KD. In one embodiment, the radiolabeled antigen binding assay is performed with a Fab form of the antibody of interest and its antigen. For example, by using a minimum concentration of (in the presence of a series of unlabeled antigen titrations: ( ¹²⁵ I) Antigen-balanced Fab was labeled and bound antigen was then captured with anti-Fab antibody coated plates to measure solution binding affinity of Fab to antigen (see, e.g., chen et al, journal of molecular biology (j.mol.biol.), 293:865-881 (1999)). To establish assay conditions, MICROTITER (registered trademark) multi-well plates (Thermo Scientific) were coated overnight with 5. Mu.g/mL of capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH 9.6). Followed by blocking with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 degrees Celsius) for 2 to 5 hours. In the non-adsorption plate (Nunc # 269620), mixing 100pM or 26pM ¹²⁵ I]Antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody Fab-12, presta et al, cancer research (Cancer res.), 57 ^TM (ii) a Packard) and place the plate in TOPCOUNT ^TM The gamma counter (Packard) counts up to ten minutes. The concentration of each Fab that produced less than or equal to 20% of the maximal binding was selected for competitive binding assays.

In one embodiment, in order to measure the binding activity of the antibody, a ligand capture method is used, for example, BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE medical, uppsala, sweden) is used, which relies on a surface plasmon resonance analysis method as a measurement principle. BIACORE (registered trademark) control software is used for the operation of the device. In one embodiment, the amine coupling kit (GE medical), uppsala, sweden) is used according to the manufacturer's instructions to immobilize molecules for ligand capture such as anti-tag antibodies, anti-IgG antibodies, protein a, etc. on a sensor chip coated with carboxymethyl dextran (GE medical), uppsala, sweden). The molecules of capture ligand are diluted with 10mM sodium acetate solution at the appropriate pH and injected at the appropriate flow rate and appropriate injection time. The binding activity was measured at a flow rate of 10 to 30. Mu.l/min using a buffer containing 0.05% polysorbate 20 (i.e., tween (registered trademark) -20) as a measurement buffer at a measurement temperature of preferably 25 ℃ or 37 ℃. For measurements with the antibody captured by the ligand capture molecule as ligand, the antibody is injected such that the target amount of antibody is captured, and then serial dilutions of the antigen and/or Fc receptor (analyte) prepared with the measurement buffer are injected. For measurements performed with antigen and/or Fc receptor captured by the ligand capture molecule as ligand, the antigen and/or Fc receptor is injected such that a target amount thereof is captured, and then serial dilutions of the antibody (analyte) prepared with the measurement buffer are injected.

In one embodiment, the measurement results are analyzed using BIACORE (registered trademark) evaluation software. By using 1:1 binding model sensorgrams for association and dissociation were fitted simultaneously, kinetic parameter calculations were performed, and association rates (kon or ka), dissociation rates (koff or KD), and equilibrium dissociation constants (KD) were calculated. For the case of weak binding activity, especially for the case of fast dissociation and difficult kinetic parameters to calculate, the equilibrium dissociation constant (KD) can be calculated using a steady state model. As an additional parameter regarding the binding activity, the "amount of binding of analyte per unit amount of ligand" can be calculated by dividing the amount of binding of analyte at a specific concentration (resonance unit: RU) by the amount of captured ligand.

[ Small molecule Compound-dependent binding Activity]

In one aspect, the anti-CD 137 antigen-binding molecule or antibody has small molecule compound-dependent CD137 binding activity. In one non-limiting embodiment, the anti-CD 137 antigen binding molecule or antibody has greater binding activity against CD137 in the presence of the small molecule compound than the binding activity against CD137 in the absence of the small molecule compoundHigh binding activity. In various embodiments, the anti-CD 137 antigen-binding molecule or antibody has a higher binding activity for CD137 in the presence of high concentrations of small molecule compounds than the binding activity for CD137 in the presence of low concentrations of small molecule compounds. In a preferred embodiment, the binding activity of the anti-CD 137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x10 times or more the binding activity in the absence of the small molecule compound ³ More than twice, 2x10 ³ More than twice, 3x10 ³ 5x10 times higher ³ 1x10 times greater ⁴ More than twice, 2x10 ⁴ More than twice, 3x10 ⁴ 5x10 times higher ⁴ More than two times or 1x10 ⁵ More than twice. In various preferred embodiments, the binding activity of the anti-CD 137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound is 2 times greater, 3 times greater, 5 times greater, 10 times greater, 15 times greater, 20 times greater, 25 times greater, 30 times greater, 50 times greater, 100 times greater, 200 times greater, 300 times greater, 500 times greater, 1x10 times greater than the binding activity in the absence of the small molecule compound ³ Height of 2x10 ³ Height of 3x10 ³ Height of 5x10 ³ Height of 1x10 ⁴ Multiple height, 2X10 ⁴ Multiple height, 3X10 ⁴ Multiple height, 5X10 ⁴ Height of 1X10 ⁵ Is high in efficiency.

The concentration of the small molecule compound may be any concentration as long as a difference in binding activity of the anti-CD 137 antigen-binding molecule or antibody is detected. In one embodiment, the concentration of the small molecule compound is, for example, 100nM or more, 500nM or more, 1 μ M or more, 3 μ M or more, 5 μ M or more, 10 μ M or more, 50 μ M or more, 100 μ M or more, 150 μ M or more, 200 μ M or more, 250 μ M or more, 300 μ M or more, 400 μ M or more, 500 μ M or more, or 1mM or more "in the presence of the small molecule compound" and/or "in the presence of a high concentration of the small molecule compound. Alternatively, the concentration may be defined as an amount sufficient for the anti-CD 137 antigen-binding molecule or antibody to show maximum binding activity. Furthermore, in one embodiment, the concentration of the small molecule compound "in the presence of a low concentration of the small molecule compound" may be, for example, 500 μ M or less, 250 μ M or less, 200 μ M or less, 150 μ M or less, 100 μ M or less, 50 μ M or less, 10 μ M or less, 1 μ M or less, 500nM or less, 100nM or less, 50nM or less or 10nM or 1nM or less. The case where the concentration of the small molecule compound is zero or the base concentration is zero may also be selected as an embodiment of a low concentration.

The term "a substantial concentration of zero" as used herein refers to, for example, a concentration that is so small that it cannot be detected by current techniques even in the presence of small molecule compounds.

In one embodiment, the binding activity to CD137 in the presence of a small molecule compound at a concentration of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M is more than 2 times, more than 5 times, more than 10 times, more than 15 times, more than 16 times, more than 17 times, more than 18 times, more than 19 times, or more than 20 times the binding activity to CD137 in the absence of the small molecule compound. In one embodiment, the binding activity of the anti-CD 137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound at 10 μ M or more is 2 times or more, 5 times or more, 10 times or more, 15 times or more, 16 times or more, 17 times or more, 18 times or more, 19 times or more, or 20 times or more the binding activity to CD137 in the absence of the small molecule compound. In one embodiment, the binding activity of the anti-CD 137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound at 100 μ M or more is 2 times or more, 5 times or more, 10 times or more, 15 times or more, 16 times or more, 17 times or more, 18 times or more, 19 times or more, or 20 times or more the binding activity to CD137 in the absence of the small molecule compound.

In one embodiment, the binding activity (KD) of the anti-CD 137 antigen-binding molecule or antibody to CD137 is 9x10 in the presence of the small molecule compound at 10 μ M or more ^-7 M below, 8x10 ^-7 M < 7x10 ^-7 M below, 6x10 ^-7 M below, 5x10 ^-7 M or less, or 4x10 ^-7 Solution of M or lessDissociation constant (KD), or preferably, 5x10 ^-7 Dissociation constant (KD) below M. In another embodiment, the binding activity (KD) of the anti-CD 137 antigen-binding molecule or antibody to CD137 is too great to be calculated by Biacore (weak binding activity) or has a dissociation constant (KD) of 1x10 in the absence of the small molecule compound ^-7 M above, 5x10 ^-7 M or more, 7x10 ^-7 M above, 8x10 ^-7 Above M, 9x10 ^-7 More than M, 1X10 ^-6 M or more, 2X 10 ^-6 More than M, 3X10 ^-6 M or more, or 4X10 ^-6 M or more, or preferably, a dissociation constant (KD) of 1x10 ^-6 M is more than M. In another embodiment, the binding activity (KD) of the anti-CD 137 antigen-binding molecule or antibody to CD137 is 9x10 when the small molecule compound is present at 100 μ M or more ^-7 M < 8x10 ^-7 M is less than, 7x10 ^-7 M below, 6x10 ^-7 M below, 5x10 ^-7 M < 4x10 ^-7 M below, 3x10 ^-7 M is less than, 2X 10 ^-7 M or less, or 1X10 ^-7 M or less, or preferably, a dissociation constant (KD) of 2X 10 ^-7 M is less than or equal to M. In another embodiment, the binding activity (KD) of the anti-CD 137 antigen-binding molecule or antibody to CD137 is too great to be calculated by Biacore (weak binding activity) or the dissociation constant (KD) is 1x10 in the absence of small molecule compounds ^-7 M above, 5x10 ^-7 M is more than, 7x10 ^-7 M above, 8x10 ^-7 Above M, 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, or preferably, a dissociation constant (KD) of 1x10 ^-6 M is more than M.

In one embodiment, the binding activity (KD) of the anti-CD 137 antigen-binding molecule or antibody to CD137 is 8x10 in the presence of the small molecule compound at 10 μ M or more ^-8 M or less, and the binding activity (KD) to CD137 in the absence of compound was too great to be calculated by Biacore (weak binding activity). In another embodiment, the anti-CD 137 antigen binding molecule or anti-CD 137 antigen binding molecule is present in 100 μ M of the small molecule compoundThe binding activity (KD) of the body to CD137 was 2x10 ^-8 M or less, and the binding activity to CD137 in the absence of small molecule compounds is too great to be calculated by Biacore (weak binding activity).

In one aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody, wherein the value of [ binding activity (binding amount) to CD137 in the presence of a low molecular compound of 10 μ M or more ]/[ binding activity (binding amount) to CD137 in the absence of a small molecular compound) ] is equal to or greater than that of a reference anti-CD 137 antigen-binding molecule. In a different aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody, wherein the value of [ binding activity (binding amount) to CD137 in the presence of a low molecular compound at 100 μ M or more ]/[ binding activity (binding amount) to CD137 in the absence of a small molecular compound) ] is equal to or greater than that of a reference anti-CD 137 antigen-binding molecule. In any of the above aspects, the reference anti-CD 137 antigen-binding molecule may be selected from an anti-CD 137 antibody that comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as a375/B167, a372/B040, a356/B040, a486/B167, a487/B167, a488/B226, a489/B223, a548/B376, a551/B256, a555/B379, a548/B256, or the HVR-H1, r-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 described in table 37.

In one embodiment, the reference anti-CD 137 antigen-binding molecule is an antibody comprising as the 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 described in table 37. In various preferred embodiments, the reference antigen binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprised in A375/B167. In another embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a375/B167 as a heavy chain variable region/light chain variable region combination. In various preferred embodiments, the reference antigen binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprised in A551/B379. In another embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment, the reference antigen binding molecule comprises heavy and light chain constant regions of human origin (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).

In one aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody, wherein the binding activity (binding amount) thereof to CD137 is the same or lower than the binding activity (binding amount) of a reference anti-CD 137 antigen-binding molecule to CD137 in the absence of a small molecule compound, and the binding activity (binding amount) to CD137 in the presence of a small molecule compound of 10 μ M or more is equal to or higher than the binding activity (binding amount) to CD137 of the reference anti-CD 137 antigen-binding molecule under the same conditions. In a different aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody, wherein the binding activity to CD137 in the absence of a small molecule compound is the same or lower than the binding activity (binding amount) to CD137 of a reference anti-CD 137 antigen-binding molecule. And, the binding activity (binding amount) to CD137 in the presence of 10 μ M or more of the small molecule compound is equal to or higher than the binding activity (binding amount) to CD137 under the same conditions of the reference anti-CD 137 antigen-binding molecule. In any of the above aspects, the reference anti-CD 137 antigen-binding molecule may be selected from an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence 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, or the HVR-H1, HVR-H2, R-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A549/B167 described in Table 37.

In one embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising the amino acid sequence of a375/B167, a372/B040, a356/B040, a486/B167, a487/B167, a488/B226, a489/B223, a548/B376, a551/B256, a551/B379, a555/B379, a548/B256, or a549/B167 described in table 37 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprised in A375/B167. In another embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a375/B167 as a heavy chain variable region/light chain variable region combination. In various preferred embodiments, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprised in A551/B379. In another embodiment, the reference anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment, the reference antigen binding molecule comprises a heavy chain constant region and a light chain constant region of human origin (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human lambda chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

In one aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody, wherein the value of [ binding activity (KD) to CD137 in the presence of 1 μ M low molecular compound) ]/[ binding activity (KD) to CD137 in the presence of 10 μ M or more low molecular compound) ] is equal to or greater than the value of the reference antigen-binding molecule. In various aspects, the present disclosure provides anti-CD 137 antigen-binding molecules or antibodies, wherein the value of [ binding activity (KD) to CD137 in the presence of 1 μ M low molecular compound) ]/[ binding activity (KD) to CD137 in the presence of 100 μ M or more low molecular compound) ] is equal to or greater than the value of the reference antigen-binding molecule. In any of the above aspects, the reference antigen binding molecule can be selected from an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-H1, HVR-H2, HVR-L3, and HVR-L3 included in A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B9, A548/B256, or A549/B167 listed in Table 37.

In one embodiment, the reference antigen binding molecule is an antibody comprising as the 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 described in Table 37. In a preferred embodiment, the reference antigen binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, wherein the amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 is identical to the amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 comprised in A375/B167. In another embodiment, the reference antigen binding molecule is an antibody comprising a375/B167 as a heavy chain variable region/light chain variable region combination. In various embodiments, the reference antigen binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 is the same as the amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551/B379. In another embodiment, the reference antigen binding molecule is an antibody comprising a551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment, the reference antigen binding molecule comprises a heavy chain constant region and a light chain constant region of human origin (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human lambda chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

In one embodiment, the binding activity of the anti-CD 137 antibody to CD137 in the presence of a small molecule compound, in the absence of a small molecule compound, at a high concentration and/or at a low concentration is measured, for example, by a ligand capture method using BIACORE (registered trademark) T200 with surface plasmon resonance spectroscopy as a measurement principle.

Details of an exemplary method for measuring the binding activity of an anti-CD 137 antibody to CD137 are described below. In one embodiment, the binding activity of the anti-CD 137 antibody to CD137 is assessed by BIACORE (registered trademark) T200. In a preferred embodiment, the assay uses 20mM ACES (pH 7.4), 150mM NaCl, 2mM MgCl ₂ And 0.05% tween 20 as running buffer and was performed at 37 ℃. In one embodiment, the measurement is performed after the antibody is captured as a ligand on the ligand capture molecule. Specifically, a suitable amount (for example, about 100ru,200ru,300ru,400ru, or 500 RU) of the antibody is captured by allowing an antibody solution prepared using a running buffer to interact with a chip first prepared by immobilizing Sure protein a (GE medical) on an S-series sensor chip CM3 (GE medical).

In a preferred embodiment, about 100RU to 500RU, preferably about 250RU to 400RU of the antibody is captured. Next, the binding activity to CD137 in the presence and absence of a small molecule compound was evaluated by interacting a CD137 solution prepared using a running buffer to which a small molecule compound was added to a target concentration (e.g., 1 μ M,10 μ M,50 μ M, or 100 μ M), or a CD137 solution prepared using a running buffer that did not contain a small molecule compound. Although the concentration of CD137 in a CD137 solution can be appropriately determined, for example, when hCD137-HisBAP (see reference example 1-1) is used as an antigen, measurement is performed using antigen concentrations of 0nM, 15.625nM, 62.5nM, 250nM, and 1000nM, respectively. In one embodiment, the dissociation constant (KD) of the anti-CD 137 antibody to human CD137 is calculated using Biacore T200 evaluation software 2.0. Specifically, the association rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) were calculated by fitting the sensorgrams obtained by measurement as a whole using a 1. From these values, the dissociation constant KD (mol/L) was calculated.

Other exemplary assays for measuring the binding activity of anti-CD 137 antibodies to CD137 will be described in detail below. Binding of anti-CD 137 antibodies to human CD137 was assessed using Biacore T200. 20mM ACES (pH 7.4), 150mM NaCl, 2mM MgCl were used ₂ And 0.05% tween 20 as running buffer, binding to human CD137 was determined and performed at 37 ℃. First, about 250 to 400RU of antibody was captured by the interaction of an antibody solution prepared using a running buffer with a chip in which Sure Protein a (GE medical) was immobilized on an S-series sensor chip CM3 (GE medical). Next, a human CD137 solution prepared using a running buffer to which ATP is added at a target concentration (e.g., 1 μ M,10 μ M,50 μ M, or 100 μ M), or a human CD137 solution prepared using a running buffer without ATP, is subjected to an interaction to evaluate the binding activity to CD137 in the presence and absence of ATP. hCD137-HisBAP prepared by the method of reference example (1-1) was used as human CD137 as an antigen, and measured at antigen concentrations of 0nM, 15.625nM, 62.5nM, 250nM and 1000nM, respectively. The chip was regenerated using 25mM NaOH and 10mM glycine-HCl (pH 1.5) and measured by repeated capture of antibody. Dissociation constants for each antibody to human CD137 were calculated using Biacore T200 evaluation software 2.0. Specifically, the association rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) were calculated by fitting the sensorgrams obtained by measurement as a whole using a 1. From these values, the dissociation constant KD (mol/L) was calculated.

In one embodiment, the binding activity of an anti-CD 137 antibody to CD137 (preferably human CD 137) may also be rewritten as "CD 137 binding amount per unit amount of antibody". Specifically, by using a sensorgram obtained by the above-described measurement method using BIACORE (registered trademark) T200, the amount of binding (RU) of CD137 to the antibody was divided by the amount of the captured antibody, thereby calculating "the amount of binding of CD137 per unit amount of the antibody". In one embodiment, the binding activity of an anti-CD 137 antibody to CD137 (preferably human CD 137) can also be measured by the method described in reference example 5-3 or 6-2.

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

The term "target tissue" refers to any tissue in vivo to which the antigen binding molecules of the present disclosure are to be delivered. The target tissue may be histologically distinguishable tissue, such as various organs, or pathologically distinguishable tissue, such as normal tissue and diseased tissue. In certain embodiments, the target tissue is tumor tissue. In contrast, "non-target tissue" refers to tissue within a living body other than the target tissue.

The term "tumor tissue" refers to a tissue comprising at least one tumor cell. Typically, tumor tissue is composed of a population of tumor cells (parenchyma) that make up the bulk of the tumor, and connective tissue and blood vessels (stroma) that exist between the tumor cells and support the tumor. In some cases these are clearly distinguishable, but in some cases they are mixed together. In some cases, there are cells, such as immune cells, that have penetrated into the tumor tissue. In contrast, "non-tumor tissue" refers to tissue in vivo other than tumor tissue. Non-diseased healthy/normal tissue is representative of such non-tumor tissue.

As a non-limiting embodiment of the cancer tissue-specific compound or the cancer tissue-specific metabolite used in the present disclosure, at least one compound selected from the compounds described in detail below may be suitably exemplified. The meaning of "at least one compound" includes: in addition to the case where the binding activity of the same antigen-binding domain to an antigen described below depends on one type of cancer tissue-specific compound or cancer tissue-specific metabolite, the case where the binding activity depends on several types of cancer tissue-specific compounds or cancer tissue-specific metabolites is also included.

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

In a specific embodiment, the tumor tissue specific compound is a metabolite produced by tumor cell specific metabolism. The metabolites may be products produced by metabolism necessary for life activities (primary metabolites) or products produced by metabolism unnecessary for life activities (secondary metabolites). Examples of major metabolites may include sugars, proteins, lipids, nucleic acids, and the like. Examples of secondary metabolites include antibiotics and dyes. The metabolite may be a biopolymer or a small molecule. In particular embodiments, the biopolymer is a molecule having a molecular weight of about 5000 or greater, which is composed of one or more types of repeating units, including, for example, polysaccharides, polypeptides, and polynucleotides. In particular embodiments, a small molecule is a molecule having a molecular weight of about 500 or less, and is a chemical species present in vivo. In a further embodiment, the tumor tissue specific compound is a small molecule metabolite produced specifically in tumor cells (Eva Gottfried, katrin Peter and Marina p. Kreutz, molecular and modular tumor therapy (2010) 3 (2), 111-132). In a further embodiment, the tumor tissue specific compound is a metabolite that is specifically produced by cells infiltrating the tumor tissue (e.g., immune cells) or stromal cells present in the tumor tissue (e.g., cancer-associated fibroblasts (CAF)). Examples of immune cells infiltrating into tumor tissue are dendritic cells, suppressor dendritic cells, regulatory T cells, depleted T cells, myeloma-derived suppressor cells (MDSCs), and the like. In further embodiments, metabolites produced by cells present in tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.) and released outside of the cells when the cells die due to apoptosis or necrosis, etc., may also be included in the tumor tissue-specific compounds of the present disclosure.

For the identification of tumor tissue specific compounds, analyses performed at the transcriptome level (e.g., dhanasekaran et al (Nature (2001) 41, 822-826), lapointe et al (Proc. Natl. Acad. Sci. USA (2004) 101, 811-816) or Perou et al (Nature (2000) 406, 747-752)) and analyses performed at the proteome level (e.g., ahrrm et al (mol. Carcinog. (2002) 33, 9-15) and Hood et al (molecular cell proteomics) (2005) 4, 1741-1753)) can be suitably used, as well as metabolic analyses centered on metabolomics (metabolomics). That is, in order to identify a metabolite in a test sample, 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)), metabolic spectrum analysis using ELISA, and the like may be used alone and/or in combination as appropriate.

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

(1) Nucleosides having a purine structure, such as Adenosine (ADO), adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), adenosine Monophosphate (AMP), and inosine;

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

(3) Metabolites of amino acids, such as kynurenine (kynurenine), anthranilic acid, 3-hydroxykynurenine, and kynurenic acid (kynurenic acid);

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

(5) Major metabolites of the glycolytic pathway or the Krebs cycle (Krebs cycle), such as lactic acid, succinic acid and citric acid; and the combination of (a) and (b),

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

(1) Nucleosides having a purine structure, such as Adenosine (ADO), adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP) and inosine

It is well known that when tumor cells die, a large amount of intracellular ATP leaks out. Therefore, ATP concentrations in tumor tissues were significantly higher than those in normal tissues (PLoS one. (2008) 3, e 2599). AMPs are metabolized by enzymes on the cell surface, such as extracellular 5 '-nucleotidase (eco-5' -nucleotidase) (CD 73) (Resta and Thompson (immunological review (immunol. Rev.), (1998) 161, 95-109) and Sadej et al (Melanoma studies (Melanoma res.), 2006) 16, 21 3-222). Adenosine is a purine nucleoside, constitutively present in the extracellular environment at low concentrations, but extracellular adenosine concentrations have been reported to increase significantly in hypoxic tissues found in solid tumors (Blay and Hoskin (Cancer res.), (1997) 57, 260 2-2605). CD73 is expressed on the surface of tumor and immune cells (Kobie et al (J.Immunol.), (2006) 177, 6780-6786)) and found to be increased in Breast Cancer (Cancer et al (Breast Cancer Res. Treat. (1996) 37, 189-193)), stomach Cancer (Durak et al (Cancer Lett.), 1994) 84, 199-202)), pancreatic Cancer (Flcke and Mannherz (Biochim. Biophys. Acta (1991), 1076, 273-281) and glioblastoma (dot et al (Br.J.cancer (1994) 70, 212-218)). It has been proposed that adenosine accumulation in tumor tissues is a result of increased dephosphorylation of AMPs by cytoplasmic 5' -nucleotidase (Headrick and Willis (J. Biochem., (1989) 261, 541-550)). Furthermore, regulatory T cells infiltrating into tumor tissue also express atpase and produce adenosine (proc.natl.acad.sci. Us (2006) 103 (35), 13132-13137 curr.med.chem. (2011) 18. It is believed that the adenosine produced maintains tumour tissue in an immunosuppressive environment via adenosine receptors such as the A2A receptor (curr. Med. Chem. (2011) 18, 5217-5223). Therefore, ATP, ADP, AMP, adenosine, and the like, which are believed to accumulate in tumor tissue at high concentrations through the metabolism of purine nucleotides, are examples of tumor tissue-specific compounds used in the present disclosure. In addition, inosine accumulates at high concentrations as adenosine is degraded to inosine by adenosine deaminase.

In a specific embodiment, the nucleoside having a purine ring structure includes an adenosine-containing compound. In particular embodiments, 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), [ gamma-thio ] adenosine triphosphate (ATP γ S), and the like. In another embodiment, the nucleoside having a purine ring structure comprises inosine, which is a metabolite of adenosine.

Further, in a specific embodiment, the nucleoside having a purine ring structure includes commercially available nucleosides having a purine ring structure, such as ADPbetaS (Sigma company) and the like.

(2) Amino acids, e.g. alanine, glutamic acid, aspartic acid, etc

In tumor cells, the uptake rate of glutamine as a nitrogen carrier in living bodies is increased, and the incorporation of glutamine and the conversion thereof into glutamic acid and lactic acid (glutamine degradation) are considered to be characteristic of tumor cells (Mazurek and Eigenbrodt (Anticancer Res.), (2003) 23, 1149-1154, and Mazurek et al (J.cell. Physiol.), (1999) 181, 136-146)). Glutamine levels in the plasma of cancer patients are reduced, while glutamate concentrations are increased (Droge et al (immunology) (1987) 174, 473-479)), in lung cancer tissues ¹³ C-labeled glucose metabolism studies have shown ¹³ C-labeled succinic acid, ¹³ C-labeled alanine, ¹³ C-labelled glutamic acid and ¹³ c-labelled citric acidThere is a correlation between salt concentrations. For these reasons, alanine, glutamic acid, aspartic acid, and the like, which are believed to accumulate in tumor tissues at high concentrations, for example, due to degradation of glutamine, are examples of tumor tissue-specific compounds used in the present disclosure.

(3) Metabolites of amino acids, e.g. kynurenine, anthranilic acid, 3-hydroxykynurenine and kynurenic acid

Indoleamine 2, 3-dioxygenase (IDO) is a tryptophan metabolizing enzyme, high expression in many cancers, e.g., melanoma, colon, kidney, etc. (Uyttenhove et al (nat. Med. (2003) 9, 1269-1274); IDO catalyzes the conversion of tryptophan to kynurenine in gliomas that do not express IDO, kynurenine is produced from tryptophan by hepatic tryptophan 2,3-dioxygenase (TDO) (Opitz et al (Nature (2011) 478 (7368), 197-203)). IDO is also expressed on dendritic cells infiltrating into the tumor tissue, and dendritic cells also produce kynurenine (immunological studies (j. Immunol.), (2008) 181, 5396-5404). Furthermore, IDO is also expressed in Myeloid Derived Suppressor Cells (MDSCs) of tumor tissues, and MDSC also produces kynurenine (Yu et al (immunological studies (j.immunol.), (2013) 190, 3783-3797)). Kynureninase converts kynurenine to anthranilic acid, kynurenine 3-hydroxylase converts kynurenine to 3-hydroxykynurenine, both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, i.e., the precursors of NAD kynurenine transaminase converts kynurenine to kynurenine for these reasons, kynurenine and its metabolites, i.e., anthranilic acid, 3-hydroxykynurenine, kynurenic acid, etc., are examples of tumor tissue specific compounds, particularly tumor cell specific metabolites, used in the present disclosure.

(4) Metabolites of arachidonic acid, e.g. prostaglandin E2

Prostaglandin E2 (PGE 2) promotes the growth and inhibits apoptosis of colon Cancer cells (Sheng et al (Cancer research (Cancer res.), (1998) 58, 362-366) among PGE2 synthetases COX-1 is mainly found to be constitutively expressed in almost all tissues, whereas COX-2 is induced by certain inflammatory cytokines and oncogenes in tumors (Warner and Mitchell (FASEB j. (2004) 18, 790-804)), it has been reported that overexpression of COX-2 is associated with poor prognosis of breast Cancer (Denkert et al (clinical breast Cancer research (clin. Breast Cancer), (2004) 4, 428-433) and rapid progression of disease in ovarian Cancer (Denker et al (mod.pathol. (2006) 19, 1261-1269). Additionally, regulatory T cells infiltrating into tumor tissues also produce PGE2 (curr. Cherm (chrom. 18, 2011.) 18, 2011. Tx1-1269) metabolic products specific to tumor tissues, especially tumor tissues, such as the metabolism of thromboxane, the tumor tissue-specific metabolic products of PGE2 (pge.g. a. C. Pcr. Cherm. 5223, these are produced by regulated T cells) in tumor tissues, inter alia, metabolic products of tumor tissue, the metabolism of colon Cancer, intracellular dna, tnf-mediated tissue (cle 2).

(5) The major metabolites of the glycolytic pathway or the Krebs cycle, such as lactic acid, succinic acid and citric acid

The glycolytic phenotype characterized by upregulation of glycolytic (Embden-Meyerhof pathway) enzymes such as pyruvate kinase, hexokinase, and Lactate Dehydrogenase (LDH) is commonly referred to as the Warburg effect, which is characteristic of solid tumors. The end products of glycolysis, lactic acid, as well as succinic acid and citric acid produced by the krebs cycle, are known to accumulate in tumor tissues (Teresa et al (molecular cancer (2009) 8, 41-59)). For these reasons, lactic acid, succinic acid, citric acid, and the like, which are major metabolites produced by glycolysis, are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in the present disclosure. In addition, it is known that succinate present in cells at high concentration leaks out of cells due to cell death (Nature Immunology), (2008) 9, 1261-1269). This is thought to be responsible for the increased concentration of succinic acid in tumor tissues where cell death frequently occurs.

(6) Metabolites of nicotinamide, e.g. 1-methylnicotinamide

Nicotinamide N-methyltransferase is known to be highly expressed in many human tumor tissues. It is also known that 1-methylnicotinamide, which is a stable metabolite of nicotinamide produced by this enzyme, is secreted outside tumor cells (Yamada et al (j.nutr.sci.vitaminol. (2010) 56, 83-86)). For this reason, 1-methylnicotinamide and the like, which is considered to accumulate in high concentrations in tumor tissues due to the metabolism of nicotinamide, are considered as examples of the tumor tissue-specific compound used in the present disclosure.

An "antigen binding molecule" of the present disclosure comprises an "antigen binding domain". As the "antigen-binding domain", a domain of any structure may be used as long as it binds to a target antigen. In one embodiment, the antigen binding domains of the present disclosure include, for example, variable regions of antibody heavy and/or light chains, avimers (international publications WO2004/044011 and WO 2005/040229) comprising a module of about 35 amino acids (a domain) contained in various cell membrane proteins of an organism, adnectins (international publication WO 2002/032925) comprising the 10Fn3 domain of fibronectin of glycoproteins expressed on cell membranes, affibodies (WO 1995/001937) using the IgG binding domain of 58 amino acids of protein a as a scaffold, darns (designed ankyrin repeat proteins) using ankyrin repeat sequences (AR) of 33 amino acid repeat sequences as a basis (international publication WO 2002/020565), anchorals (e.g., neutrophil gelatinase-associated lipocalin (NGAL)) based lipocalin (international publication WO 2003/940262), variable Lymphocyte Receptors (VLR), which are amalgams such as amaurognals and contain modules of eel mucin (WO 2008, WO 016854) and the like, which serve as amalgams in amalgamatory systems of acanthra and eel (WO 2008). In particular embodiments, the antigen binding domains of the present disclosure comprise the heavy and light chain variable regions of an antibody. In further embodiments, the antigen binding domains of the present disclosure include, for example, scFv (single chain Fv), single chain antibodies, fv, scFv2 (single chain Fv 2), fab, or F (ab') 2.

[ HVR and variable region]

In one aspect, the disclosure provides an anti-CD 137 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 an amino acid sequence selected from any one of SEQ ID NOs 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (c) HVR-H3 comprising an amino acid sequence selected from any one of SEQ ID NOs 17, 18, 19 or 20. In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising an amino acid sequence selected from any one of SEQ ID NOs 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (c) HVR-H3 comprising an amino acid sequence selected from any one of SEQ ID NOs 17, 18, 19 or 20.

In one embodiment, the anti-CD 137 antigen-binding molecule is an antibody comprising as the 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 described in Table 37. In a preferred embodiment, the antigen binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 included in A375/B167. In further embodiments, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a375/B167 as a heavy chain variable region/light chain variable region combination. In various preferred embodiments, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 included in A551/B379. In further embodiments, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising a551/B379 as a heavy chain variable region/light chain variable region combination.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In a different aspect, the present disclosure provides an anti-CD 137 antigen binding molecule or antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising an amino acid sequence selected from any one of SEQ ID NOs 21, 22, 23, 24 and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising an amino acid sequence selected from any one of SEQ ID NOs 27, 28 and 29. In one embodiment, an anti-CD 137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising an amino acid sequence selected from any one of SEQ ID NOs 21, 22, 23, 24, and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising an amino acid sequence selected from any one of SEQ ID NOs 27, 28 and 29.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In one embodiment, the anti-CD 137 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.

In another aspect, an anti-CD 137 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), (ii), and (iii) below: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) HVR-H2 comprising an amino acid sequence selected from any one of SEQ ID NOs 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (iii) HVR-H3 comprising an amino acid sequence selected from any one of 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), (ii), and (iii) below: (i) HVR-L1 comprising an amino acid sequence selected from any one of 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 an amino acid sequence selected from any one of SEQ ID NOs 27, 28 and 29.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In another aspect, the present disclosure provides an anti-CD 137 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.

In specific embodiments, any one or more amino acids of the above anti-CD 137 antibodies are substituted at the following HVR positions:

in HVR-H2 (SEQ ID NO: 30): position 5, 6, 7, 10, 13, 14 and/or 17;

in HVR-H3 (SEQ ID NO: 31): position 3 and/or 6;

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

in HVR-L3 (SEQ ID NO: 33): positions 6, 7 and/or 8.

In particular embodiments, the substitutions provided herein are conservative substitutions. In particular embodiments, any one or more of the following substitutions may be made in any combination:

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

in HVR-H3 (SEQ ID NO: 17): A3P, K or I; F6E;

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

in HVR-L3 (SEQ ID NO: 27): E6P; H7A; Q8I

For HVR-H2, HVR-H3, HVR-L1 and HVR-L3, all possible combinations of the above substitutions are contained in the consensus sequences of SEQ ID NOS 30, 31, 32 and 33, respectively.

In any of the above embodiments, the anti-CD 137 antigen binding molecule or antibody is humanized. In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises any of the HVRs as in the embodiments described above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-CD 137 antigen-binding molecule or antibody comprises any of the HVRs of the embodiments described above, and further comprises a heavy chain variable region (VH) or a light chain variable region (VL) comprising a Framework (FR) sequence. In one embodiment, FR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 35, FR2 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 36, FR3 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 37, and FR4 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 38. In one embodiment, FR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:39, FR2 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:40, FR3 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:41 and FR4 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42.

In another aspect, the anti-CD 137 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 NOs 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-CD 137 antigen-binding molecule or antibody comprising the sequence retains the ability to bind to CD 137. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-CD 137 antibody comprises the VH sequence of SEQ ID NO 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, including post-translational modifications of that sequence. In particular 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 an amino acid sequence selected from any one of SEQ ID NOs 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (c) HVR-H3 comprising an amino acid sequence selected from any one of SEQ ID NOs 17, 18, 19 or 20. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In another aspect, an anti-CD 137 antigen binding molecule or antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs 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 comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-CD 137 antigen-binding molecule or antibody comprising the sequence retains the ability to bind to CD 137. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs 54, 55, 56, 57, 58, 59 or 60. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-CD 137 antigen binding molecule or antibody comprises the VL sequence of SEQ ID NO 54, 55, 56, 57, 58, 59 or 60, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising an amino acid sequence selected from any one of SEQ ID NOs 21, 22, 23, 24 and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising an amino acid sequence selected from any one of SEQ ID NOs 27, 28 and 29. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In another aspect, an anti-CD 137 antigen binding molecule or antibody is provided, wherein said antigen binding molecule or antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID No. 43 and SEQ ID No. 54, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID No. 44 and SEQ ID No. 55, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID No. 45 and SEQ ID No. 55, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO 46 and SEQ ID NO 54, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO 47 and SEQ ID NO 54, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID No. 48 and SEQ ID No. 56, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID No. 49 and SEQ ID No. 57, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:50 and SEQ ID NO:58, respectively, including post-translational modifications of these sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:51 and SEQ ID NO:59, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:51 and SEQ ID NO:60, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:52 and SEQ ID NO:60, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:50 and SEQ ID NO:59, respectively, including post-translational modifications of those sequences.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO:53 and SEQ ID NO:54, respectively, including post-translational modifications of those sequences.

Such post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

For each anti-CD 137 antigen binding molecule or antibody of the present disclosure, the amino acid sequences corresponding to the preferred heavy chain variable region and light chain variable region and the SEQ ID NOs of HVR1, HVR2 and HVR3 thereof are shown in the table below.

[ Table 1]

When the anti-CD 137 antigen-binding molecules or antibodies provided herein have glutamine as the heavy or light chain N-terminal amino acid, that amino acid may be substituted with glutamic acid. When the anti-CD 137 antibody provided herein has glutamic acid as the heavy or light chain N-terminal amino acid, the amino acid may be substituted with glutamine.

In a preferred embodiment, an anti-CD 137 antigen-binding molecule or antibody comprising the HVRs, heavy chain variable region and/or light chain variable region described above, all have low molecular weight compound-dependent binding activity for CD137 as described above.

[ constant region]

In another aspect, the anti-CD 137 antigen binding molecule or antibody comprises a constant region. The constant region can be a heavy chain constant region (including an Fc region), a light chain constant region, or both. In another aspect, the anti-CD 137 antigen binding molecule or antibody comprises an Fc region. In some embodiments, the constant region is a constant region having a native sequence. Examples of heavy chain constant regions derived from natural antibodies include, for example, the heavy chain constant regions of human IgG1 (SEQ ID NOS: 61, 62), human IgG2, human IgG3, human IgG4, and the like. Examples of light chain constant regions derived from natural antibodies include, for example, human kappa chains, human lambda chains (e.g., SEQ ID NO: 63), and the like.

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 alterations described herein. By "parent antigen binding molecule" is meant an antigen binding molecule comprising a parent constant region or a parent Fc region. In some embodiments, the parent Fc region is an Fc region having a native sequence (or an Fc region of a native antibody). Antibodies include, for example, igA (IgA 1, igA 2), igD, igE, igG (IgG 1, igG2, igG3, igG 4), igM and the like. The antibody may be derived from a human or monkey (e.g. cynomolgus monkey, rhesus monkey, marmoset monkey, chimpanzee or baboon). Natural antibodies may also include naturally occurring mutations. A number of IgG allotype sequences arising from genetic polymorphisms are described in NIH publication No. 91-3242, "protein sequences of immunological significance", and any of them can be used in the present disclosure. In one embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of human IgG1, as shown in SEQ ID NO 61, 62, or 182.

In one aspect, the anti-CD 137 antigen-binding molecule or antibody has an increased isoelectric point (pI) as compared to an anti-CD 137 antigen-binding molecule or antibody comprising a native sequence Fc region or a parent Fc region. In some embodiments, the variant Fc region comprises at least one amino acid alteration. In a further embodiment, the amino acid change results in an increase in the isoelectric point (pI) of the variant Fc region as compared to the parent Fc region. Without being bound by a particular theory, it is believed that the pH of the biological fluid (e.g., plasma) is in the neutral pH range. In biological fluids, the net positive charge of the antigen binding molecule or antibody with increased pI is increased due to the increased pI, and as a result, the physicochemical coulombic interaction more strongly attracts the antigen binding molecule or antibody to the surface of endothelial cells, which have a net negative charge compared to antigen binding molecules or antibodies without increased pI. In this way, an agonistic antigen binding molecule (or antibody) or an agonistic antigen binding molecule (or antibody) that binds antigen may be brought closer to the surface of a cell expressing an Fc-gamma receptor, resulting in increased binding of the antigen binding molecule or antibody to the Fc-gamma receptor expressing cell. For those anti-CD 137 agonistic antigen-binding molecules or antibodies that exhibit CD137 agonistic activity based on their contribution to the binding activity of Fc-gamma receptors, anti-CD 137 agonistic antigen-binding molecules or antibodies that increase binding to Fc-gamma receptor-expressing cells due to the amino acid alteration that increases pI may exhibit greater CD137 agonistic activity as compared to anti-CD 137 agonistic antigen-binding molecules or antibodies that do not have the amino acid alteration that increases pI.

In the present disclosure, the pI may be a theoretically or experimentally determined pI. The value of pI can be determined, for example, by isoelectric focusing as known to those skilled in the art. The value of the theoretical pI can be calculated using, for example, gene and amino acid sequence analysis software (Genetyx, etc.). In the calculation, the properties of the antibody can be reflected in the calculation formula. For example, (i) typically, cys, conserved in antibodies, forms disulfide bonds and does not carry the charge of side chains; thus, such Cys may be excluded from the calculation, and only free form Cys that does not form a disulfide bond may be included in the calculation. Or, (ii) the charge state or isoelectric point of the antibody may change due to post-translational modification; thus, in view of this post-translational modification, the calculation formula can be modified as follows: (a) When the N-terminus of the heavy chain is Q (glutamine), the N-terminal amino group is not counted assuming pyroglutamyl acylation to occur; (b) When the C-terminus of the heavy chain is K (lysine), K (only one residue) is excluded from the calculation, assuming truncation occurs; and (C) assuming that all these C's form disulfide bonds within the molecule, the side chains of all C's (cysteines) present at commonly conserved positions are excluded from the calculation. In a preferred embodiment, both of the above (i) and (ii) can be reflected in the calculation formula.

In one embodiment, the pI value may be increased, e.g., 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, as compared to before modification.

In one embodiment, the amino acid changes associated with increased pI and methods of increasing pI of an antigen binding molecule or antibody are described in detail herein "iii compositions and methods (agonistic antigen binding molecules comprising a variant Fc region with increased isoelectric point (pI)". One skilled in the art will understand that any of the amino acid changes and methods described in "iii. Compositions and methods (agonistic antigen binding molecules comprising a variant Fc region with increased isoelectric point (pI)" for increasing pI can be applied to an anti-CD 137 antigen binding molecule or antibody.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody has a variant Fc region with an increased pI, and the variant Fc region comprises at least one amino acid change at least one position selected from the group consisting of: positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 according to EU numbering. In a further embodiment, the variant Fc-region with increased pI comprises Arg or Lys at each selected position.

In further embodiments, the anti-CD 137 antigen binding molecule or antibody has a variant Fc region with an increased pI, and the variant Fc region comprises at least one amino acid change at least one position selected from the group consisting of: positions 311, 343 and 413 according to EU numbering. In a further embodiment, the variant Fc-region having an increased pI comprises an amino acid change at position 311, 343 or 413 according to EU numbering. In a further embodiment, the variant Fc-region with increased pI comprises Arg or Lys at each selected position.

In another aspect, the present disclosure provides an anti-CD 137 antigen binding molecule or antibody comprising a variant Fc region having an increased pI, the variant Fc region comprising an amino acid change of any one of the following (1) to (3): according to EU numbering, (1) at positions 311 and 343; (2) at positions 311 and 413; and (3) at positions 343 and 413. In a further embodiment, the variant Fc-region with increased pI comprises Arg or Lys at each selected position.

In one embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure comprise a variant Fc region comprising the amino acid changes identified in table 2 below.

Amino acid changes to increase pI of Fc region

[ Table 2]

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises a variant Fc region prepared by making amino acid changes to an Fc region having the native sequence. In one embodiment, the variant Fc region has increased binding activity to at least one Fc- γ receptor selected from the group consisting of Fc- γ RIa, fc- γ RIIa, fc- γ RIIb, fc- γ RIIIa and Fc- γ RIIIb, as compared to an Fc region having the native sequence or a parent Fc region. Preferably, the variant Fc region has increased binding activity to Fc- γ RIIb as compared to an Fc region having the native sequence or a parent Fc region. anti-CD 137 antibodies comprising a variant Fc region having increased binding activity to Fc- γ RIIb are reported to have increased agonistic activity compared to anti-CD 137 antibodies comprising an Fc region having the native sequence. In one embodiment, as amino acid changes that increase the binding activity to Fc- γ RIIb, for example, the amino acid changes taught in WO2012/115241, WO2014/030728, WO2014/163101 and/or WO2017/104783 may be used. In a preferred embodiment, the alteration that increases binding activity to Fc- γ RIIb is an amino acid alteration at least one position selected from the group consisting of: positions 234, 235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbering.

"Fc γ receptor" (referred to herein as Fc γ receptor, fc γ R, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, igG2, igG3, and IgG4 monoclonal antibodies, and specifically refers to any member of the family of proteins encoded by the Fc γ receptor gene. In humans, this family includes Fc γ RI (CD 64), which includes isoforms Fc γ RIa, fc γ RIb and Fc γ RIc; fc γ RII (CD 32), which includes isoforms Fc γ RIIa (including allotypes H131 (H-type) and R131 (R-type)), fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; and Fc γ RIII (CD 16), which includes isoforms Fc γ RIIIa (including allotypes V158 and F158) and Fc γ RIIIb (including allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA 2), as well as any human Fc γ R, an Fc γ R isoform or allotype not yet found, but is not so limited. Fc γ RIIb1 and Fc γ RIIb2 are reported to be splice variants of human Fc γ RIIb. In addition, splice variants known as Fc γ RIIb3 have been reported (J Exp Med,1989, 170, 1369-1385. In addition to these splice variants, human Fc γ RIIb includes AAI46679.1 registered in NCBI and all splice variants registered in NCBI, which are NP _001002273.1, NP _001002274.1, NP _001002275.1, NP _001177757.1 and NP _003992.3. In addition, human Fc γ RIIb includes each of the genetic polymorphisms previously reported, as well as Fc γ RIIb (Arthritis and rheumatism (Arthritis Rheum.) 48:3242-3252 (2003); kono et al, hum. Mol. Genet.14:2881-2892 (2005); and Kyogoju et al, arthritis and rheumatism (Arthritis Rheum.), 46.

In Fc γ RIIa, there are two allotypes, one with histidine at amino acid position 131 of Fc γ RIIa (type H) and one with arginine substituted at amino acid position 131 (type R) (Warrerdam, J.Exp.Med.172:19-25 (1990)).

Fc γ R includes Fc γ R of human, mouse, rat, rabbit and monkey origin, but is not limited thereto, and may be derived from any organism. Mouse Fc γ rs include Fc γ RI (CD 64), fc γ RII (CD 32), fc γ RIII (CD 16), and Fc γ RIII-2 (CD 16-2), as well as any mouse Fc γ R or Fc γ R isotype, but are not so limited.

In another aspect, the present disclosure provides an anti-CD 137 antigen binding molecule or antibody comprising a variant Fc region having increased binding activity to Fc- γ RIIb, said variant Fc region comprising an amino acid alteration of any one of the following (1) to (8): according to EU numbering, (1) at positions 234, 238, 264 and 330; (2) at positions 234, 238 and 330; (3) at positions 234, 237, 238, and 330; (4) at positions 236, 268, and 330; (5) at positions 235, 236, 268, 295, 326 and 330.

In one embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present invention comprise a variant Fc region comprising the amino acid changes identified in table 3 below. In another embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure comprise a variant Fc region comprising any one of the combinations of amino acid alterations identified in table 3 below, in addition to the amino acid alterations described in table 2 (the amino acid alterations involved in increasing the pI of the Fc region).

Amino acid changes for increasing Fc- γ RIIb binding activity in Fc region

[ Table 3]

Numbering	Amino acid substitutions (EU numbering)
		1	L234Y/P238D/V264I/A330K
2	L234Y/P238D/A330K
		3	L234Y/G237D/P238D/A330K
4	G236N/H268D/A330K
		5	L235W/G236N/H268D/Q295L/K326T/A330K

In one embodiment, the present disclosure provides variant Fc regions, including those having at least one amino acid change and having equal or greater binding activity to Fc- γ RIIb than the reference Fc region. In one embodiment, the reference Fc region is an Fc region comprising any one of the combinations of amino acid changes identified in table 3 above. In a preferred embodiment, the reference Fc region is an Fc region comprised in heavy chain constant region TT14 (SEQ ID NO: 149), TT16 (SEQ ID NO: 150), MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154). In a preferred embodiment, the reference Fc region is an Fc region comprised in the heavy chain constant region MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154).

In another aspect, the present disclosure provides an isolated agonistic antigen binding molecule or antibody comprising a variant Fc region having increased binding activity to an Fc-gamma receptor (preferably, fc-gamma RIIb) and increased pI. In a certain embodiment, a variant Fc region described herein comprises at least two amino acid alterations in a parent Fc region. As described above, antigen binding molecules or antibodies with increased pI are more strongly attracted to endothelial cell surfaces with a net negative charge by physicochemical coulombic interactions than antigen binding molecules or antibodies without increased pI. Thus, for those agonistic antigen-binding molecules or antibodies that exhibit agonistic activity based on their contribution to the binding activity of an Fc-gamma receptor, preferably Fc-gamma RIIb, the agonistic activity of the antigen-binding molecule or antibody may be increased by a combination of amino acid changes that increase the Fc-gamma receptor, preferably Fc-gamma RIIb, or amino acid changes that increase the pI.

In one embodiment, the anti-CD 137 antigen binding molecule or antibody comprises a variant Fc region comprising both amino acid changes that increase binding activity to an Fc-gamma receptor (e.g., fc-gamma RIIb) and amino acid changes that increase the isoelectric point (pI) as described above. As described above, antigen binding molecules or antibodies with increased pI are attracted more strongly to endothelial cell surfaces with a net negative charge by physicochemical coulombic interactions than antigen binding molecules or antibodies without increased pI. Thus, for those anti-CD 137 agonist antigen-binding molecules or antibodies that show CD137 agonistic activity based on their contribution to the binding activity of an Fc-gamma receptor (preferably Fc-gamma RIIb), the agonistic activity of the anti-CD 137 antigen-binding molecule or antibody may be increased by a combination of increasing the amino acid alteration of the Fc-gamma receptor (preferably Fc-gamma RIIb) and increasing the amino acid alteration of the pI.

In one aspect, the disclosure provides a polypeptide comprising a variant Fc region having increased binding activity to Fc- γ RIIb and having an increased pI, said variant Fc region comprising at least three amino acid alterations comprising (a) at least one amino acid alteration at least one position selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbering, and (b) at least two amino acid alterations at least two positions selected from the group consisting of positions 311, 343 and 413 according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a variant Fc region having increased binding activity to Fc- γ RIIb and increased pI, comprising an amino acid change of any one of the following (1) to (26): according to 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.

In one embodiment, a variant Fc region of the present disclosure comprises any combination of the amino acid alterations identified in table 4 below.

[ Table 4]

Number of	Amino acid substitutions (EU numbering)
		1	L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K
2	K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K
		3	L234Y/P238D/T250V/V264I/T307P/A330K/P343R/D413K
4	L234Y/P238D/V264I/A330K/P343R/D413K
		5	L234Y/G237D/P238D/T250V/T307P/A330K/P343R/D413K
6	L234Y/G237D/P238D/A330K/P343R/D413K
		7	L235W/G236N/H268D/Q295L/K326T/A330K/Q311R/P343R
8	L234Y/P238D/T250V/V264I/T307P/A330K/Q311R/P343R
		9	L234Y/P238D/V264I/A330K/Q311R/P343R
10	L234Y/G237D/P238D/T250V/T307P/A330K/Q311R/P343R
		11	L234Y/G237D/P238D/A330K/Q311R/P343R
12	L235W/G236N/H268D/Q295L/K326T/A330K/P343R
		13	K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R
14	L235W/G236N/H268D/Q295L/K326T/A330K/D413K
		15	K214R/G236N/H268D/A330K/P343R
16	K214R/L235W/G236N/H268D/A330K/P343R
		17	K214R/G236N/H268D/A330K/D413K
18	K214R/G236N/H268D/A330K/P343R/D413K
		19	K214R/L235W/G236N/H268D/A330K/P343R/D413K
20	K214R/G236N/H268D/A330K/Q311R
		21	K214R/L235W/G236N/H268D/A330K/Q311R
22	K214R/G236N/H268D/A330K/Q311R/P343R
		23	K214R/L235W/G236N/H268D/A330K/Q311R/P343R
24	K214R/G236N/H268D/A330K/Q311R/D413K
		25	K214R/L235W/G236N/H268D/A330K/Q311R/D413K
26	K214R/L235W/G236N/H268D/Q295L/K326T/A330K/Q311R

In one embodiment, a variant Fc region comprising any combination of the amino acid alterations described in table 4 above lacks the amino acid at position 447 according to EU numbering. In a preferred embodiment, a variant Fc region comprising any combination of the amino acid alterations described in table 4 above lacks the amino acids at positions 446 and 447 according to EU numbering.

The skilled person will understand that in addition to the alterations exemplified above, at least one amino acid alteration that increases the binding activity to Fc- γ R (including Fc- γ RIIb) compared to the parent Fc region as described or suggested, for example, in WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101 or WO2017104783, and at least one amino acid alteration that increases the pI compared to the parent Fc region as described or suggested, for example, in WO2017/104783, WO2017/046994, and any combination of these amino acid alterations may be used.

In addition, amino acid changes made for other purposes may be incorporated into the variant Fc regions described herein. For example, the following amino acid substitutions may be added: amino acid substitutions that increase FcRn binding activity (Hinton et al, j.immunol.176 (1): 346-356 (2006); dall' Acqua et al, j.biol.chem.281 (33): 23514-23524 (2006); petkova et al, intl.immunol.18 (12): 1759-9 (2006); zalevsky et al, nat.biotechnol.17628 (2): 157-159 (2010); WO 2006/019447, WO 2006/053301; and WO 2009/086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO 2009/041613). Alternatively, polypeptides having the property of facilitating antigen clearance described in WO 2011/122011, WO 2012/132067, WO 2013/046704, or WO 2013/180201, polypeptides having the property of specifically binding to a target tissue described in WO 2013/180200, polypeptides having the property of repeatedly binding to multiple antigenic molecules described in WO 2009/125825, WO 2012/073992, or WO 2013/047752 may be combined with the variant Fc regions described herein. Alternatively, the amino acid changes disclosed in EP1752471 and EP1772465 may be combined in CH3 of the variant Fc regions described herein in order to confer binding activity to other antigens.

In one embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure comprise a heavy chain constant region comprising any one of the amino acid sequences selected from SEQ ID NOs 64-85. Preferably, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO 75 or 82.

In a preferred embodiment, an anti-CD 137 antigen binding molecule or antibody comprising a variant Fc region as described above has the CD137 binding activity as described above in relation to small molecule-dependent compounds.

In one embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure comprise the following variable and constant regions: a variable region comprising the HVR, heavy chain variable region and/or light chain variable region described above; and the variant Fc region described above. In a preferred embodiment, the anti-CD 137 antigen binding molecule or antibody of the present disclosure may be any anti-CD 137 antibody selected from the group consisting of the antibodies described in table 72.

In another aspect, the present disclosure provides antigen binding molecules or antibodies that bind to the same epitope on CD137 of an anti-CD 137 antigen binding molecule or antibody provided herein in the presence of a low molecular weight compound (e.g., in the presence of 10 μ Μ or more, 50 μ Μ or more, 100 μ Μ or more, 150 μ Μ or more, 200 μ Μ or more, or 250 μ Μ or more of the low molecular weight compound). For example, in certain embodiments, antigen binding molecules or antibodies are provided that bind to the same epitope as an anti-CD 137 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 37 as a heavy chain variable region/light chain variable region combination. In one embodiment, an anti-CD 137 antigen-binding molecule or antibody of the present disclosure having CD137 binding activity that is dependent on the antigen-binding activity of a small molecule compound recognizes an epitope formed by a complex formed by an antigen (e.g., CD 137) and a low molecular weight compound (e.g., ATP).

In another aspect, the present disclosure provides antigen binding molecules or antibodies that compete with anti-CD 137 antigen binding molecules or antibodies provided herein for binding to CD137 in the presence of a low molecular weight compound (e.g., in the presence of 10 μ Μ or more, 50 μ Μ or more, 100 μ Μ or more, 150 μ Μ or more, 200 μ Μ or more, or 250 μ Μ or more of the low molecular weight compound). For example, in certain embodiments, the anti-CD 137 antigen-binding molecules or antibodies compete for the site of binding to CD137 with anti-CD 137 antigen-binding molecules or antibodies that comprise 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 37 as heavy chain variable region/light chain variable region combinations.

In another aspect of the disclosure, the anti-CD 137 antigen binding molecule or antibody according to any of the embodiments above is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-CD 137 antibody is an antibody fragment, such as an Fv, fab ', scFv, diabody, or F (ab') ₂ And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody type or isotype as defined herein.

In another aspect, an anti-CD 137 antigen-binding molecule or antibody according to any of the above embodiments may comprise any feature, alone or in combination, as described in sections 1-7 below:

1. agonistic activity of anti-CD 137 antigen-binding molecules or antibodies

In particular embodiments, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure have CD137 agonistic activity. CD137 signaling not only stimulates IFN- γ secretion and proliferation of NK cells (buechelle et al, 2012, lin et al, 2008, melero et al, 1998), but also enhances their survival and DC activation as indicated by upregulated costimulatory molecules and cytokine secretion (Choi et al, 2009, futagawa et al, 2002. However, CD137 is best characterized as CD4 on T cells ⁺ And CD8 ⁺ Co-stimulatory molecules in the subset that modulate TCR-induced activation. anti-CD 137 agonist antibodies enhance T cell proliferation in combination with TCR activationStimulating lymphokine secretion and reducing the sensitivity of T lymphocytes to activation-induced cell death (Snelet et al, reviewed in 2011). Among these phenomena, the physiological phenomena observed on T cells following CD137 signaling are mediated by downstream signals activated by CD137 signaling, such as TRAF2, TRAF1, in particular NF-. Kappa.B, JNK, erk, akt, survivin (survivin), bcl-XL and/or Bcl-2 (Ward-Kavanagh et al, immunity, 44 (2016)).

In one embodiment, an "anti-CD 137 agonist antigen binding molecule" or "anti-CD 137 agonist antibody" is an antigen binding molecule or antibody that: it transduces CD137 signals by binding to CD137 and significantly induces or enhances IFN- γ secretion, proliferation and increased survival of NK cells; DC activation, indicated by cytokine secretion and upregulation of costimulatory molecules; TCR induction; t cell proliferation; and/or lymphokine secretion. In various embodiments, an "anti-CD 137 agonistic antigen binding molecule" or "anti-CD 137 agonistic antibody" is an antigen binding molecule or antibody that transduces CD137 signals by binding to CD137 on T cells and significantly induces NF- κ B that activates T cells. Furthermore, the antigen binding molecule or antibody "showing CD137 agonistic activity" means that any of the above-described physiological phenomena is observed when the antigen binding molecule or antibody binds to CD 137. The method of measuring CD137 agonist activity is described in detail in the "c.

In particular embodiments, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure have small molecule compound-dependent CD137 agonistic activity. In one non-limiting embodiment, the CD137 agonistic activity of the anti-CD 137 antigen-binding molecule or antibody against CD137 is greater in the presence of the small molecule compound than in the absence of the small molecule compound. In various embodiments, the CD137 agonistic activity of the anti-CD 137 antigen-binding molecule or antibody is greater in the presence of high concentrations of the small molecule compound compared to the CD137 agonistic activity in the presence of low concentrations of the small molecule compound. In a further embodiment, the CD137 agonistic activity of the anti-CD 137 antigen-binding molecule or antibody in the presence of the small molecule compound is in the absence of the small molecule compound More than 2 times, more than 3 times, more than 5 times, more than 10 times, more than 20 times, more than 30 times, more than 50 times, more than 100 times, more than 200 times, more than 300 times, more than 500 times, 1x10 times of the next CD137 agonistic activity ³ More than twice, 2x10 ³ More than twice, 3x10 ³ More than twice, 5x10 ³ More than twice, 1x10 ⁴ More than twice, 2x10 ⁴ More than twice, 3x10 ⁴ More than twice, 5x10 ⁴ More than twice, or 1x10 ⁵ More than twice.

Any suitable concentration may be selected as the concentration of the small molecule compound as long as a difference in binding activity of the anti-CD 137 antigen-binding molecule or antibody is detected. In one embodiment, the anti-CD 137 antigen binding molecule or antibody transduces CD137 signals by binding to CD137 on the surface of a cell. Thus, one skilled in the art would understand that an anti-CD 137 antigen-binding molecule or antibody having small molecule compound-dependent CD137 binding activity has CD137 agonistic activity that is dependent on the small molecule compound. However, on the other hand, since the methods of measuring binding activity and agonistic activity are different, one skilled in the art will appreciate that the concentration of the small molecule compound at which a difference in binding activity is detected may be different from the concentration of the small molecule compound at which a difference in agonistic activity is detected (e.g., for an anti-CD 137 antigen-binding molecule or antibody in which CD137 binding activity is more than 2 times that in the absence of the small molecule compound in the presence of 10 μ M of the small molecule compound, CD137 agonistic activity (measured value) in the presence of 10 μ M of the small molecule compound may be less than 2 times that in the absence of the small molecule compound). Furthermore, it will be understood by those skilled in the art that the determination of agonistic activity may vary depending on the assay for CD137 agonistic activity (see "c. Assay").

In one embodiment, the anti-CD 137 antigen-binding molecule or antibody (i) shows agonistic activity on CD137 in the presence of a small molecule compound of 10 μ Μ, 50 μ Μ, 100 μ Μ, 150 μ Μ, 200 μ Μ or 250 μ Μ, and (ii) has substantially no agonistic activity on CD137 in the absence of the small molecule compound, or has low agonistic activity on CD137 in the absence of the small molecule compound (compared to the presence of the small molecule compound).

In one embodiment, when the agonist activity of the anti-CD 137 antigen-binding molecule or antibody is assessed by "a) an agonist activity assay (PBMC)" described in detail in "c. In a further embodiment, the anti-CD 137 antigen-binding molecule or antibody exhibits (i) agonistic activity against CD137 in the presence of 250 μ Μ of the small molecule compound, and (ii) substantially no agonistic activity against CD137 in the absence of the small molecule compound.

In one embodiment, when the agonistic activity of the anti-CD 137 antigen-binding molecule or antibody is evaluated by the "b) agonistic activity assay (reporter assay)" described in detail in the "c. Assay," the anti-CD 137 antigen-binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of 10 μ Μ,50 μ Μ, 100 μ Μ, 150 μ Μ, 200 μ Μ or 250 μ Μ small molecule compound, and (ii) has substantially no agonistic activity or has lower agonistic activity against CD137 in the absence of small molecule compound (as compared to in the presence of small molecule compound). The concentration of antibody in the reporter gene assay can be selected arbitrarily, for example, the final concentration of antibody is 0, 0.001, 0.01, 0.1, 1, or 10. Mu.g/mL. In preferred embodiments, the final concentration of antibody is 0.1. Mu.g/mL or 1. Mu.g/mL.

In one embodiment, in the "b.agonistic activity assay (reporter gene assay)" described in detail in the "c.assay", when the final concentration of the antibody is 0.1 μ g/mL, (i) the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the presence of 10 μ M of the small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than ii) the CD137 agonistic activity (relative light units) in the absence of the small molecule compound. In one embodiment, when the final concentration of the antibody in the "b. Agonistic activity assay (reporter gene assay)" described in detail in the "c. Assay" is 0.1 μ g/mL, (i) the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the presence of 100 μ M small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than ii) the CD137 agonistic activity (relative light units) in the absence of small molecule compound. In one embodiment, when the final concentration of the antibody in the "b.agonistic activity assay (reporter gene assay)" described in detail in the "c.assay" is 0.1 μ g/mL, (i) the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the presence of 250 μ M small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than ii) the CD137 agonistic activity (relative light units) in the absence of small molecule compound. In any of the embodiments above, further, in the absence of the small molecule compound, 0.1 μ g/mL of the anti-CD 137 antigen-binding molecule or antibody shows substantially no CD137 agonistic activity.

In one embodiment, when the final concentration of the antibody is "1 μ g/mL", in the "b.agonistic activity assay (reporter gene assay)" described in detail in the "c.assay" (i) the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the presence of 10 μ M small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than ii) the CD137 agonistic activity (relative light units) in the absence of small molecule compound. In one embodiment, in the "b.agonistic activity assay (reporter gene assay)" described in detail in the "c.assay", when the final concentration of the antibody is 0.1 μ g/mL, (i) the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the presence of 100 μ M of the small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than ii) the CD137 agonistic activity (relative light units) in the absence of the small molecule compound. In one embodiment, the CD137 agonistic activity (relative light units) of the anti-CD 137 antigen-binding molecule or antibody in the "b.agonistic activity assay (reporter gene assay)" described in detail in the "c.assay" is (i) 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more higher than that of the CD137 agonistic activity (relative light units) in the presence of 250 μ M of a small molecule compound in (i) the final concentration of the antibody is 0.1 μ g/mL. In any of the above embodiments, further, in the absence of the small molecule compound, 1 μ g/mL of the anti-CD 137 antigen-binding molecule or antibody shows substantially no CD137 agonistic activity.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, F (ab') ₂ Fv and scFv fragments, as well as other fragments described below. For a review of some antibody fragments, see Hudson et al, nat. Med.,9:129-134 (2003). For reviews on scFv fragments, see, e.g., pluckthun, pharmacology of monoclonal antibodies, volume 113, rosenburg and Moore eds., (Springer-Verlag, new York), pp.269-315 (1994); see also WO 93/16185; U.S. Pat. nos. 5,571,894 and 5,587,458. Relating to Fab and F (ab') containing rescue receptor binding epitope residues and having increased half-life in vivo ₂ See U.S. Pat. No. 5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen-binding sites and may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, nat. Med.,9:129-134 (2003); and Hollinger et al, proc.natl.acad.sci.usa,90:6444-6448 (1993). Tri-and tetrad antibodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part 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. Pat. No. 6,248,516B1).

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

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,56; 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 mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, front.biosci.13:1619-1633 (2008), and further described, for example, in Riechmann et al, nature 332:323-329 (1988); queen et al, proc.nat' l Acad.Sci.USA86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, methods 36 (2005) (specificity determining region (SDR) transplantation is described); padlan, mol.Immunol.28:489-498 (1991) (describe "resurfacing"); dall' Acqua et al, methods 36 (Methods) 43-60 (2005) (describing "FR shuffling"); and Osbourn et al, methods 36 (2005) and Klimka et al, br.J. cancer,83, 252-260 (2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach (see, e.g., sims et al, J.Immunol.151:2296 (1993)); and framework regions derived from the consensus sequence of a human antibody of a particular subgroup of light chain variable regions or heavy chain variable regions (see, e.g., carter et al, proc. Natl. Acad. Sci. USA,89 4285 (1992); and Presta et al, J.Immunol.,151 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)); the framework regions derived from screening FR libraries (see, e.g., baca et al, J.biol.chem.272:10678-10684 (1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are commonly described in van Dijk and van de Winkel, curr. Opin. Pharmacol.5:368-74 (2001) and Lonberg, curr. 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the chromosome of the animal. In such a transgenic mouse, the expression of the gene,the endogenous immunoglobulin loci have typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, e.g., the description XENOMOUSE ^TM U.S. Pat. nos. 6,075,181 and 6,150,584 to technology; U.S. Pat. nos. 5,770,429 describing HuMab (registered trademark) technology; U.S. Pat. No. 7,041,870, which describes K-M MOUSE (registered trademark) technology, and U.S. patent application publication No. US 2007/0061900, which describes VelociMouse (registered trademark) technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example by conjugation with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (see, e.g., kozbor J.Immunol.,133 (1984); brodeur et al, monoclonal antibody production techniques and applications, pp 51-63 (Marcel Dekker, inc., new York, 1987); and Boerner Li et al, J.Immunol.,147 (1991)). Human antibodies produced by human B-cell hybridoma technology are described in Li et al, proc.natl.acad.sci.usa,103, 3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. 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). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, histology and histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, methods and findings of experimental and clinical pharmacology, 27 (3): 185-91 (2005).

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from a library of antibodies are described below.

5. Antibodies derived from libraries

Antibodies of the disclosure can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding properties. Such methods are described, for example, in Hoogenboom et al, methods in molecular biology, 178:1-37 (edited by O' Brien et al, human Press, totorwa, N.J., 2001) and described further below: for example, mcCafferty et al, nature, 348:552 to 554; clackson et al, nature, 352:624-628 (1991); marks et al, j.mol.biol.222:581-597 (1992); marks and Bradbury, methods in Molecular Biology,248:161-175 (Lo, ed., human Press, totorwa, new Jersey, 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).

In some phage display methods, VH and VL gene libraries are cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in the phage library, which can then be amplified according to Winter et al, ann. Screening for antigen binding phage as described in 433-455 (1994). Phage typically display antibody fragments, either as single chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to an immunogen without the need to construct hybridomas. Alternatively, a suitable material may be used such as Griffiths et al, EMBO j.,12:725-734 (1993), to clone naive libraries (e.g., from humans) to provide a single source of antibodies against a wide range of non-self as well as self-antigens without any immunization. Finally, naive libraries can also be synthesized by cloning unrearranged V gene fragments from stem cells and using PCR primers comprising random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, j.mol.biol., 227. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. patent publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

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

Antigen binding molecules or antibodies of the present disclosure having antigen binding activity that is dependent on low molecular weight compounds can be selected by screening on a library of antigen binding molecules. As such a library, the above-mentioned combinatorial library can be used. The library of antigen binding molecules may be a library with unbiased antigen binding molecules (naive library), or may be a library with biased antigen binding molecules. Examples of the latter type of libraries include libraries of antigen binding molecules that have been previously endowed with binding activity against a particular compound. In a certain embodiment, the library of antigen binding molecules is a library of antigen binding molecules into which amino acid changes conferring binding activity of a particular compound have been previously introduced. Examples of libraries of this type include, for example, the libraries described in international publication WO 2015/083764.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD137, while the other is for any other antigen. In certain embodiments, a bispecific antibody can bind two different epitopes of CD 137. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing CD 137. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

In one embodiment, the anti-CD 137 antigen-binding molecules or antibodies of the present disclosure are bispecific antibodies, one arm of which has CD 137-binding activity that is dependent on a small molecule compound, while the other arm binds to an antigen other than CD 137. The "antigen" other than CD137 is not particularly limited in structure. In other words, the antigen may be an inorganic substance or an organic substance. Exemplary antigens are disclosed below. In one embodiment, the antigen is preferably an antigen expressed in cancer cells, immune cells, stromal cells, and the like in cancer tissues or inflammatory tissues.

Herein, the "antigen" structures are not particularly limited as long as they comprise the epitope to which the antigen binding molecules of the present invention bind. The antigen may be an inorganic or organic substance. In some embodiments, examples of antigens include: 17-IA,4-1BB,4Dc, 6-keto-PGF 1a, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin (activin), activin A, activin AB, activin B, activin C, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressen, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, ang, APAF-1, APE, APJ, APP, APRIL, ARC, ART, atrix, arimin, anti-natriuretic, alpha-V/beta-1 antagonist, ADAM 3, ADAM/beta-1 antagonist, PARG, PARAF-1, APE, ADAM 3, axl, B2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, bad, BAFF-R, bag-1, BAK, bax, BCA-1, BCAM, bcl, BCMA, BDNF, B-ECGF, bFGF, BID, bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenic protein, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8 a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, B-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, <xnotran> CAD-8, , cAMP, (CEA), , A, B, C/DPPI, D, E, H, L, O, S, V, X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10,CCR,CCR1,CCR10,CCR10,CCR2,CCR3,CCR4,CCR5,CCR6,CCR7,CCR8,CCR9,CD1,CD2,CD3,CD3E,CD4,CD5,CD6,CD7,CD8,CD10,CD11a,CD11b,CD11c,CD13,CD14,CD15,CD16,CD18,CD19,CD20,CD21,CD22,CD23,CD25,CD27L,CD28,CD29,CD30,CD30L,CD32,CD33 (p 67 ), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, , , CKb8-1, (Claudin) -6,CLC,CMV,CMV UL,CNTF,CNTN-1,COX,C-Ret, CRG-2,CT-1,CTACK,CTGF,CTLA-4,PD1,PDL1,LAG3,TIM3, (galectin) -9,CX3CL1,CX3CR1,CXCL,CXCL1,CXCL2,CXCL3,CXCL4,CXCL5,CXCL6,CXCL7,CXCL8,CXCL9,CXCL10,CXCL11,CXCL12,CXCL13,CXCL14,CXCL15,CXCL16,CXCR,CXCR1,CXCR2,CXCR3,CXCR4,CXCR5,CXCR6, , DAN, DCC, dcR3, DC-SIGN, ( ), des (1-3) -IGF-I ( IGF-1), dhh, , DLL3, DNAM-1,DNA , dpp, DPPIV/CD26, dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, , , eNOS, eot, 1,EpCAM, B2/EphB4, </xnotran> EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast Activation Protein (FAP), fas, fcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR-3, fibrin, FL, FLIP, flt-3, flt-4, follicle stimulating hormone, CXXXC chemotactic molecule (fractalkine), FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, gas6, GCP-2, GCSF, GDD 2, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), CDMP-6 (BMP-13, CDMP-2), GDF-7 (BMP-12), myostatin (GDF-8), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR- α 2, GFR- α 3, GITR, glucagon, glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic Growth Factor (HGF), hepB gp120, heparanase, her2, her2/neu (ErbB-2), her3 (ErbB-3), her4 (ErbB-4), herpes Simplex Virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, hrk, human cardiac myosin, human Cytomegalovirus (HCMV), human Growth Hormone (HGH), HVEM, I-309, IAP, ICAM-1, ICAM-3, ICE, ICOS, IFNg, ig, igA receptor, igE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF) - α, INF- β, INF- γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α 2, integrin α 3, integrin α 4, integrin α 4/β 1, integrin α 4/β 7, integrin α 5 (α V), integrin α 5/β 1, integrin α 5/β 3, integrin α 6, integrin β 1, integrin β 2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP (TGF-1), latent TGF-1, LBP, LDGF, LECT2, lefty, lewis-Y antigen, lewis-Y associated antigens, LFA-1, LFA-3, lfo, LIF, LIGHT, lipoproteins, LIX, LKN, lptn, L-selectin, LT-a, LT-B, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, mac-1, MAdCAM, MAG, MAP2MARC, MCAM, MCK-2, MCP, M-CSF, MDC, mer, METALLOPROTEASS, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, mpo, MSK, MSP, mucin (Muc 1), MUC18, muller' S tube inhibitory substance, mug, muSK, NAIP, NAP, NCAD, N-C adhesin (adherin), NCA90, NCAM, NCAM, enkephalinase (neprilysin), neurotrophic factor-3, -4, or-6, neural RANK protein (neurturin), nerve Growth Factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), plGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory Syncytial Virus (RSV) F, RSV Fgp, ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, stat, STEAP-II, TACE, TACI, TAG-72 (tumor associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α/β), tdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, tfR, TGF, TGF- α, TGF- β, TGF- β pan-specificity, TGF- β RI (ALK-5), TGF- β RII, TGF- β RIIb, TGF- β RIII, TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, TGF- β 5, thrombin, thymic Ck-1, thyroid stimulating hormone, tie, TIMP, TIQ, tissue factor, TMEFF2, tmpo, TMPRSS2, TNF, TNF- α, TNF- β 2, TNFC, TNF-RI, TNF-RII, TNFRSF10A (TRAIL 1-2, DRApoApoR 4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 Dcr1, LIT, TRID), TNFRSF10D (TRAIL R4 Dcr2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR 1), TNFRSF12 (TWEAK R FN 14), TNFRSF13B (TACI), TNFRSF13C (BATR), TNFRSF14 (HVEM ATAR, hveA, LIGHT R, TR 2), TNFRSF16 (NGFR p75 NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TNFRY TAJ, TRAFF), TNFRSF19L (RELT), TNFRSF1A (TNFRRI CD120A, p 55-60), TNFRSF1B (TNFRSF 3-R), TNFRSF 80 (TNFRSF 3-R), TNFRSF4 (OX 40ACT35, TXGP 1R), TNFRSF5 (CD 40p 50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR 3M68, TR 6), TNFRSF7 (CD 27), TNFRSF8 (CD 30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR 6), TNFRSF22 (DcTRAIL R2TNFRH 2), TNFRST23 (DcRST 1 TNFRRH 1), TNFRSF25 (DR 3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFRSF10 (TRAIL Apo-2 ligand, TL 2), TNFRSF11 (TRANCE/RANK ligand, OPG ligand), TNFRSF12 (EAK-3 ligand, DR3 ligand, TNFRSF13 (APRIL TL 2), TNFRSF13B (BAYS 1, THYS 1, THLU-L14), TNFRSF18 (TNFRSF-1), TNFRSF6 (TNFRSF 15, TNFRSF-1, TNFRSF 15, TNFRSF 6), TNFSF1A (TNF-a selectin, DIF, TNFSF 2), TNFSF1B (TNF-B LTa, TNFSF 1), TNFSF3 (LTb TNFC, p 33), TNFSF4 (OX 40 ligand gp34, TXGP 1), TNFSF5 (CD 40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD 27 ligand CD 70), TNFSF8 (CD 30 ligand CD 153), TNFSF9 (4-1 BB ligand CD137 ligand), TP-1, t-PA, tpo, TRAIL, TRAIL-R1, TRANCE, transferrin receptor, TRF, trk, TROP-2, TLR1 (Toll-like receptor 1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TRAIL 8, TLR9, TREF 10, TLR 125, TSG 125, TSY antigen-related tumor antigen-associated carbohydrate-expressing compounds, TWEAK, TXB2, ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrins, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XEDAR, XIAP, XPD, HMGB1, igA, abeta, CD81, CD97, CD98, DDR1, DKK1, EREG, hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prokallikrein, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kinins, IL-31, IL-31R, nav1.1, nav1.2, nav1.3, nav1.4, nav1.5, nav1.6, nav1.7, nav1.8, nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin (sclerostin), fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, and S1P. In some embodiments, examples of antigens include hormone receptors and growth factors. In certain embodiments, antigens are those expressed or secreted in cells (e.g., tumor cells, immune cells, stromal cells, etc.) present in tumor tissue.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, nature, 305 (1983), WO 93/08829 and Traunecker et al, EMBO J.10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by: engineered electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, science, 229 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., kostelny et al, J.Immunol.148 (5): 1547-1553 (1992)); bispecific antibody fragments are prepared using the "diabody" technique (see, e.g., hollinger et al, journal of the american national academy of sciences (proc.natl.acad.sci.usa), 90 6444-6448 (1993)); and the use of single chain Fv (scFv) dimers (see, e.g., gruber et al, j.immunol.,152, 5368 (1994)); and as for example Tutt et al, journal of immunology (j. Immunol.), 147:60 (1991) the trispecific antibody was prepared as described.

Also included herein are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576 A1).

The antibodies or fragments herein also include "dual-acting Fab" or "DAF" comprising an antigen binding site that binds to CD137 as well as another, different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions 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 properties, e.g., antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 5 under the heading "preferred substitutions". Table 5 provides more substantial variations under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the product screened for a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

[ Table 5]

Original residues	Exemplary permutations	Preference is given to substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobic: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acidic: asp and Glu;

(4) Basic: his, lys, arg;

(5) Residues that influence chain orientation: gly, pro;

(6) Aromatic compounds: trp, tyr, phe.

Non-conservative substitutions will result in the exchange of members of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modification (e.g., improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hotspots", i.e., residues encoded by codons that mutate at high frequency during somatic maturation (see, e.g., chowdhury, methods in molecular biology (Methods mol. Biol., 207-179 (2008)), and/or residues that are contacted with antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al, methods in Molecular Biology, 178:1-37 (O' Brien et al, human Press, toltowa, N.J. (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, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR targeting methods in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular CDR-H3 and CDR-L3 are generally targeted.

In certain embodiments, a substitution, insertion, or deletion can occur within one or more HVRs, so long as the alteration does not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) can be made in HVRs. Such changes may be, for example, outside of antigen-contacting residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either remains unchanged or comprises no more than one, two, or three amino acid substitutions.

Such as Cunningham and Wells (1989), science, 244:1081-1085, a useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis". In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody interaction with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for replacement. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertional variants of the antibody molecule include the fusion of an enzyme (e.g., for ADEPT) or polypeptide that increases the plasma half-life of the antibody to the N-terminus or C-terminus of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise a branched bi-antennary oligosaccharide, which is typically attached by an N-bond to Asn297 of the CH2 domain of the Fc region. See, e.g., wright et al, TIBTECH,15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications may be made to the oligosaccharides in the antibodies of the present disclosure to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the fucose content in such antibodies may be 1% to 80%,1% to 65%,5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008/077546. Asn297 refers to the asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues). However, due to minor sequence variations of the antibody, asn297 may also be located about +/-3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. US 2003/0157108 (Presta, l.); US 2004/0093621 (Kyowa Hakko Kogyo Co.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotechnology bioengineering (Biotech. Bioeng.), 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, arch. Biochem. Biophys.249:533-545 (1986); U.S. patent application No. US 2003/0157108A 1, presta, L.; and WO 2004/056312A 1, adams et al, especially example 11), and knock-out cell lines, such as the alpha-1, 6-fucosyltransferase gene FUT8, knock-out CHO cells (see, e.g., yamane-Ohnuki et al, biotech Bioeng., 87 614 (2004); kanda, Y. Et al, biotech. Bioeng., 94 (4): 680-688 (2006); and WO 2003/085107).

Antibody variants are further provided with branched oligosaccharides, e.g., wherein the bi-antennary oligosaccharides attached to the Fc region of the antibody are branched by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al); U.S. Pat. No. 6,602,684 (Umana et al); and US2005/0123546 (Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant (which can also be referred to as an "altered Fc region"). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4Fc region) comprising an amino acid alteration (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the present disclosure contemplates antibody variants with some, but not all, effector functions, which make them desirable candidates for applications in which the in vivo half-life of the antibody is important and certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding activity. The primary cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9:457-492 (1991) page 464, table 3 summarizes FcR expression on hematopoietic cells. Non-in vitro assay for evaluating ADCC Activity of molecules of interest Limiting examples are described in U.S. Pat. nos. 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); 5,821,337 (see Bruggemann, M. Et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACT1 for flow cytometry) ^TM Non-radioactive cytotoxicity assays (CellTech, inc.; and CytoTox 96 (registered trademark)) and the nonradioactive cytotoxicity assay (Promega, michigan Madison.) useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells or, in addition, ADCC activity of the target molecule can be assessed in vivo, e.g., in animal models disclosed in Clynes et al, proc. Nat' l Acad. Sci.USA 95.

Antibodies with reduced effector function include those with substitutions in one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants that have increased or decreased binding to FcR are described (see, e.g., U.S. Pat. nos. 6,737,056, wo 2004/056312, and shelds et al, j.biol. Chem.9 (2): 6591-6604 (2001))

In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region, resulting in altered (i.e., increased or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. patent nos. 6,194,551, WO 99/51642 and Idusogie et al, journal of immunology (j. Immunol., 164).

Antibodies with prolonged half-life and increased binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of maternal IgG to the fetus (Guyer et al, journal of immunology (j.immunol.; 117 (1976) and Kim et al, journal of immunology (j.immunol.; 24 (1994)) are described in US2005/0014934A1 (Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those in which one or more of the following Fc region residues are replaced: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 of the Fc region (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, nature, 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351 for other examples of variants of the Fc region.

In one embodiment, the binding activity of an antibody Fc region (including a variant Fc region (the same applies hereinafter)) to each human Fc- γ receptor (Fc- γ R) can be measured by, for example, a ligand capture method using BIACORE (registered trademark) T200, which relies on a surface plasmon resonance analysis method as a measurement principle.

Details of exemplary methods for measuring the binding activity of the Fc region of an antibody to various human Fc-gamma receptors (Fc-gamma Rs) are described below. In one embodiment, the binding activity of the Fc region of an antibody to Fc- γ R is assessed using BIACORE (registered trademark) T200. In a preferred embodiment, the measurement is performed at 25 ℃ using a measurement buffer of 50mM phosphate, 150mM NaCl, 0.05w/v% -P20, pH 7.4. Specifically, approximately 1000RU of an antibody containing a variant Fc region was first captured onto a sensor chip, and CaptureSelect (trademark) human Fab- λ kinetic biotin conjugate (zemer fly science) was immobilized as a molecule that captures the ligand. Human Fc- γ R was diluted with measurement buffer to a Fc- γ RIA concentration of 8nM and other Fc- γ R concentrations of 1000nM and allowed to bind to the captured antibody. The binding activity of each antibody to each Fc- γ R was evaluated by calculating the amount of bound Fc- γ R per unit amount of antibody (RU) using Biacore T200 evaluation software 2.0. In one embodiment, the binding activity of the Fc region of an antibody to various human Fc-gamma receptors (Fc-gamma R) can be measured by the method described in reference example 7-4.

In a preferred embodiment, the Fc- γ R used in the above measurement method may be an extracellular domain of Fc- γ R prepared by the following method. First, genes of the extracellular domain of Fc- γ R were synthesized by methods known to those skilled in the art. For this synthesis, the sequence of each Fc- γ R was prepared based on the information registered at NCBI. More specifically, the sequence of Fc- γ RI was prepared based on the sequence of NCBI accession # NM _000566.3, the sequence of Fc- γ RIIa was prepared based on the sequence of NCBI accession # NM _001136219.1, the sequence of Fc- γ RIIb was prepared based on the sequence of NCBI accession # NM _004001.3, and the sequence of Fc- γ RIIIa was prepared according to the sequence of NCBI accession # NM _001127593.1, and a His tag was added to the C-terminus. Polymorphic sites of Fc- γ RIIa were prepared with reference to J.Exp.Med.,1990, 172, 19-25, and polymorphic sites of Fc- γ RIIIa were prepared with reference to J.Clin.invest.,1997, 100, 1059-1070. The obtained gene fragment is inserted into an expression vector of an animal cell to prepare an expression vector. The prepared expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells, and the protein of interest was expressed. The culture supernatant was collected and filtered through a 0.22 micron filter, then purified essentially by the four steps described below. Firstly, performing cation exchange column chromatography (SP Sepharose FF); secondly, performing affinity column chromatography (HisTrap HP) on the His label; third, gel filtration column chromatography (Superdex 200); fourthly, sterile filtration is carried out. Note that for Fc- γ RI, the first step is anion exchange column chromatography using Q Sepharose FF. The concentration of the purified Protein was calculated based on the absorption coefficient calculated by measuring the absorbance at 280nm using a spectrophotometer and using PACE or such a method for measurement (Protein Science, 1995,4, 2411-2423).

In one embodiment, the binding activity of the antibody Fc region to human FcRn can be measured by using a ligand capture method such as BIACORE (registered trademark) T200 which relies on a surface plasmon resonance analysis method as a measurement principle.

Details of an exemplary method of measuring the binding activity of an antibody Fc region to human FcRn are described below. In one embodiment, the binding activity of the Fc region of an antibody to human FcRn is assessed using BIACORE (registered trade mark) T200. In a preferred embodiment, the measurement is performed using a measurement buffer of 50mM phosphate, 150mM NaCl,0.05w/v% -P20, pH 6.0 at 25 ℃. Specifically, about 400RU of an antibody containing an Fc region was first captured onto a sensor chip on which CaptureSelect (trademark) human Fab- λ kinetic biotin conjugate (seemer femtology) as a molecule of a capture ligand was immobilized, and then human FcRn diluted with a measurement buffer was bound thereto. The binding activity of each antibody to FcRn was assessed by calculating KD (M) using the steady state model in Biacore T200 assessment software 2.0. In a preferred embodiment, the human FcRn protein used for this measurement is prepared according to the method described in reference example 2 of WO 2010107110. In one embodiment, the binding activity of the antibody Fc region to various human FcRn can be measured by the method described in reference examples 7-5.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thiomabs," in which one or more residues of the antibody are replaced with a cysteine residue. In particular embodiments, the substituted residues occur at accessible positions of the antibody. By replacing those residues with cysteine, the reactive thiol group is thus located at a accessible site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or linker-drug moiety, to produce an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted 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). Cysteine engineered antibodies can be produced as described, for example, in U.S. Pat. No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to include other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. In general, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a defined treatment, and the like.

In another embodiment, conjugates of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al, proc.natl.acad.sci.usa, 102. The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein portion to a temperature that kills cells adjacent to the antibody-non-protein portion.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-CD 137 antigen binding molecules or antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp2/0 cell). In one embodiment, a method of making an anti-CD 137 antigen binding molecule or antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody provided above under conditions suitable for expression of the anti-CD 137 antigen binding molecule or antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of the anti-CD 137 antigen binding molecules or antibodies, for example, as described above, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, methods in Molecular Biology, vol.248 (B.K.C.Lo, eds., human Press, totorwa, N.J., 2003), pp.245-254, which describes the expression of antibody fragments in E.coli). After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See gernggross, natural biotechnology (nat. Biotech.), 22:1409-1414 (2004), and Li et al, natural biotechnology (nat. Biotech.), 24:210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES that produce antibodies in transgenic plants are described ^TM A technique).

Vertebrate cells can also be used as hosts. For example, are suitable forMammalian cell lines grown in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); and human embryonic kidney lines (293 or 293 cells, as described by Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse trophoblasts (TM 4 cells, e.g., as described in Mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV 1); vero kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); for example, in Mather et al, annals n.y.acad.sci.,383: TRI cells described in 44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al, proc.Natl.Acad.Sci.USA 77; and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., yazaki and Wu, methods in Molecular Biology, vol.248 (B.K.C.Lo, eds., human Press, totorwa, N.J.), pp.255-268 (2003).

C. Measurement of

The anti-CD 137 antigen binding molecules or antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding assays and other assays

In one aspect, the antigen binding activity of the antigen binding molecules or antibodies of the present disclosure is tested, for example, by known methods such as ELISA, western blot, and the like.

In another aspect, competition assays in the presence of small molecule compounds can be utilized to identify such antigen binding molecules or antibodies: which competes for binding to CD137 with an anti-CD 137 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 37 as a combination of a heavy chain variable region/light chain variable region in the presence of a low molecular weight compound (e.g., in the presence of 10 μ M or more, 50 μ M or more, 100 μ M or more, 150 μ M or more, 200 μ M or more, or 250 μ M or more of the low molecular weight compound). In certain embodiments, such competitive antigen-binding molecules or antibodies bind to the same epitope (e.g., a linear epitope or a conformational epitope) bound by an anti-CD 137 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 as a combination of heavy chain variable region/light chain variable region. Morris (1996) "Epitope Mapping protocol," Methods in Molecular Biology, "Vol.66 (human Press, totorwa, N.J.), provides detailed exemplary Methods for Mapping epitopes bound by antigen binding molecules or antibodies. In one embodiment, an anti-CD 137 antigen-binding molecule or antibody of the present disclosure having CD137 binding activity that is dependent on the antigen-binding activity of a small molecule compound recognizes an epitope formed by a complex formed by an antigen (e.g., CD 137) and a low molecular weight compound (e.g., ATP).

In an exemplary competition assay using an antibody, immobilized CD137 is incubated in the presence of a low molecular weight compound (e.g., in the presence of 10 μ M or more, 50 μ M or more, 100 μ M or more, 150 μ M or more, 200 μ M or more, or 250 μ M or more of a low molecular weight compound) in a solution containing a first labeled antibody that binds to CD137 (e.g., an anti-CD 137 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 37 as a combination of heavy chain variable region/light chain variable region) and a second unlabeled antibody (tested for its ability to compete with the first antibody for binding to CD 137). The second antibody may be present in the hybridoma supernatant. As a control, immobilized CD137 was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD137, excess unbound antibody is removed and the amount of label associated with the immobilized CD137 is measured. If the amount of label associated with immobilized CD137 is substantially reduced in the test sample relative to the control sample, it is indicative that the second antibody competes with the first antibody for binding to CD137. See Harlow and Lane (1988) antibodies: chapter 14 of the Laboratory Manual (Antibodies: A Laboratory Manual ch.14) (Cold spring harbor Laboratory, cold spring harbor, N.Y.). One skilled in the art will appreciate that assays can be performed similarly to antigen binding molecules other than antibodies.

2. Activity assay

In one aspect, assays for identifying biological activity of an anti-CD 137 antigen-binding molecule or antibody having biological activity are provided. Biological activities may include, for example, CD137 agonist activity; a plasma half-life; anti-tumor activity; and low or suppressed systemic responses in tissues other than tumors. Antigen binding molecules or antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, antigen binding molecules (e.g., anti-CD 137 antigen binding molecules) or antibodies of the present disclosure are tested for such biological activity.

a) Agonist activity assay (PBMC)

In one embodiment, agonistic activity on CD137 is measured by contacting a cell expressing CD137 with an anti-CD 137 antigen binding molecule or antibody in solution with or without the addition of a small molecule compound. In one embodiment, agonistic activity on CD137 in a solution to which a small molecule compound is added and agonistic activity on CD137 in a solution to which a small molecule compound is not added are assessed by the amount of cytokine production (e.g., the amount of IL-2, IFN- γ, and/or IL-6 produced) measured within 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours after the CD137 expressing cells and the CD137 antigen binding molecule or antibody are contacted in solution, respectively. In one embodiment, the solution to which the small molecule compound is added is adjusted such that the adjusted concentration of the small molecule compound is 10. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, or 250. Mu.M. In further embodiments, the CD137 expressing cells to be used are isolated human Peripheral Blood Mononuclear Cells (PBMCs) or T cells expanded from isolated human PBMCs.

In one embodiment, human PBMCs isolated from blood collected from healthy individuals by centrifugation at 400 × g for 30 minutes at room temperature are used. Preferably, human PBMCs isolated in the following two steps are used. In a first step, leucosep (Greiner Bio-One) supplemented with Ficoll-Paque PLUS (GE medical) was centrifuged at 1000Xg for 1 min at room temperature, then blood diluted with PBS was added and centrifuged at 400Xg for 30 min at room temperature. In the second step, the buffy coat was collected from the tube after centrifugation and then washed with 60mL PBS (Wako).

Details of an exemplary method for measuring CD137 agonist activity using human PBMCs are described below. Note that even though the following examples illustratively use ATP as the small molecule compound, this does not exclude other small molecule compounds. In one embodiment, isolated human PBMCs are diluted to a cell density of 5X10 with medium (5% human Serum (SIGMA), 95% AIM-V (Sammerfeihl technology)) ⁶ and/mL. The isolated human PBMCs are then contacted with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody to induce CD137 expression in the human PBMCs. Preferably, 50. Mu.L of anti-human CD 3. Epsilon. Antibody (BD Co., clone SP 34) diluted in medium and 20. Mu.g/mL of anti-human CD28 antibody (BD, clone: CD 28.2) are added to isolated human PBMC (100. Mu.L, cell density 5X 10) ⁶ /mL)。

Then, to human PBMCs to which an anti-human CD3 epsilon antibody and/or an anti-human CD28 antibody are added are further added: (i) medium with or without ATP; (ii) an anti-CD 137 antigen binding molecule or antibody. Preferably, 25 μ L of medium with or without ATP is added. Preferably, 25. Mu.L of 40. Mu.g/mL of the anti-CD 137 antigen-binding molecule or antibody is added. More preferably, the above (i) and (ii) are added about 6 hours after contacting human PBMC with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody. In one embodiment, the amount of IL-2 produced is preferably measured prior to the amount of IFN- γ produced. In one embodiment, the amount of IL-2 produced is measured within about 24 hours after contacting human PBMCs with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody. Preferably, the amount of IL-2 production is measured about 24 hours after contacting human PBMCs with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody, and about 18 hours after addition of anti-CD 137 antigen binding molecule or antibody.

In another embodiment, the amount of IFN- γ production is measured within about 48 hours after contacting human PBMCs with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody. Preferably, the amount of IFN- γ production is measured about 48 hours after contacting human PBMCs with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody, and about 42 hours after addition of anti-CD 137 antigen binding molecule or antibody. In one embodiment, the amount of IL-2 production and/or IFN- γ production is determined by measuring the amount of IL-2 production and/or IFN- γ production in the collected culture supernatant. In one embodiment, human PBMC supplemented with anti-human CD3 epsilon antibody and/or anti-human CD28 antibody is 5% CO at 37 ℃ ₂ The incubator was left until all measurements were completed.

Details of another exemplary method for measuring CD137 agonist activity using human PBMCs are described below. Isolated human PBMC were diluted to 5X10 with medium (5% human Serum (SIGMA), 95% AIM-V (Saimer Feishell technology)) ⁶ Cell density in/mL. Human PBMC were then adjusted to 5x10 ⁶ Cell density per mL and 100 μ L each of human PBMCs were seeded into 96 well multi-well plates (flat bottom, covered) (Corning). Thereafter, human PBMCs were subjected to manipulations that induced CD137 expression. For example, CD137 expression in human PBMC is induced by adding 50. Mu.L of 0.04. Mu.g/mL anti-human CD3 epsilon antibody (BD Co., clone SP 34) and 20. Mu.g/mL anti-human CD28 antibody (BD, clone: CD 28.2) diluted with medium.

After induction of CD137 expression in human PBMCs, the plates were shaken and the plates were concentrated in 5% CO ₂ The incubator was allowed to stand at 37 ℃ for 6 hours. Then, 25. Mu.L of 2mM ATP (SIGMA), diluted with medium or ATP-free medium alone, respectively, and 25. Mu.L of 40. Mu.g/mL of each antibody were added to each well, followed by shaking the plate, 5% CO ₂ The incubator was allowed to stand at 37 ℃ for 18 hours. Then, a part of the culture supernatant was collected, and the culture was usedSupernatant of the product was collected using human IL-2DuoSet ELISA kit (R) &D System) or human IL-2ELISA Set (BD bioscience) quantitated the amount of IL-2 contained in the culture supernatant. The plate after collection of the culture supernatant is again 5% CO ₂ The incubator was left standing at 37 ℃ for 24 hours. Then, part of the culture supernatant was collected and used as human IFN-. Gamma.DuoSet ELISA kit (R)&D system) or human IFN- γ ELISA development kit (PeproTech) to quantify IFN- γ in culture supernatants. The ELISA was performed essentially according to the protocol attached to the kit. For human IL-2 DuoSet ELISA kit (R)&D System) and human IFN-. Gamma.DuoSet ELISA kit (R)&D System), according to the protocol, using a catalyst containing H ₂ O ₂ Tetramethylbenzidine and 1 NH ₂ SO ₄ (Wako) substrate solution (R)&D system) for color development and color termination. For human IL-2ELISASet (BD Biosciences), 1N H was used ₂ SO ₄ (Wako) color termination.

For the IFN-. Gamma.ELISA development kit (PeproTech), TMB chromogen solution (Saimer Feishel technology) and 1N H were used ₂ SO ₄ (Wako) color development and color termination. Then, absorbance measurements were performed with EnVision (PerkinElmer) and the amounts of IL-2 and IFN-. Gamma.in the culture supernatant (pg/mL) were calculated separately using a calibration curve prepared according to the protocol. In this PBMC assay, CD137 agonist activity can be expressed as fold change in the amount of IL-2 and IFN- γ in the culture supernatant relative to a negative control antibody (an antibody that does not bind CD 137). In one embodiment, CD137 agonistic activity is measured according to the method described in reference examples 5-5-1 and 5-5-2.

In one embodiment, when agonistic activity against CD137 is assessed by the amount of cytokine production in the human PBMC assay (e.g., the amount of IL-2, IFN- γ, and/or IL-6 production), in the presence of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M of a small molecule compound, if the amount of cytokine production when an anti-CD 137 antigen-binding molecule or antibody is added is 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.05-fold or more, 1.06-fold or more, 1.07-fold or more, 1.08-fold or more, 1.09-fold or more, 1.1-fold or more, 1.11-fold or more, 1.12-fold or more, 1.13-fold or more, 1.14-fold or more, or 1.15-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold higher than the amount of cytokine production when a negative control antibody is added, it can be judged that the anti-CD 137 antigen-binding molecule exhibits agonistic activity against CD137 in the presence of a small molecule compound.

In one embodiment, when agonistic activity against CD137 is evaluated by the amount of IL-2 production in a human PBMC assay, in the presence of a small molecule compound of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M, if the amount of IL-2 production when an anti-CD 137 antigen-binding molecule or antibody is 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, or 1.05-fold or more higher than the amount of IL-2 production when a negative control antibody is added, and in a preferred embodiment 1.05-fold or more, it may be judged that the anti-CD 137 antigen-binding molecule or antibody exhibits agonistic activity against CD137 in the presence of a small molecule compound.

In one embodiment, when agonistic activity on CD137 is evaluated by the amount of IL- γ production in a human PBMC assay, if the amount of IL- γ production when an anti-CD 137 antigen-binding molecule or antibody is added is 1.01-fold or more, 1.11-fold or more, 1.12-fold or more, 1.13-fold or more, 1.14-fold or more, or 1.15-fold or more higher than the amount of IL- γ production when a negative control antibody is added in the presence of a small molecule compound of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M or 250 μ M, it can be judged that the anti-CD 137 antigen-binding molecule or antibody exhibits agonistic activity on CD137 in the presence of a small molecule compound.

In a further embodiment, of the anti-CD 137 antigen-binding molecules or antibodies that are judged to exhibit agonistic activity against CD137 in the presence of a small molecule compound in the above comparison with a negative control, an antibody that is judged not to exhibit CD137 agonistic activity or to have lower CD137 agonistic activity in the absence of a small molecule compound (as compared with the presence of a small molecule compound) is further determined. Specifically, the anti-CD 137 antigen-binding molecule or antibody is judged to exhibit no or low CD137 agonist activity in the absence of the small molecule compound when (ii) is greater than (i) below:

(i) [ amount of cytokine production in the absence of a small molecule compound when an anti-CD 137 antigen-binding molecule or antibody is added ]/[ amount of cytokine production in the absence of a small molecule compound when a negative control antibody is added ]

(ii) [ amount of cytokine production in the presence of a Small molecule Compound when an anti-CD 137 antigen-binding molecule or antibody is added ]/[ amount of cytokine production in the presence of a Small molecule Compound when a negative control antibody is added ]

In various embodiments, when comparing the CD137 agonistic activity of a first anti-CD 137 antigen-binding molecule or antibody and a second anti-CD 137 antigen-binding molecule or antibody using the amount of cytokine production in a human PBMC assay (e.g., the amount of IL-2, IFN- γ, and/or IL-6 production), if (i) in the presence of 10. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, or 250. Mu.M small molecule compound, when a first anti-CD 137 antigen-binding molecule or antibody is added, the amount of cytokine production is (ii) under the same conditions, when the amount of cytokine produced by the addition of the second anti-CD 137 antigen-binding molecule or antibody is 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more, or 1.15 times or more, 1.5 times or more, 2 times or more, or 3 times or more higher than the amount of cytokine produced by the addition of the second anti-CD 137 antigen-binding molecule or antibody, it can be determined that the activation activity of the first anti-CD 137 antigen-binding molecule or antibody is higher than the activation activity of the second CD137 antigen-binding molecule or antibody.

In one embodiment, when agonistic activity on CD137 is evaluated by the amount of IL-2 production in a human PBMC assay, if (i) the amount of cytokine production when a first anti-CD 137 antigen-binding molecule or antibody is added is 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, or 1.04-fold or more higher than the amount of IL-2 production when a second anti-CD 137 antigen-binding molecule or antibody is added under the same conditions, or 1.04-fold or more higher in a preferred embodiment, in the presence of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M small molecule compound, the activation activity of the first anti-CD 137 antigen-binding molecule or antibody can be judged to be higher than the activation activity of the second CD137 antigen-binding molecule or antibody.

In one embodiment, when the agonistic activity on CD137 is evaluated by the amount of production of IL- γ in the human PBMC assay, if (i) the amount of production of cytokine when the first anti-CD 137 antigen-binding molecule or antibody is added in the presence of a small molecule compound of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M is 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, or 1.1 times or more higher, or in a preferred embodiment 1.1 times or more, the amount of production of IL- γ when the second anti-CD 137 antigen-binding molecule or antibody is added under the same conditions, it may be judged that the activation of the first anti-CD 137 antigen-binding molecule or antibody is higher than the activation activity of the second CD137 antigen-binding molecule or antibody. In one embodiment, the first anti-CD 137 antigen binding molecule or antibody is an antigen binding molecule or antibody comprising a parent Fc region and the second anti-CD 137 antigen binding molecule or antibody is an antigen binding molecule or antibody comprising a variant Fc region.

In one embodiment, the methods described in reference examples 5-5-1 and 5-5-2 can be used to measure CD137 agonist activity using human PBMCs, and the methods described in reference examples 2-6 can be used to measure CD137 agonist activity using T cells expanded from isolated human PBMCs.

In one embodiment, agonistic activity on CD137 may be measured by contacting CD 8-positive T cells or CD 4-positive T cells isolated from human PBMCs with an anti-CD 137 antigen binding molecule or antibody in a solution with or without the addition of a small molecule compound. At this time, the Fc γ RIIb-expressing cells may be further added to the solution. Alternatively, agonist activity on CD137 can be measured by contacting B cells (which can be isolated from human PBMCs, or can use known B cell lines) with an anti-CD 137 antigen binding molecule or antibody. In one embodiment, agonistic activity on CD137 may be assessed by measuring the amount of cytokine production (e.g., IL-2, ifn- γ, and/or IL-6 production) after contacting CD 8-positive T cells, CD 4-positive T cells, or B cells with an anti-CD 137 antigen-binding molecule or antibody in solution.

In one embodiment, the agonist activity assay uses human Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood collected from healthy individuals. Thus, in any of the above embodiments, one skilled in the art will appreciate that the assay results using the assay may vary for each donor of a blood sample. In view of this, an antibody that does not exhibit agonist activity for a portion or a majority of human PBMCs isolated from multiple donors is not judged to exhibit CD137 agonist activity even though another portion of the human PBMCs meets the criteria for agonist activity. Further, when an assay according to the above-described method is performed on human PBMCs isolated from a plurality of donors and the criterion of CD137 agonist activity is satisfied in most of the donors, it can be judged that CD137 agonist activity is exhibited. Alternatively, when assays according to the above-described methods are performed on human PBMCs isolated from multiple donors, the mean or median value of the assay (e.g., IL-2, IFN- γ, and/or IL-6 production) can be used to determine the presence or absence of CD137 agonist activity. In one embodiment, when human PBMCs isolated from multiple donors are assayed according to the methods described above, the number of donors is, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 or more.

b) Determination of agonistic Activity (reporter Gene assay)

In one embodiment, agonist activity to CD137 is assessed by a reporter assay in solution with or without the addition of a small molecule compound. In one embodiment, after contacting a T cell expressing the NF- κ B luciferase reporter construct and CD137 with an anti-CD 137 antigen-binding molecule and standing in the corresponding solution for a certain time, the agonistic activity on CD137 in a solution to which a small molecule compound is added and the agonistic activity on CD137 in a solution to which a small molecule compound is not added are evaluated by the measured luciferase luminescence signal, respectively. In one embodiment, the solution to which the small molecule compound is added is adjusted such that the adjusted small molecule compound concentration is 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M. Construction of reporter gene for expressing NF-kB-luciferaseT cells of somatic and CD137 are preferably GloResponse ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line (Promega, CS 196004).

Details of an exemplary method for measuring CD137 agonist activity using a reporter gene assay are described below. In the following examples ATP is used as an example of a small molecule compound, but other small molecule compounds are not excluded. In one embodiment, first, the concentration of Fc γ RIIB-expressing cells is adjusted to 5x10 with medium (CHO medium (90 ham's f12, 10% fbs)) (CHO medium) ⁴ mL, and at 37 ℃ in 5% CO ₂ The incubator was left overnight. In one embodiment, as the cells expressing FcgRIIB, not only cells forcibly expressing FcgRIIB but also cell lines endogenously expressing FcgRIIB, such as B cell lines, B cells isolated from a living body, and the like can be used. In a preferred embodiment, the cells expressing Fc γ RIIB are Fc γ RIIB CHO-K1 cells (Promega). Next, after removal of the medium by aspiration, the GloResponse was treated with a different medium (99% RPMI,1% FBS) ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line (hereinafter referred to as "4-1BB Jurkat") was adjusted to 2X10 ⁶ The volume is/mL. In a preferred embodiment, every 200. Mu.L of cells expressing FcyRIIB conditioned with culture medium, 25. Mu.L of GloResponse conditioned with culture medium is added ^TM NF-kB-Luc 2/4-1BB Jurkat cell line cells. Subsequently, 25. Mu.L of each anti-CD 137 antigen-binding molecule diluted to a desired concentration (e.g., a final concentration of 0, 0.001, 0.01, 0.1, 1, 10. Mu.g/mL) with the above-mentioned medium (99% RPMI,1% FBS) was added, and further, 25. Mu.L of an ATP solution diluted to a desired concentration (e.g., a final concentration of 0, 10, 50, 100, 150, 200, 250. Mu.M) with the above-mentioned medium (99% RPMI,1% FBS) was added. In one embodiment, the luciferase luminescence signal is measured after 2 hours or less, 4 hours or less, 6 hours or less, 24 hours or less after addition of the anti-CD 137 antigen binding molecule to 4-1BB Jurkat. In a preferred embodiment, 4-1BB Jurkat is 5% CO ₂ The incubator was allowed to stand at 37 ℃ for 6 hours. After standing, 75. Mu.L of Bio-Glo reagent was added, and luminescence was measured with a microplate reader. In a preferred embodiment, the Bio-Glo reagent is Bio-Glo luciferaseAssay system (buffer and substrate). In one embodiment, 4-1BB Jurkat may be left to stand at room temperature for 5 minutes, 10 minutes, 15 minutes, or 20 minutes after being taken out of the incubator in order to keep the temperature during the reaction constant. In a preferred embodiment, 4-1BB Jurkat is removed from the incubator and allowed to stand at room temperature for 15 minutes. In one embodiment, the value of the light emission of 4-1BB Jurkat with the addition of anti-CD 137 antigen binding molecule is divided by the value of the light emission of 4-1BB Jurkat without the addition of anti-CD 137 antigen binding molecule, and this value is taken as the fold induction (relative light units) and used as an index for assessing the CD137 agonistic activity of each antigen binding molecule.

Another exemplary method of measuring CD137 agonist activity using a reporter gene assay is described below. Each 200. Mu.L of Fc γ RIIB CHO-K1 cells (Promega) was conditioned to 5X10 with medium ⁴ Concentration of/mL, then added to each well of a 96-well plate and at 5% CO ₂ The incubator was left overnight at 37 ℃. As the medium, CHO medium (90% Ham' F12, 10% FBS) was used. Then, after completely removing the medium by aspiration, 25. Mu.L of GloResponse was added ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell lines were adjusted to 2X10 with assay medium (99% RPMI,1% FBS) ⁶ mL, then added to each well. Subsequently, 25. Mu.L of each antigen solution diluted with assay medium to a final concentration of 0, 0.001, 0.01, 0.1, 1 or 10. Mu.g/mL was added, and finally 25. Mu.L of ATP solution diluted with assay medium to 0 or 250. Mu.M was added. The plate was 5% CO ₂ After standing in the incubator at 37 ℃ for 6 hours, the plate was left at room temperature for 15 minutes, and then 75. Mu.L of Bio-Glo reagent was added to each well. As the Bio-Glo reagent, for example, a Bio-Glo luciferase assay system (buffer and substrate) can be used. Thereafter, luminescence of each well was measured with a microplate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of the well to which no antibody was added was defined as fold induction. In reporter assays, CD137 agonistic activity can be assessed by the fold change (relative light units) in luminescence of wells to which each antibody was added relative to the luminescence of wells to which no antibody was added.

The following descriptionAnother exemplary method of measuring CD137 agonist activity using a reporter gene assay is described. To each well of the 384-well plate, 10. Mu.L of the assay medium (99% RPMI,1% FBS) was added to adjust its concentration to 2X10 ⁶ Fc γ RIIB CHO-K1 cells (Promega) per mL. Subsequently, 10 μ L of an antibody solution containing ADP or an antibody solution containing ATP, or an antibody solution containing no ATP or ADP was added to each well. Then, 10. Mu.L of the assay medium (99% RPMI,1% FBS) was added to each well and adjusted to 2X 10 ⁶ GloResponse/mL ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line. The final concentration of ADP was 50. Mu.M, and the final concentration of ATP was 50. Mu.M. The plate was 5% CO ₂ After being left at 37 ℃ for 6 hours in the incubator and left at room temperature for 15 minutes, 30. Mu.L of Bio-Glo reagent was added to each well. As the Bio-Glo reagent, the Bio-Glo luciferase assay system (buffer and substrate) was used. Thereafter, luminescence of each well was measured with a microplate reader. CD137 agonistic activity can be assessed by the relative light units (also referred to as fold or fold change in luminescence) of the luminescence of wells to which each antibody was added relative to the luminescence of wells to which no antibody was added.

Another exemplary method of measuring CD137 agonist activity using a reporter gene assay is described below. To each well of the 384-well plate, 10. Mu.L of the assay medium (99% RPMI,1% FBS) was added to adjust the concentration to 4X10 ⁵ Fc γ RIIB CHO-K1 cells (Promega) per mL. Subsequently, 10 μ L of an antibody solution containing ADP or an antibody solution containing ATP, or an antibody solution containing no ATP or ADP was added to each well. Then, 10. Mu.L of the assay medium (99% RPMI,1% FBS) was added to each well and adjusted to 2X 10 ⁶ GloResponse/mL ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line. The final concentration of ADP was 10. Mu.M, and the final concentration of ATP was 10. Mu.M. The plate was 5% CO ₂ After being left at 37 ℃ for 6 hours in the incubator and left at room temperature for 15 minutes, 30. Mu.L of Bio-Glo reagent was added to each well. As the Bio-Glo reagent, a Bio-Glo luciferase assay system (buffer and substrate) was used. Thereafter, luminescence of each well was measured with a microplate reader. CD137 agonistic activity may be measured by the phase of luminescence of wells to which each antibody was added versus wells to which no antibody was addedLight units (also referred to as fold or fold change in luminescence) were evaluated.

In one embodiment, when agonist activity to CD137 is measured using a reporter gene assay, if (i) in the presence of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, or 250 μ M of a small molecule compound, the agonist activity (relative light units) to CD137 is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.5-fold or more, 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 agonist activity (relative light units) to CD137 in the absence of a small molecule compound, it can be determined that the anti-CD 137 antigen-binding molecule or antibody exhibits agonist activity to CD137 in the presence of a small molecule compound. In one embodiment, the final concentration of antibody in (i) and (ii) above is 0, 0.001, 0.01, 0.1, 1 or 10. Mu.g/mL, and in preferred embodiments, 0.1. Mu.g/mL or 1. Mu.g/mL.

In further embodiments, when the anti-CD 137 antigen-binding molecule or antibody is added at a particular concentration (e.g., a final concentration of 0.001, 0.01, 0.1, 1, or 10 μ g/mL, or in preferred embodiments 0.1 μ g/mL or 1 μ g/mL), if the fold induction (relative light units) is 10 or less, 9 or less, 8 or less, or in preferred embodiments 5 or less, in more preferred embodiments substantially 1 in the absence of the small molecule compound, the anti-CD 137 antigen-binding molecule or antibody is judged to exhibit substantially no CD137 agonistic activity in the absence of the small molecule compound. In one embodiment, "fold induction (relative light units) is substantially 1 in the absence of a small molecule compound" means that the fold induction (relative light units) is less than 2 fold, less than 1.9 fold, less than 1.8 fold, less than 1.7 fold, less than 1.6 fold, less than 1.5 fold, less than 1.4 fold, less than 1.3 fold, less than 1.2 fold, or less than 1.1 fold in the absence of a small molecule compound.

c) Plasma concentration

In one embodiment, hemodynamics of the anti-CD 137 antigen binding molecules or antibodies of the present disclosure are measured and/or compared using human CD137 knock-in mice. For example, a human CD137 knock-in mouse is prepared by introducing a human CD137 gene substitution vector into a mouse embryonic stem cell (ES cell) and substituting the mouse CD137 gene with the human CD137 gene. In one embodiment, a CD137 antigen-binding molecule or antibody of the present disclosure is administered to a human CD137 knock-in mouse by a single intravenous administration, and blood is collected multiple times from immediately after administration to about 5 days, 10 days, 15 days, 20 days, 25 days, or 30 days after administration. In a preferred embodiment, the CD137 antigen-binding molecules or antibodies of the present disclosure are administered to human CD137 knock-in mice by a single intravenous administration, and blood is collected multiple times over a period of 5 minutes to 28 days after administration. Plasma was rapidly separated from the collected blood, and the antibody concentration in the plasma was measured by Electrochemiluminescence (ECL). In one embodiment, the concentration of the antibody in plasma can be measured by the method described in reference example 6-3-2.

In the measurement of plasma half-life of a mouse knocked in with human CD137, if the anti-CD 137 antigen-binding molecule or antibody disappears from plasma at a slower rate than the reference molecule, the anti-CD 137 antigen-binding molecule or antibody is determined to have improved hemodynamics compared to the reference molecule. In addition, if the speed of disappearance of the anti-CD 137 antigen-binding molecule or antibody (switch molecule or switch antibody) having the binding activity depending on the small molecule compound from the plasma is slower than that of the anti-CD 137 antigen-binding molecule or antibody (non-switch molecule or non-switch antibody) not having the binding activity depending on the small molecule compound, it can be judged that the switch molecule (or switch antibody) does not bind to CD137 expressed in the non-tumor tissue as compared with the non-switch molecule (or non-switch antibody).

d) Antitumor activity

In one aspect, anti-CD 137 antigen binding molecules or antibodies thereto are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-CD 137 antigen-binding molecules or antibodies thereof are tested for their ability to inhibit tumor growth in vivo. In vivo model systems, such as allograft or xenograft models, can be used for this test. In an exemplary xenograft system, human tumor cells are introduced into appropriately immunocompromised non-human animals, such as athymic "nude" mice. Administering to an animal an antibody of the disclosure. The ability of the antibody to inhibit or reduce tumor growth was measured. In certain embodiments of the above xenograft system, the human tumor cell is a tumor cell from a human patient. Such xenograft models are commercially available from Oncotest corporation (Frieberg, germany). In certain embodiments, the human tumor cells are introduced into a suitable immunocompromised non-human animal by subcutaneous injection or by transplantation into a suitable site, such as a milk fat pad.

In one embodiment, the anti-CD 137 antigen binding molecules or antibodies of the present disclosure can be measured and/or compared for anti-tumor activity using a syngeneic tumor cell transplantation model based on the human CD137 knock-in mice described above. The cancer cell line used in the test may be appropriately selected, but a mouse colon cancer cell line MC38 cell line is preferred. In one embodiment, the MC38 cell line is transplanted under the skin of the abdominal region of a mouse and when the tumor volume reaches about 50-300mm ³ Then, the model is considered to be established. After modeling, MC38 cell-transplanted mice were grouped and then received administration of each anti-CD 137 antigen binding molecule or antibody. In one embodiment, tumor volume measurements are made at a frequency of once to twice a week to assess anti-tumor activity. Tumor volume was calculated using the following equation: tumor volume = (long axis × short axis)/2. In one embodiment, the anti-CD 137 antigen binding molecule or antibody may be tested and evaluated for anti-tumor activity according to the methods described in reference examples 6-4.

In one embodiment, an anti-CD 137 antigen-binding molecule or antibody is considered to exhibit anti-tumor activity when the tumor volume in the anti-CD 137 antigen-binding molecule or antibody administration group is less than the tumor volume in the vehicle group, or the tumor volume in the anti-CD 137 antigen-binding molecule or antibody administration group increases less than the vehicle group.

e) Systemic reaction

In one embodiment, a syngeneic tumor cell transplantation model based on the human CD137 knock-in mice described above is used to measure and/or compare systemic responses to the anti-CD 137 antigen binding molecules or antibodies of the present disclosure. The organ for measuring the systemic response may be appropriately selected, but the liver, spleen and/or lymph node is preferable. In one embodiment, the systemic response is assessed by removing the liver, spleen and/or lymph nodes from a human CD137 knockin mouse at an appropriate time after administration of the anti-CD 137 antigen binding molecule or antibody. When the organ to be removed is the spleen and/or lymph nodes, the organ is weighed and/or cells in the lymphocyte fraction are counted. In the counting, it is preferable that the hemolyzed lymphocyte fraction is applied to the spleen, and the lymphocyte fraction obtained by the homogenization is applied to the lymph node. When the organ to be removed was the liver, the cells in the lymphocyte fraction obtained with the liver dissociation kit mouse (Milteny Biotec) were counted. In addition, T cell analysis using Flow Cytometry (FCM) can be performed on lymphocyte fractions from each organ (liver, spleen, and/or lymph nodes). In the FCM assay, for example granzyme B expression or PD-1 expression or ICOS expression or the ratio of CD8- α positive T cells to CD45 positive cells in CD8- α positive T cells is used. In one embodiment, the systemic response to anti-CD 137 antigen-binding molecules or antibodies may be tested and evaluated as described in reference examples 6-4.

In one embodiment, in the evaluation of the various indicators described above, an anti-CD 137 antigen-binding molecule or antibody is considered to inhibit a systemic response and/or inhibit activation of immune cells in non-tumor tissue (e.g., liver, spleen, and/or lymph nodes) as compared to a reference molecule when the value of the group administered with the anti-CD 137 antigen-binding molecule or antibody is lower than the value of the group receiving the same amount of the reference molecule. Further, in the evaluation of the above-described various indexes, when the value of the administration group receiving the anti-CD 137 antigen-binding molecule or antibody (switch molecule or switch antibody) having the binding activity depending on the small molecule compound is lower than that of the administration group receiving the same amount of the anti-CD 137 antigen-binding molecule or antibody (non-switch molecule or non-switch antibody) not having the binding activity depending on the small molecule compound, it is considered that the switch molecule (switch antibody) inhibits the systemic reaction and/or inhibits the activation of immune cells in non-tumor tissues (for example, liver, spleen and/or lymph node) as compared with the non-switch molecule (or non-switch antibody).

It is to be understood that any of the above-described measurement methods can be performed using the immunoconjugates of the disclosure in place of, or in addition to, the anti-CD 137 antigen-binding molecules or antibodies.

D. Immunoconjugates

The present disclosure also provides immunoconjugates comprising an anti-CD 137 antigen-binding molecule or antibody of the present disclosure conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs including, but not limited to, maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064 and european patent EP 0 425 235B1); auristatins (auristatins), such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 Hinman et al, cancer research (Cancer Res.), 53; anthracyclines such as daunorubicin or doxorubicin (see Kratz et al, current pharmaceutical chemistry (Current Med. Chem.), 13; methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel and otaxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, physcosin II a chain, alpha-sarcin, aleurites fordii (Aleurites fordii) protein, dianthin protein, phytolacca americana (PAPI, PAPII, and PAP-S) protein, momordica charantia inhibitory factor, curcin, crotin, gelonin inhibitor, gelonin, mitosin, restrictocin, phenomycin, enomycin, and trichothecene.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for producing radioconjugates. Examples include ²¹¹ At、 ¹³¹ I、 ¹²⁵ I、 ⁹⁰ Y、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹² Bi、 ³² P、 ²¹² Pb and Lu radioisotopes. When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example Tc-99m or ¹²³ I, or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine 123, iodine 131, indium 111, fluorine 19, carbon 13, nitrogen 15, oxygen 17, gadolinium, manganese, or iron.

Conjugates of an antibody and a cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiophene (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e.g., bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and bis-active fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). For example, the ratio of vietta et al, science, 238:1098 (1987) preparation of ricin immunotoxin. Carbon-14 labeled 1-isothiocyanato benzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to antibodies. See WO94/11026. The linker may be a "cleavable linker" which facilitates the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al, cancer research (Cancer Res.), 52.

Immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate), for example, from Piercs Biotechnology, inc., of Rockford, ill., U.S.A.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-CD 137 antigen binding molecules or antibodies provided herein can be used to detect the presence of a CD137 antigen binding molecule in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue.

In one embodiment, an anti-CD 137 antigen binding molecule or antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of CD137 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-CD 137 antigen-binding molecule or antibody described herein under conditions that allow the anti-CD 137 antigen-binding molecule or antibody to bind to CD137, and detecting whether a complex is formed between the anti-CD 137 antigen-binding molecule or antibody and CD 137. Such methods may be in vitro methods or in vivo methods. In one embodiment, the anti-CD 137 antigen binding molecule or antibody is used to select a subject suitable for treatment with the anti-CD 137 antigen binding molecule or antibody, e.g., where CD137 is a biomarker for selecting patients.

Exemplary diseases that can be diagnosed using the antibodies of the present disclosure include cancer.

In certain embodiments, labeled anti-CD 137 antigen binding molecules or antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties that are detected indirectly (e.g., enzymes or ligands), for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P， ¹⁴ C， ¹²⁵ I， ³ H and ¹³¹ i, fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase and glucose 6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled with enzymes using hydrogen peroxide oxidation dye precursors such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical preparation

The pharmaceutical formulations of the anti-CD 137 antigen binding molecules or antibodies described herein are prepared by mixing the antibody of the desired purity with one or more optional pharmaceutically acceptable carriers in lyophilized formulations or in aqueous solution (Remington's pharmaceutical sciences, 16 th edition, osol, a. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); small molecule (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), baxter international). Certain exemplary sHASEGP and methods of use are described in U.S. patent publication Nos. 2005/0260186 and 2006/0104968, including rHuPH20. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including histidine-acetate buffers.

The active ingredient may be embedded in microcapsules or macroemulsions of colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), for example prepared by coacervation techniques or by interfacial polymerization, respectively, such as hydroxymethylcellulose microcapsules or gelatin-microcapsules and poly (methylmethacylate) microcapsules. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, osol, A. Eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and therapeutic compositions

Any of the anti-CD 137 antigen binding molecules or antibodies provided herein can be used in a method of treatment.

In one aspect, in the present disclosure, an anti-CD 137 antigen-binding molecule or antibody is provided for use as a medicament. In the present disclosure, examples of the drug specifically include drugs for inducing an anti-tumor effect, for example, cell activation by binding of an anti-CD 137 antigen-binding molecule or antibody to CD137 expressed on an immune cell such as a T cell, inhibition of neovascularization in a tumor, inhibition of tumor cell proliferation, depletion of B cells promoting a tumor, and the like. In other aspects, in the present disclosure, anti-CD 137 antigen binding molecules or antibodies are provided for the treatment of tumors. In certain embodiments, anti-CD 137 antigen-binding molecules or antibodies are provided for use in methods of treatment. In certain embodiments, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody for use in a method of treating an individual having a tumor, the method comprising administering to the individual an effective amount of an anti-CD 137 antigen-binding molecule or antibody. In further embodiments, in the present disclosure, examples of tumors include solid tumors in which B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells and/or regulatory T cells (Treg cells) are infiltrated.

In other aspects, the present disclosure provides anti-CD 137 antigen-binding molecules or antibodies for use in a method of activating immune cells in an individual, the method comprising administering to the individual an effective amount of an anti-CD 137 antigen-binding molecule or antibody to activate immune cells, such as B cells, dendritic cells, natural killer cells, macrophages, and/or CD8 positive T cells (more particularly, these immune cells have infiltrated tumor tissue).

In a further aspect, the present disclosure provides an anti-CD 137 antigen-binding molecule or antibody for use in a method of destroying a cell (e.g., a tumor cell) in an individual, the method comprising administering to the individual an effective amount of the anti-CD 137 antigen-binding molecule or antibody. In certain embodiments, examples of tumors include solid tumors in which B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells) are infiltrated. The "individual" according to any of the above embodiments is preferably a human.

In a further aspect, the present disclosure provides the use of an anti-CD 137 antigen-binding molecule or antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a tumor (which may be more appropriate (and is also applicable hereinafter) depending on the context, referred to as "cancer"). In a further embodiment, the medicament is for use in a method of treating a tumor (or cancer, depending on the context), the method comprising administering to an individual having a tumor (or cancer, depending on the context) an effective amount of the medicament. In further embodiments, the medicament is for inducing an anti-tumor effect, e.g., cell activation by binding of an anti-CD 137 antigen binding molecule or antibody to CD137 expressed on immune cells (e.g., T cells), inhibiting neovascularization in a tumor, inhibiting tumor cell proliferation, depleting B cells that promote a tumor, and the like. In further embodiments, the medicament is for use in a method of inhibiting neovascularization in a tumor, inhibiting tumor cell proliferation, depleting tumor-promoting B cells, or the like (e.g., by cellular activation resulting from binding of an anti-CD 137 antigen binding molecule or antibody to CD137 expressed on immune cells (e.g., T cells) in an individual), the method comprising administering to the individual an effective amount of the medicament therefor. An "individual" according to any of the above embodiments may be a human.

In a further aspect, the present disclosure provides a method for treating a tumor. In one embodiment, the method comprises administering to an individual having such a tumor an effective amount of an anti-CD 137 antigen binding molecule or antibody. In further embodiments, in the present disclosure, examples of tumors include solid tumors in which B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells) are infiltrated.

In a further aspect, the present disclosure provides a method for activating an immune cell in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CD 137 antigen binding molecule or antibody. In further embodiments, in the present disclosure, immune cells include immune cells such as B cells, dendritic cells, natural killer cells, macrophages, and/or CD8 positive T cells (more specifically, these immune cells have infiltrated into tumor tissue). In the method, when the expression level of at least one gene selected from the group consisting of CD8b1, gzmb, prf1 and Ifng in the tumor tissue is increased, it can be judged that the activation of T cells in the tumor tissue is enhanced.

In a further aspect, the present disclosure provides a method for activating or promoting immune cells, in particular CD8, in a tumor tissue of an individual ⁺ Methods of T cell proliferation. In one embodiment, such methods include a method comprising administering to an individual an effective amount of an anti-CD 137 antigen-binding molecule or antibody. In the activation of CD8 ⁺ In the method for T-cell therapy, when at least one CD8 selected from the group consisting of OVA tetramer, granzyme B, PD-1, DLRG-1 and ICOS is used ⁺ When the positive rate of the T cell activation marker increases (positive rate (%) = CD8 positive for the activation marker in tumor tissue) ⁺ T cell count/CD 8 in tumor tissue ⁺ Number of T cells × 100), CD8 in tumor tissue can be judged ⁺ The T cells are activated.

In a further aspect, the present disclosure provides methods for destroying cells (particularly tumor cells) in an individual. In one embodiment, the methods include those comprising administering to the individual an effective amount of an anti-CD 137 antigen binding molecule or antibody. In certain embodiments, examples of tumors include solid tumors in which B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells) are infiltrated. An "individual" according to any of the above embodiments may be a human.

In some embodiments, the individual is a patient having cancer cells or tumor tissue containing cancer cells. Preferred types of cancer in the present disclosure include, for example, stomach cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myeloid leukemia, pediatric cancer (wilms, neuroblastoma, sarcoma, hepatoblastoma, and germ cell tumor), but are not limited thereto. More preferred types of Cancer include gastric Cancer, colorectal Cancer, lung Cancer, mesothelioma, liver Cancer, breast Cancer, skin Cancer, lymphoma and myeloid leukemia, but are not limited to this (Tumori et al (2012) 98,478-484 Tumor Biol (2015) 36,4671-4679 am J Clin Pathol (2008), 224-230 Adv Ant Pathol (2014) 21,450-460 Med Oncol (2012) 29,663-669 clinical Cancer Research (2004), 6612-6621, applied Immunohistochem Mol Morphol (2009) 17,40-46 Eur J Peiatr Surg (2015) 25, 138-144J Clin Pathol (2011) 64,587-591 Ditro J Pathol (2006) 30,1570-1575, pp — 76, pp — 1576 (1576). More preferred types of cancer include gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

The anti-cancer agents comprising anti-CD 137 antigen binding molecules of the present disclosure are useful for treating patients with cancers refractory to treatment with immune checkpoint inhibitors. For example, a patient to whom administration of an immune checkpoint inhibitor does not produce the desired efficacy may be treated with an anti-cancer agent of the present disclosure. "cancer refractory to treatment with an immune checkpoint inhibitor" may also be indicated as "cancer that has been treated with an immune checkpoint inhibitor", "cancer that has deteriorated during or after treatment with an immune checkpoint inhibitor", "cancer for which an immune checkpoint inhibitor is ineffective", "cancer that has relapsed after treatment with an immune checkpoint inhibitor", "cancer that is resistant to treatment with an immune checkpoint inhibitor", "cancer that is poorly treated with an immune checkpoint inhibitor", or "cancer that has not been successfully treated with an immune checkpoint inhibitor".

Cancers refractory to treatment with the above immune checkpoint inhibitors include, but are not limited to, cancers having Mutations in the JAK1, JAK2 and/or B2M genes (see, e.g., shin, daniel Sanghoon et al, "Primary Resistance to PD-1 Block media by JAK1/2 Mutations," Cancer Discovery Vol.7,2 (2017): 188-201; or Sade-Feldman, moshe et al, "Resistance to checkpoint block therapy through activation of anti-gen presentation," Nature Communications Vol.8 (1): 6.2017, 26.10).

In a further aspect, in the present disclosure, pharmaceutical formulations comprising anti-CD 137 antigen-binding molecules or antibodies for use in the above-described methods of treatment, therapeutic uses, and medicaments may comprise an effective amount of an anti-CD 137 antigen-binding molecule or antibody having a lower level of immune activation in non-tumor tissue as compared to an anti-CD 137 antigen-binding molecule that does not have small molecule compound-dependent CD137 binding activity.

In one embodiment, the non-tumor tissue comprises lymph nodes, spleen, and/or liver.

In further embodiments, pharmaceutical formulations comprising anti-CD 137 antigen-binding molecules or antibodies for use in the above-described methods of treatment, therapeutic uses and medicaments may comprise an effective amount of an anti-CD 137 antigen-binding molecule or antibody that does not substantially bind to CD137 expressed in non-tumor tissue.

In further embodiments, pharmaceutical formulations comprising anti-CD 137 antigen-binding molecules or antibodies for use in the above-described methods, therapeutic uses, and medicaments may comprise an effective amount of an anti-CD 137 antigen-binding molecule or antibody having an extended blood half-life compared to an anti-CD 137 antigen-binding molecule that does not have CD137 binding activity that is dependent on a small molecule compound.

In further embodiments, pharmaceutical formulations comprising anti-CD 137 antigen-binding molecules or antibodies for use in the above-described methods, therapeutic uses, and medicaments may comprise an effective amount of an anti-CD 137 antigen-binding molecule or antibody that has a low level of side effects compared to an anti-CD 137 antigen-binding molecule that does not have CD137 binding activity that is dependent on a small molecule compound.

In further embodiments, the side effects include elevated AST, elevated ALT, fever, nausea, acute hepatitis, liver injury, splenomegaly, enteritis, purulent inflammation of the skin, neutropenia, lymphopenia, thrombocytopenia, transaminase expression, and/or hyperbilirubinemia.

In one embodiment, the anti-CD 137 antigen-binding molecules of the present disclosure have low side effects, and thus can be increased in dose without fear of side effects, and as a result, they can exhibit stronger drug efficacy (cytotoxic activity or anti-tumor activity).

In another aspect, the present disclosure provides a pharmaceutical formulation comprising any of the anti-CD 137 antigen binding molecules or antibodies provided herein, e.g., for use in any of the methods of treatment above. In one embodiment, the pharmaceutical formulation comprises any of the anti-CD 137 antigen binding molecules or antibodies provided herein and a pharmaceutically acceptable carrier.

The antigen binding molecules or antibodies of the present disclosure may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if local treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether short-term or long-term administration is employed. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administrations, and pulsed infusions.

The antibodies of the present disclosure will be formulated, administered and administered according to the manner of clinical medical guidelines. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the condition. The effective amount of these other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the present disclosure will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease (whether the antibody is used for prophylactic or therapeutic purposes), previous treatment methods, the patient's clinical history and response to the antibody and the discretion of the attending physician. The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 microgram/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of the antibody may serve as an initial candidate dose for administration to the patient, e.g., by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dosage may range from about 1 microgram/kg to 100 mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will generally be continued until the desired suppression of disease symptoms occurs. An exemplary dose of antibody will range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., with the patient receiving about two to about twenty doses, or, for example, about six doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. The progress of the therapy is readily monitored by conventional techniques and assays.

It is understood that the immunoconjugates of the disclosure can be used in place of, or in addition to, the anti-CD 137 antigen binding molecules or antibodies of the disclosure to perform any of the above-described formulations or methods of treatment. The anti-CD 137 antigen-binding molecule used is an antibody comprising the amino acid sequence of A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256, or A549/B167 as a heavy chain variable region/light chain variable region combination shown in Table 37. In a preferred embodiment, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, which has the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375/B167. In further embodiments, the anti-CD 137 antigen-binding molecule can be an anti-CD 137 antibody comprising a375/B167 as a combination of heavy chain variable region/light chain variable region. In various preferred embodiments, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, which has the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551/B379. In further embodiments, the anti-CD 137 antigen-binding molecule can be an anti-CD 137 antibody comprising a551/B379 as a combination of a heavy chain variable region/a light chain variable region. Furthermore, the anti-CD 137 antibody used may be any one anti-CD 137 antibody selected from the antibodies listed in table 72.

H. Article of manufacture

In another aspect of the present disclosure, an article of manufacture is provided comprising materials for treating, preventing and/or diagnosing the above-mentioned conditions. The article includes a container and a label or package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container itself contains the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the present disclosure. The label or package insert indicates that the composition is for use in treating the selected condition. The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. From a commercial and user perspective, it may also include other materials, including other buffers, diluents, filters, needles, and syringes.

The term "package insert" is used to refer to instructions typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings for use of such therapeutic products.

It is to be understood that any of the above articles of manufacture can include an immunoconjugate of the disclosure in place of, or in addition to, an anti-CD 137 antigen binding molecule or antibody.

Combination therapy

A. Combination therapy and therapeutic compositions for combination therapy

The present disclosure relates to methods of treating and/or preventing cancer comprising using a combination of the above-described anti-CD 137 antigen binding molecules and another anti-cancer agent. In one embodiment, the present disclosure relates to pharmaceutical compositions comprising an anti-CD 137 antigen binding molecule for use in combination therapy with another anti-cancer agent. In further embodiments, the disclosure relates to pharmaceutical compositions comprising another anti-cancer agent for use in combination therapy with the anti-CD 137 antigen binding molecules of the disclosure. The present disclosure relates to methods of treating or preventing cancer and/or activating immunity within tumor tissue in an individual comprising administering an anti-CD 137 antigen binding molecule and another anti-cancer agent. The anti-CD 137 antigen binding molecules and/or other anti-cancer agents are used in combination to treat cancer and/or to exert a synergistic or additive effect in treating cancer.

In some embodiments, the combination therapies of the present disclosure provide a method of enhancing the therapeutic or prophylactic effect of another anti-cancer agent in treating or preventing cancer by using the other anti-cancer agent together with the above-described anti-CD 137 antigen-binding molecule. In addition, the combination therapies of the present disclosure provide methods of enhancing the therapeutic or prophylactic effect of the above-described anti-CD 137 antigen-binding molecules in treating cancer by using them together with another anticancer agent. Here, the enhancement of the therapeutic or prophylactic effect means, for example, an increase in the response rate of treatment, a decrease in the amount of an anticancer agent to be administered for treatment, and/or a reduction in the treatment period of an anticancer agent, but is not limited thereto. In addition, the combination therapies of the present disclosure provide a method of increasing progression-free survival in an individual comprising administering an effective amount of the above-described anti-CD 137 antigen-binding molecule and another anti-cancer agent.

In some embodiments, the combination therapies of the present disclosure comprise administering the above-described anti-CD 137 antigen-binding molecule and another anti-cancer agent. The anti-CD 137 antigen binding molecule and other anti-cancer agents may be administered by any suitable method known in the art. For example, the anti-CD 137 antigen-binding molecule and the other anti-cancer agent may be administered concurrently (i.e., simultaneously) or sequentially (i.e., at different times). When the anti-CD 137 antigen-binding molecule and the other anticancer agent are administered sequentially (i.e., at different times), the administration interval between the anti-CD 137 antigen-binding molecule and the other anticancer agent is not particularly limited, and may be set in consideration of the administration route, dosage form, and the like. For example, 0 to 168 hours, preferably 0 to 72 hours, preferably 0 to 24 hours, more preferably 0 to 12 hours, but is not limited thereto.

The anti-CD 137 antigen-binding molecule described above and other anti-cancer agents may be co-administered. The anti-CD 137 antigen-binding molecule may also be administered intermittently (i.e., at irregular intervals). The anti-CD 137 antigen-binding molecule can be administered prior to the administration of the other anti-cancer agents. Alternatively, the anti-CD 137 antigen-binding molecule may be administered after administration of the other anti-cancer agent.

In addition, other anti-cancer agents may be administered intermittently (i.e., at irregular intervals). Other anti-cancer agents may also be administered prior to administration of the anti-CD 137 antigen binding molecule. Alternatively, the other anti-cancer agents may be administered after administration of the anti-CD 137 antigen binding molecule.

In some embodiments, the anti-CD 137 antigen-binding molecules described herein and other anti-cancer agents known or described herein can be used in combination therapy with the above-described anti-CD 137 antigen-binding molecule and another anti-cancer agent.

In addition to the combination therapy of the anti-CD 137 antigen-binding molecule described above with other anti-cancer agents, additional therapies may be performed. Therapies added to the combination therapies of the present disclosure may include additional administration of anti-CD 137 antigen binding molecules and/or other anti-cancer agents.

The present invention provides, as a non-limiting aspect, an immune response activator, a cancer therapeutic agent, or a cancer prophylactic agent (hereinafter referred to as a pharmaceutical composition or the like) against a cancer cell or a tumor tissue containing a cancer cell, which comprises the above-described anti-CD 137 antigen-binding molecule, another anti-cancer agent, or a combination of an anti-CD 137 antigen-binding molecule and another anti-cancer agent. The pharmaceutical compositions and the like of the present disclosure can be used in the combination therapies of the present disclosure. In some embodiments, by using the above-described anti-CD 137 antigen-binding molecule in combination with another anticancer agent, the pharmaceutical composition or the like of the present disclosure has a stronger effect in inhibiting cell proliferation, activating immunity against cancer cells or tumor tissues containing cancer cells, or treating or preventing cancer than treatment using these alone. In addition, by using the above-described anti-CD 137 antigen-binding molecule in combination with another anticancer agent, the pharmaceutical composition of the present invention has a synergistic or additive effect in inhibiting cell growth, activating immunity against cancer cells or tumor tissues containing cancer cells, or treating or preventing cancer.

In some embodiments, a "pharmaceutical composition comprising a combination of an anti-CD 137 antigen-binding molecule and another anti-cancer agent" or the like of the present disclosure refers to a pharmaceutical composition or the like in which the above-described anti-CD 137 antigen-binding molecule and another anti-cancer agent are combined for simultaneous, separate or sequential administration in the treatment or prevention of a disease. For example, the pharmaceutical composition of the present disclosure and the like may be provided in the form of a combination drug comprising both an anti-CD 137 antigen-binding molecule and another anti-cancer agent. Further, for example, in the pharmaceutical composition of the present disclosure and the like, a drug containing an anti-CD 137 antigen-binding molecule and a drug containing another anticancer agent may be separately provided, and these drugs may be used simultaneously or sequentially. The disease is not particularly limited, but cancer is preferred.

The present disclosure provides a pharmaceutical composition or the like for use in combination with other anticancer agents, which comprises the above-described anti-CD 137 antigen-binding molecule as an active ingredient. The present disclosure also provides a pharmaceutical composition or the like comprising another anticancer agent as an active ingredient, for use in combination with the above-described anti-CD 137 antigen-binding molecule.

The present invention provides a pharmaceutical composition and the like for improving the therapeutic effect of another anticancer agent in treating cancer with another anticancer agent by combining the above-mentioned anti-CD 137 antigen-binding molecule with another anticancer agent. In addition, the present disclosure provides a pharmaceutical composition or the like that improves the therapeutic effect of an anti-CD 137 antigen-binding molecule in treating cancer with an anti-CD 137 antigen-binding molecule by combining another anticancer agent with the above-described anti-CD 137 antigen-binding molecule.

In some embodiments, the present disclosure provides the use of an anti-CD 137 antigen-binding molecule and/or another anti-cancer agent in the preparation of the above-described pharmaceutical composition and the like comprising the anti-CD 137 antigen-binding molecule and/or the other anti-cancer agent as an active ingredient.

In the present invention, containing the above-mentioned anti-CD 137 antigen-binding molecule and/or another anticancer agent as an active ingredient means containing the anti-CD 137 antigen-binding molecule and/or other anticancer agent as a main active ingredient. Therefore, the content ratio of the anti-CD 137 antigen-binding molecule and/or the other anticancer agent is not limited.

In some embodiments, the anti-CD 137 antigen binding molecules described herein and other known or described anti-cancer agents described herein can be used in the aforementioned pharmaceutical compositions, and the like.

In one non-limiting embodiment, it is used in combination with an anti-CD 137 antigen binding moleculeExamples of anticancer agents include nitrogen mustard analogs, alkyl sulfonates, ethyleneimine, nitrosourea, epoxides, other alkylating agents, folic acid analogs, purine analogs, pyrimidine analogs, other metabolic antagonists, vinca alkaloids or analogs, podophyllotoxin derivatives, camptothecin analogs, colchicine derivatives, taxanes, other plant alkaloids or natural substances, actinomycin, anthracyclines or related substances, other cytotoxic antibiotics, platinum compounds, methylhydrazine (methylhydrazine), kinase inhibitors, angiogenesis inhibitors, hormones, DNA modifying enzyme inhibitors, immunostimulants, proteasome inhibitors, enzymes, histone deacetylase inhibitors, DNA modifying enzyme inhibitors, cytokine preparations, retinoids (retinoids), T cell activation agonists, immune checkpoint inhibitors, indoleamine 2, 3-dioxygenase (IDO) inhibitors, co-stimulatory molecule activators, natural killer cell activators, T cell redirecting antigen binding molecules, anti-fibrotic agents, regulatory T cell depleting agents, CD4 agents, and so-called "B" antagonists "or" antagonists ⁺ T cell depleting agents, B cell depleting agents, natural killer cell depleting agents, macrophage depleting agents, monoclonal antibodies, other molecularly targeted drugs or other anti-cancer agents, but are not limited thereto.

An "immune checkpoint" in the present disclosure refers to a molecule that is expressed on immunocompetent cells (including T cells) and that transmits a signal to the immunocompetent cells to suppress an immune response by binding to a ligand. Immune checkpoints and ligands thereof include, for example, PD-1, CTLA-4, TIM3, LAG3, PD-L1, PD-L2, BTNL2, B7-H3, B7-H4, CD48, CD80,2B4, BTLA, CD160, CD60, CD86, VISTA or TIGIT molecules, but are not limited thereto. In some embodiments, an "immune checkpoint inhibitor" in the present disclosure refers to an agent that inhibits signaling of an immune checkpoint by inhibiting binding of the immune checkpoint to its ligand. Preferably, the immune checkpoint inhibitor is an antigen binding molecule, preferably an antibody or antibody fragment, that binds to an immune checkpoint or its ligand and inhibits signaling of the immune checkpoint.

In one non-limiting embodiment, a pharmaceutical composition is provided wherein the additional anti-cancer agent is a chemotherapeutic agent, a T cell activation agonist, an immune checkpoint inhibitor Agents, T cell redirecting antigen binding molecules, anti-fibrotic agents, angiogenesis agent inhibitors, regulatory T cell depleting agents, CD4 ⁺ A T cell depleting agent, a B cell depleting agent, a natural killer cell depleting agent, or a macrophage depleting agent.

In one non-limiting embodiment, the chemotherapeutic agent includes, but is not limited to, an antimetabolite, a plant alkaloid, or a platinum compound. Preferred examples of antimetabolites include, but are not limited to, enocitabine, capecitabine, carmofur, gemcitabine, cytarabine, tegafur, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, and methotrexate. A particularly preferred example of an antimetabolite is capecitabine. Preferred examples of plant alkaloids include, but are not limited to, irinotecan, etoposide, sobuzosin, notitazone, paclitaxel, vinorelbine, vincristine, vindesine, and vinblastine. Examples of particularly preferred plant alkaloids include paclitaxel. Preferred examples of platinum compounds include, but are not limited to, oxaliplatin, carboplatin, cisplatin, and nedaplatin. Examples of particularly preferred platinum compounds include cisplatin, carboplatin, or a combination thereof.

In one non-limiting embodiment, the T cell activation agonist includes, but is not limited to, an agonistic antibody of the TNF receptor superfamily (TNFRSF) or an agonistic antibody of a co-stimulatory molecule.

"agonistic antibodies to co-stimulatory molecules" targeted molecules include TMIGD2, HHLA2, ICOS ligands, CD28, CD80, CD86, and the like. An example of a preferred co-stimulatory molecule agonistic antibody is an anti-CD 28 antibody.

The molecule targeted by the "agonistic antibody of TNF receptor superfamily" is not particularly limited as long as it is a factor that activates cells expressing TNF receptor superfamily (e.g., T cells, NK cells), but is preferably a factor belonging to the "TNF superfamily" or the "TNF receptor superfamily". As factors belonging to the "TNF superfamily" or the "TNF receptor superfamily", a ligand having a trimeric structure and a receptor having a trimeric structure, which contributes to activation of various immune cells, to which the ligand binds are known (nat. Rev. Immunol.,2012,12, 339-51). Factors belonging to the TNF superfamily or TNF receptor superfamily include, for example, CD137L, CD40L, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, GITRL, TNFRSF25 and TL1A. Examples of OX40 agonist antibodies include MOXR0916, MEDI6469, MEDI0562, MEDI6383, PF-04518600, GSK-3174998, and various known OX40 agonist antibodies. Examples of CD40 agonist antibodies include RG-7876, ADC-1013, SEA-CD40, APX005M, dacemazumab (Dacetuzumab), and various known CD40 agonist antibodies. Examples of GITR agonistic antibodies include AMG228, AMK-1248, MK-4166, BMS-986156, TRX518, and various known GITR agonist antibodies. Examples of CD27 agonist antibodies include vallizumab (Varllumab) (CAS registry number 1393344-72-3) and various known CD27 agonist antibodies. Examples of CD137 agonist antibodies include Urelumab (Urelumab) (CAS registry number: 934823-49-1), PF-05082566, and various known CD137 agonist antibodies. The CD137 agonistic antibody may be a bispecific antigen-binding molecule that binds to CD137 (e.g., a bispecific antibody that binds to CD137 and a cancer antigen, or a fusion protein that binds to CD137 ligand (4-1 BBL) and a cancer antigen, etc.).

In one non-limiting embodiment, examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM 3 antibodies, anti-LAG 3 antibodies, or anti-TIGIT antibodies. For example, examples of anti-PD-1 antibodies include pembrolizumab (CAS registry number: 1374853-91-4), nivolumab (CAS registry number: 946414-94-4), MEDI0680, PDR001, BGB-A317, REGN2810, SHR-1210, PF-06801591, and various known anti-PD 1 antibodies. Examples of anti-PD-L1 antibodies include astuzumab (CAS registry number: 1380723-44-3), avizumab (Avelumab) (CAS registry number: 1537032-82-8), DOVALUMAb (CAS registry number: 1428935-60-7), MDX-1105, and various known anti-PD-L1 antibodies. Examples of anti-CTLA-4 antibodies include Ipilimumab (Ipilimumab) (CAS registry No.: 477022-00-9), tremelimumab (Tremelimumab) (CAS registry No.: 745013-59-6), and various known anti-CTLA-4 antibodies. Examples of anti-TIM 3 antibodies include MBG452 and various known anti-TIM 3 antibodies. Examples of anti-LAG 3 antibodies include BMS-986016, LAG525, and various known anti-LAG 3 antibodies. Examples of anti-TIGIT antibodies include Tiragolumab (RG 6058) and various known anti-TIGIT antibodies. The immune checkpoint inhibitor can also be a bispecific antigen binding molecule that binds to (1) an immune checkpoint molecule and (2) a TNF receptor superfamily (TNFRSF) member or costimulatory molecule. In one embodiment, the combination therapy of the present disclosure may be a three-agent combination using a CD137 antigen binding molecule, an anti-PD-1 antibody, and an anti-TIGIT antibody, or a three-agent combination using a CD137 antigen binding molecule, an anti-PD-L1 antibody, and an anti-TIGIT antibody.

In one embodiment, the anti-CD 137 antigen binding molecules of the present disclosure and immune checkpoint inhibitors (preferably anti-PD-L1 antibodies) when used in combination treat cancer and/or exert a synergistic or additive effect in treating cancer with temporary or mild side effects. Although this is not intended to be bound by a particular theory, administration of an anti-CD 137 antigen binding molecule to an individual activates immune cells within the tumor tissue of the individual. Expression of PD-L1 is induced by this activation of immune cells, PD-L1 ⁺ The cells have the effect of suppressing an activated immune response. Thus, in combination therapy of an anti-CD 137 antigen-binding molecule with an anti-PD-L1 antibody, PD-L1 ⁺ The action of the cells is inhibited by the anti-PD-L1 antibody, and a stronger anti-tumor effect can be exerted. Examples of side effects include, but are not limited to, weight loss and liver dysfunction (elevated ASL and/or ALT levels).

In one non-limiting embodiment, a "T cell redirecting antigen binding molecule" is a multispecific antigen binding molecule comprising "a domain comprising an antibody variable region having T cell receptor complex binding activity" and "a domain comprising an antibody variable region having cancer antigen binding activity". Multispecific antibodies are preferred, and bispecific antibodies are more preferred. Multispecific (or bispecific) antibodies may have a single chain antibody structure, e.g., one in which the variable regions of the antibody are bound by a linker. The T cell redirecting antigen binding molecule may also contain an Fc region with reduced binding activity to Fc γ receptors.

"domain comprising an antibody variable region having cancer antigen binding activity" refers to a portion of an antibody comprising a region that specifically binds to and is complementary to a portion of a cancer antigen or the entire cancer antigen. As used herein, the term "cancer-specific antigen" refers to an antigen expressed by a cancer cell that is capable of distinguishing between cancer cells and healthy cells, for example, including antigens expressed as the cell becomes malignant, or abnormal sugar chains that appear on the cell surface and on protein molecules when the cell becomes cancerous. Examples are the various antigens disclosed herein (e.g., in "ii. Compositions and methods (anti-CD 137 agonistic antigen binding molecules), a. Exemplary anti-CD 137 antigen binding molecules or antibodies, 6. Multispecific antibodies"), but are not so limited. The cancer specific antigens targeted by the cancer specific antigen binding domains of the present disclosure are particularly preferably antigens expressed on the cell surface, such antigens including, for example, GPC3, CD19, CD20, EGFR, HER2, epCAM, EREG, FAP, CEA, DLL3, claudin (Claudin) -6 and FGFR3.

In one embodiment, the anti-T cell antigen binding molecule is a multispecific antibody, preferably a bispecific antibody, comprising (1) a domain comprising an antibody variable region having glypican 3 binding activity, (2) a domain comprising an antibody variable region having T cell receptor complex binding activity, and (3) a domain comprising an Fc region having reduced binding activity to an fey receptor. In one embodiment, with respect to the antibody L chain variable region contained in the antibody variable region having a glypican 3-binding activity and the antibody variable region having a T cell receptor complex-binding activity, it is preferable to obtain a common L chain capable of imparting a binding ability to both the H chain having a glypican 3-binding activity and the H chain having a T cell receptor complex-binding activity, and use it as the common L chain variable region of the above-mentioned bispecific antibody.

In one embodiment, the anti-T cell antigen binding molecule is ERY974.ERY974 is a bispecific antibody comprising (1) a domain comprising an antibody variable region having a glypican 3 binding activity, (2) a domain comprising an antibody variable region having a T cell receptor complex binding activity, and (3) a domain comprising an Fc region having a reduced binding activity to an fey receptor, and comprising:

TR01H113, disclosed in WO2016/047722 as a CD 3-side heavy chain variable region,

e2702sKsc, disclosed in WO2016/047722 as the CD3 side constant region of the heavy chain,

GCH065, disclosed in WO2016/047722 as the GPC 3-side heavy chain variable region,

e2704sEpsc, disclosed in WO2016/047722 as the GPC3 side heavy chain constant region,

l0011 disclosed in WO2016/047722 as a common light chain variable region, and

-k0, disclosed in WO2016/047722 as a common light chain constant region.

The "domain containing an antibody variable region having a T cell receptor complex binding activity" is preferably a domain containing an antibody variable region having a T cell receptor binding activity, more preferably a domain containing an antibody variable region having a CD3 binding activity. In addition, the above-mentioned "antibody variable region-containing domain" is provided by one or more antibody variable domains, and preferably, the antibody variable region-containing domain contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of domains containing such antibody variable regions include various antibody fragments such as "scFv (single chain Fv)", "single chain antibody", "Fv" and "scFv2 (single chain Fv 2)", "Fab" or "F (ab') 2", and the like.

In one non-limiting embodiment, an "anti-fibrotic drug" is a drug that inhibits or suppresses fibrosis, examples include, but are not limited to, pirfenidone and nintedanib. In addition, the anti-fibrotic drug may be a hyaluronidase drug, such as PEGPH20.

In one non-limiting embodiment, examples of angiogenesis inhibitors include, but are not limited to, anti-VEGF antibodies and anti-VEGFR 2 antibodies. Examples of angiogenesis inhibitors include bevacizumab, ramucirumab (ramucirumab), sorafenib, everolimus, sirolimus (temsirolimus), lenvatinib (lentitinib), aflibercept (aflibercept), and various known angiogenesis inhibitors.

In one non-limiting embodiment, a "regulatory T cell depleting agent" is an agent that depletes regulatory T cells, including, but not limited to, anti-CD 4 antibodies, anti-CD 25 antibodies, anti-CCR 4 antibodies, and anti-CTLA 4 antibodies.

In one non-limiting embodiment, "CD4 ⁺ The cell depleting agent is CD4 ⁺ Cellular agents, examples of which include, but are not limited to, anti-CD 4 antibodies.

In one non-limiting embodiment, a "B cell depleting agent," "natural killer cell depleting agent," and "macrophage depleting agent" are agents that deplete B cells, natural killer cells, and macrophages, respectively. For example, B cell depleting agents include, but are not limited to, rituximab and obituzumab (obinutuzumab), while macrophage depleting agents include, but are not limited to, anti-CSFlR antibodies.

In one non-limiting embodiment, "monoclonal antibody" includes, for example, anti-TGF-beta antibodies, anti-latent TGF-beta antibodies, anti-IL-8 antibodies, and the like, but is not limited thereto.

In some embodiments, the other anticancer agent of the present invention may be any anticancer agent, without particular limitation, as long as its therapeutic or prophylactic effect is enhanced or it enhances the therapeutic or prophylactic effect of the anti-CD 137 antigen-binding molecule of the present invention when used in combination with the anti-CD 137 antigen-binding molecule.

In one non-limiting embodiment, the combination therapies of the present disclosure may include, but are not limited to, the above-described anti-CD 137 antigen binding molecules and at least one other therapeutic agent, an immunomodulator, a cancer treatment vaccine, adoptive (adopted) T cell therapy, and the like. Suitable cancer therapy vaccines include, but are not limited to, whole tumor cell vaccines, tumor antigen vaccines, vector-based vaccines, oncolytic virus vaccines and dendritic cell vaccines. In addition to the above-mentioned therapies, multidisciplinary therapy combined surgery, radiotherapy, and the like can be performed.

In one non-limiting embodiment, the combination therapies of the present disclosure can be performed by combining the above-described anti-CD 137 antigen binding molecules with cytokine therapy using cytokines as anti-tumor immune response enhancers. Examples of such cytokines include, but are not limited to, IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, GM-CSF, IFN α (interferon- α), IFN α -2b, IFN β, and IFN γ.

In one non-limiting aspect, the present invention provides a cell growth suppressing agent, an immune response activating agent, a cancer therapeutic agent or a cancer preventing agent, which comprises the above-mentioned pharmaceutical composition.

The "individual" to whom the above-described anti-CD 137 antigen-binding molecule and/or another anti-cancer agent is administered may be a human. In some embodiments, the individual is a patient having cancer cells or tumor tissue comprising cancer cells. Preferred types of cancer in the present disclosure include, for example, stomach cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, pediatric cancers (wilms, neuroblastoma, sarcoma, hepatoblastoma, and germ cell tumor), but are not limited thereto. More preferred types of Cancer include gastric Cancer, colorectal Cancer, lung Cancer, mesothelioma, liver Cancer, breast Cancer, skin Cancer, lymphoma and myeloid leukemia, but are not limited to this (Tumori et al (2012) 98,478-484 Tumor Biol (2015) 36,4671-4679 am J Clin Pathol (2008), 224-230 Adv Ant Pathol (2014) 21,450-460 Med Oncol (2012) 29,663-669 clinical Cancer Research (2004) 10,6612-6621, applied Immunohistochem Mol Morphol (2009) 17,40-46 Eur J Pediatr Surg (2015) 25, 138-144J Clin Pathol (2011) 64, 587-591J Dithol Pathol (2015J) 30, 1570-1576, pp — 76. More preferred types of cancer include gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

In the present disclosure, the individual may be a patient who has received treatment with the anti-CD 137 antigen-binding molecule described above and/or any anti-cancer agent prior to the combination therapy of the anti-CD 137 antigen-binding molecule and another anti-cancer agent. Alternatively, the individual may be one who is unable to receive standard therapy or for whom standard therapy is not effective. In some embodiments, the individual has cancer at an early or late stage.

Combination therapy or pharmaceutical compositions for combination therapy comprising an anti-CD 137 antigen binding molecule of the present disclosure and another anti-cancer agent may be used to treat patients with cancer refractory to treatment with an immune checkpoint inhibitor. For example, the combination therapies (pharmaceutical compositions) of the present disclosure can be used to treat patients for which the desired efficacy is not observed by administration of immune checkpoint inhibitors. "cancer refractory to an immune checkpoint inhibitor" may also be indicated as "cancer that has been treated with an immune checkpoint inhibitor", "cancer that has worsened during or after treatment with an immune checkpoint inhibitor", "cancer for which an immune checkpoint inhibitor is ineffective", "cancer that has relapsed after treatment with an immune checkpoint inhibitor", "cancer that is resistant to treatment with an immune checkpoint inhibitor", "cancer that is poorly treated with an immune checkpoint inhibitor", or "cancer that has not been successfully treated with an immune checkpoint inhibitor". Preferred examples of other anti-cancer agents for inclusion in a combination therapy (pharmaceutical composition) include, but are not limited to, immune checkpoint inhibitors or T-cell redirecting antigen binding molecules.

Cancers refractory to treatment with the above immune checkpoint inhibitors include, but are not limited to, cancers having a gene mutation in JAK1, JAK2 and/or B2M (Shin, daniel Sanghoon et al, "Primary Resistance to PD-1 Block media by JAK1/2 Mutations". Cancer discovery vol.7,2 (2017): 188-201; or Sade-Feldman, moshe et al, "Resistance to checkpoint block therapy approach actuation of antigen presentation," Nature communications vol.8,1 1136.26 Oct.2017, etc.).

Alternatively, the combination therapy (pharmaceutical composition) of the present disclosure may be used to treat an individual having a cancer refractory to treatment with the anticancer agent of the present disclosure. For example, the combination therapy (pharmaceutical composition) of the present disclosure is used to treat an individual who becomes resistant to the anticancer agent of the present disclosure after administration of the anticancer agent, or an individual for which no desired efficacy is observed by administration of the anticancer agent of the present disclosure. In other words, a cancer that has been treated with the anticancer agent therapy of the present disclosure can be treated with the combination therapy (pharmaceutical composition) of the present disclosure. Preferred examples of another anti-cancer agent included in the pharmaceutical composition include, but are not limited to, immune checkpoint inhibitors or T-cell redirecting antigen binding molecules.

In another aspect, the present disclosure provides a method of activating immune cells in tumor tissue of an individual by the above-described combination therapy. In one embodiment, the method comprises administering to the individual an effective amount of an anti-cancer agent, wherein the anti-cancer agent comprises an anti-CD 137 antigen binding molecule and another anti-cancer agent. In further embodiments, in the present disclosure, such immune cells include B cells, dendritic cells, natural killer cells, macrophages and/or CD8 ⁺ T cells (more specifically, these immune cells invade tumor tissue). In this method, when the expression level of at least one gene selected from Cd3e, cd8b1, gzmb, prf1 and Ifng in the tumor tissue is increased, it can be judged that the activation of T cells in the tumor tissue is enhanced.

In another aspect, the present disclosure provides a method of activating immune cells, particularly CD8, in a tumor tissue of an individual by the above-described combination therapy ⁺ T cells or a method for promoting proliferation thereof. In one embodiment, the method comprises administering to the individual an effective amount of an anti-cancer agent, wherein the anti-cancer agent comprises an anti-CD 137 antigen binding molecule and another anti-cancer agent. In activating CD8 ⁺ In the method of T-cell therapy, when at least one CD8 selected from the group consisting of OVA tetramer, granzyme B, PD-1, KLRG-1 and ICOS is used ⁺ Increase in the positive rate of T cell activation marker (positive rate (%) = CD8 positive for activation marker in tumor tissue ⁺ T cell count/CD 8 in tumor tissue ⁺ Number of T cells × 100), CD8 in tumor tissue can be judged ⁺ The T cells are activated.

It is to be understood that the immunoconjugates of the disclosure can be used in place of, or in addition to, the anti-CD 137 antigen-binding molecules or antibodies of the disclosure to perform any of the above-described combination therapies or therapeutic compositions for combination therapies. The anti-CD 137 antigen-binding molecule used is an antibody comprising the amino acid sequence of A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256, or A549/B167, which are heavy chain variable region/light chain variable region combinations shown in Table 37. In a preferred embodiment, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, which has the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375/B167. In further embodiments, the anti-CD 137 antigen-binding molecule can be an anti-CD 137 antibody comprising a375/B167 as a combination of heavy chain variable region/light chain variable region. In various preferred embodiments, the anti-CD 137 antigen-binding molecule is an anti-CD 137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, which has the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551/B379. In further embodiments, the anti-CD 137 antigen-binding molecule can be an anti-CD 137 antibody comprising a551/B379 as a combination of a heavy chain variable region/a light chain variable region. Furthermore, the anti-CD 137 antibody used may be any one selected from the antibodies listed in table 72.

B. Reagent kit

In another aspect, the present disclosure provides kits for treating or preventing a plurality of diseases or disorders exemplified by tumors or cancers as detailed in "g. Methods of treatment or prophylactic compositions" above, in which the anti-CD 137 antigen-binding molecules (or immunoconjugates thereof) or pharmaceutical compositions comprising them of the present disclosure are combined with at least one other anti-cancer agent as detailed in "a. Combination therapy and therapeutic compositions for combination therapy" above. Such kits in the present disclosure may be provided, for example, as kits for treating or preventing cancer (or tumor).

The kits of the present disclosure comprise as an active ingredient an anti-CD 137 antigen-binding molecule of the present disclosure (or an immunoconjugate thereof) or a pharmaceutical composition (or pharmaceutical formulation) comprising the same, and (2) instructions or labeling indicating administration of at least one additional anti-cancer agent prior to, concurrently with, or after administration of the pharmaceutical composition to a patient.

In the kit of the present disclosure, the active ingredient may be filled in a container.

Kits of the present disclosure may include a label on the container or a package insert attached to the container.

The kits of the present disclosure are provided as commercial packages, for example, as therapeutic products.

The containers in the present disclosure preferably include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of various materials, such as glass and plastic. The container may contain the active ingredient alone or may have a sterile access port (e.g., the container may be a bag or vial containing a solution for intravenous administration with a stopper pierceable by a hypodermic injection needle).

The label or package insert in the present disclosure is typically included in a commercial package of the above-described therapeutic product, which is one example of a kit of the present disclosure, used to refer to instructions for use of such therapeutic product, which includes information about the indications, methods of use, dosages, methods of administration, combination therapies, contraindications and/or warnings. For combination therapy, for example, it may comprise instructions for the combined use of an anti-CD 137 antigen binding molecule (or immunoconjugate thereof) or a pharmaceutical composition (or pharmaceutical formulation) comprising the same in the present disclosure and at least one other anti-cancer agent as described in detail above in "a.

The kits of the present disclosure may also include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. Kits of the present disclosure may also include other equipment as needed from a commercial perspective or from a user perspective, such as other buffers, diluents, filters, needles, and syringes.

[ examples ]

The following are examples of the methods and compositions of the present disclosure. It is to be understood that various other aspects can be implemented in accordance with the above general description.

Example 1 homology by Using hCD137KI mice (LLC 1/OVA/GPC3 cloning of C5 cells) Evaluation of antitumor Activity of anti-CD 137 antibody for (syngeneic) tumor cell transplantation model

(1-1) preparation of cell line and syngeneic tumor transplantation mouse model and evaluation of antitumor Activity

In this study, the internally established mouse lung cancer cell lines LLC1/OVA/GPC3 cloned C5 cells (as described in reference example 9-4-1) and hCD137KI mice (as described in reference example 6-3) were used. LLC1/OVA/GPC3 clone C5 cell line was transplanted subcutaneously to the abdomen of mice until the tumor volume reached 164-465mm ³ Mice were grouped at time. Grouping was performed 10 days after transplantation. As shown in Table 6 below, A551-MB110/B379-ml0r antibody was administered to the tail vein of mice 11 days and 14 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 6]

Tumor volumes were measured 10, 14, 17 and 21 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = Length (mm) x Width (mm)/2

Tumor growth inhibition (TGI (%)) was calculated according to the following formula.

TV variation (mm) ³ ) = tumor volume at measurement-tumor volume at grouping

TGI (%) = (1- (mean of TV change per group/mean of TV change of vehicle control group)) × 100

As a result, the A551-MB110/B379-ml0r antibody showed tumor growth inhibition rates of 103, 101 and 106% at 7.5, 2.5 and 0.83mg/kg against LLC1/OVA/GPC3 clone C5 cells (FIG. 1, table 7). That is, the A551-MB110/B379-ml0r antibody showed comparably high antitumor activity at all doses of this example. One individual in the 2.5mg/kg administration group had to be euthanized because the overall condition deteriorated due to the diffusion of tumor cells in the peritoneum 15 days after tumor transplantation.

[ Table 7]

(1-2) preparation of cell line and syngeneic tumor transplantation mouse model, and reaction (Gene expression level) in tumor Evaluation of

In this study, internally established LLC1/OVA/GPC3 clones C5 cells (as described in reference example 9-4-1) and hCD137 KI mice (as described in reference example 6-3) were used. The LLC1/OVA/GPC3 clone C5 cell line is transplanted to the abdomen of mouse subcutaneously, and when the tumor volume reaches 100-180mm ³ Mice were grouped at time. Grouping was performed 10 days after transplantation. As shown in Table 8 below, vehicle control and A551-MB110/B379-ml0r antibody were administered to the tail vein of mice 11 days and 14 days after transplantation. PBS containing 0.05% tween 20 was used as the excipient.

[ Table 8]

Tumors were harvested 15 days after transplantation. The collected tumors were immersed in RNA-later (QIAGEN) and stored. Total RNA was extracted from tumor samples. Gene expression levels of total RNA were measured using nCounter punch Mouse Profiling Panel (NanoString Technologies) and nCounter Digital analyzer (NanoString Technologies).

The measured gene expression levels were analyzed using nSolver (NanoString Technologies) and Microsoft Excel 2013 (Microsoft corporation). Among the genes whose expression levels were measured, the expression levels of Cd8b1, gzmb, prf1, ifng, which were correlated with the cytotoxic activity of T cells, were plotted (fig. 2). The group treated with a551-MB110/B379-ml0r alone showed an increase in the expression level of all genes compared to the vehicle control group (Dunnett test, p <0.05, p <0.01, p < 0.001)).

As shown above, A551-MB110/B379-ml0r induced a strong intratumoral response to LLC1/OVA/GPC3 clone C5 cancer-bearing human CD137 knock-in mice under the conditions of this study.

⁺ (1-3) evaluation of intratumoral Effect (Positive Rate of various activation markers in CD8T cells)

(1-3-1) preparation of cell line and syngeneic tumor transplantation mouse model, and method for evaluating intratumoral effects

In this study, the internally established mouse lung cancer cell line LLC1/OVA/GPC3 cloned C5 cell line (as described in reference example 9-4-1) and hCD137 KI mice (as described in reference example 6-3) were used. LLC1/OVA/GPC3 clone C5 cell line was subcutaneously transplanted into the abdomen of mice until the tumor volume reached 69-195mm ³ Mice were grouped at time. Grouping was performed 12 days after transplantation. As shown in Table 9 below, A551-MB110/B379-ml of 0r was administered into the tail vein of the mice 13 days and 16 days after the transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

4 days after the first administration, tumor tissue was removed, lymphocyte fractions were prepared by the method detailed below, and CD8 was assessed by Flow Cytometry (FCM) ⁺ The positive rate of various activation markers in T cells is used to assess intratumoral responses. In the evaluation of each of these indices, when the index showing an intratumoral response was higher in the A551-MB110/B379-ml0 r-administered group than in the vehicle-administered group, it was judged that the activation of T cells in the tumor tissue was enhanced.

[ Table 9]

(1-3-2) excision and lymphocyte fraction of tumor tissue from mice transplanted with LLC1/OVA/GPC3 clone C5 cell line Preparation of

For excised tumor tissue, the number of cells in the lymphocyte fraction was measured. The lymphocyte fraction obtained by using a mouse tumor dissociation kit (Miltenyi Biotec) was used to measure the number of cells in the lymphocyte fraction.

(1-3-3) evaluation of lymphocyte fraction derived from mice transplanted with LLC1/OVA/GPC3 clone C5 cell line by FCM Activation marker positivity in (1)

For evaluation by FCM, CD8 was used ⁺ Positive rates for various activation markers in T cells. For this purpose, fluorescently labeled anti-CD 8 antibody (MBL), OVA-tetrameric reagent (MBL), anti-granzyme B antibody (BioLegend), anti-PD-1 antibody (BioLegend), anti-KLRG-1 antibody (BD Biosciences) and anti-ICOS antibody (Invitrogen) were used in the FCM assay. In BD LSRFortessa ^TM Measurements were performed on X-20 (BD Biosciences).

CD8 Using FCM ⁺ Evaluation of the positive rates of the various activation markers in T cells revealed that the positive rates of the various activation markers were increased in the tumor tissues of the A551-MB110/B379-ml0 r-administered group compared to the vehicle-administered group (FIG. 3).

As a result, A551-MB110/B379-ml0r enhanced CD8 in tumors ⁺ Activation of T cells.

Example 2 syngeneic (syngeneic) tumor cell transplantation model by using hCD137KI mouse (E.G7- OVA cells) of anti-CD 137 antibody

(2-1) preparation of cell line and syngeneic tumor transplantation mouse model toAnd A551-MB110/B379-ml0r antibody Evaluation of antitumor Activity of

In this study, the mouse lymphoma cell line E.G7-OVA cells (ATCC, CRL-2113) and hCD137KI mice (described in reference example 6-3) were used. The E.G7-OVA cell line was transplanted subcutaneously into the abdomen of mice when the tumor volume reached about 200mm ³ Mice were grouped together. Grouping was performed 7 days after transplantation. As shown in Table 10 below, A551-MB110/B379-ml0r antibody was administered to the tail vein of mice 7 days and 10 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 10]

Tumor volumes were measured 7 days, 10 days, 14 days, 17 days, 21 days, 24 days and 28 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, in the E.G7-OVA cell transplantation model, the mean tumor volume of the vehicle-control group was 2611.8mm at 21 days after the transplantation ³ . The average tumor volumes of the A551-MB110/B379-ml0r 0.83 and 2.5mg/kg groups were 1755.7mm ³ And 1322.1mm ³ . The A551-MB110/B379-ml0r 0.83 and 2.5mg/kg groups showed significantly lower values at 28 days after transplantation compared to the vehicle control group (Williams test). The change in tumor volume of the vehicle control group and the A551-MB110/B379-ml0r2.5mg/kg group is shown in FIG. 4.

(2-2) preparation of cell line and syngeneic tumor transplantation mouse model, and evaluation of A375-mIgG1/B167-ml0r Method for anti-tumor activity of antibody

In this study, the mouse lymphoma cell line E.G7-OVA cells (CRL-2113) and hCD137 KI mice (described in reference example 6-3) from ATCC were used. Transplanting E.G7-OVA cells into abdomen of mouse, and allowing the tumor volume to reach about 200-400mm ³ Mice were grouped together. Grouping was performed 7 days after transplantation. A375-mIgG1/B167-ml0r antibody was administered to the tail vein of mice 7 days and 10 days after transplantation, as shown in Table 11 below. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 11]

Tumor volumes were measured 7 days, 10 days, 15 days, 18 days, 21 days, 25 days, 29 days and 36 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = Length (mm) x Width (mm)/2

As a result, a375-mIgG1/B167-ml0r antibody showed strong antitumor effect on e.g. 7-OVA cells, and complete tumor regression was observed in 4 out of 5 mice (fig. 5).

Example 3 anti-syngeneic tumor cell transplantation model (C1498 cells) by Using hCD137KI mice Evaluation of anti-tumor Activity of CD137 antibody

In this study, mouse AML (acute myeloid leukemia) cell line C1498 cells (ATCC, ITEM No.: TIB-49) from ATCC and hCD137KI mouse (described in reference example 6-3) were used. The C1498 cell line was transplanted subcutaneously into the abdomen of mice when the tumor volume reached about 270mm ³ Mice were grouped together. Grouping was performed 7 days after transplantation. As shown in Table 12 below, A551-MB110/B379-ml0r antibody was administered to the tail vein of mice 7 days and 10 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 12]

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, in the C1498 cell transplantation model, the mean tumor volume of the vehicle-controlled group was 3405.3mm at 17 days after the transplantation ³ . The mean tumor volume in the A551-MB110/B379-ml0r2.5mg/kg group was 2181.3mm ³ . The A551-MB110/B379-ml0r2.5mg/kg group showed a lower value at 17 days after transplantation, compared to the vehicle control group. The change in tumor volume for the vehicle control group and the A551-MB110/B379-ml0r2.5mg/kg group is shown in FIG. 6.

Example 4 syngeneic tumor cell transplantation model by Using hCD137KI mouse (Hepa 1-6/hGPC3 cells) Evaluation of antitumor Activity of anti-CD 137 antibody

(4-1) preparation of cell line and syngeneic tumor transplantation mouse model, and A551-MB110/B379-ml0r antibody Evaluation of antitumor Activity of

In the present study, mouse hepatoma cell lines Hepa1-6/hGPC3 cells (Science relative Medicine 04Oct 2017, vol.9, issue 410, eaal4291) prepared by introducing an expression plasmid of human glypican-3 (GPC 3) into Hepa1-6 cells (CRL-1830) from ATCC and hCD137KI mice (described in reference to example 6-3) were used. The Hepa1-6/hGPC3 cell line was subcutaneously transplanted into the abdomen of mice when the tumor volume reached about 200mm ³ Mice were grouped at time. Grouping was performed 10 days after transplantation. As shown in Table 13 below, A551-MB110/B379-ml0r antibody was administered to the tail vein of mice 10 days and 13 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 13]

Tumor volumes were measured at 10, 13, 17, 20, 24, 27 and 31 days post-transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, in the Hepa1-6/hGPC3 cell transplantation model, the mean tumor volume of the vehicle-control group was 1525.3mm at 31 days after transplantation ³ . The average tumor volume of the A551-MB110/B379-ml0r 2.5mg/kg group was 471.6mm ³ . The A551-MB110/B379-ml0r 2.5mg/kg group showed a lower value at 31 days after transplantation compared to the vehicle control group. The change in tumor volume for the vehicle control group and the A551-MB110/B379-ml0r 2.5mg/kg group is shown in FIG. 7.

(4-2) preparation of cell line and syngeneic tumor transplantation mouse model, and A375-mIgG1/B167-ml0r antibody Evaluation of antitumor Activity of

In the present study, mouse hepatoma cell lines Hepa1-6/GPC3 cells (Science relative Medicine 2017, 10/4/l, vol.9, issue 410, eaal 4291) prepared by introducing an expression plasmid for human glypican-3 (GPC 3) into Hepa1-6 cells (CRL-1830) from ATCC and hCD137KI mice (described in reference example 6-3) were used. Hepa1-6/GPC3 cells were subcutaneously transplanted into the abdomen of mice when the tumor volume reached about 200-300mm ³ Mice were grouped at time. Grouping was performed 7 days after transplantation. As shown in Table 14 below, A375-mIgG1/B167-ml0r antibody was administered to the tail vein of mice 7 days and 10 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 14]

Tumor volumes were measured 7, 10, 13, 18 and 20 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, the A375-mIgG1/B167-ml0r antibody showed an antitumor effect on Hepa1-6/GPC3 cells (FIG. 8).

Example 5 model of homologous tumor cell transplantation by Using hCD137KI mice (LLC 1/OVA/GPC3 clone) C5 Evaluation of the Effect of various immune cells on the antitumor Activity of anti-CD 137 antibodies

In this study, the internally established mouse lung cancer cell lines LLC1/OVA/GPC3 cloned C5 cells (as described in reference example 9-4-1) and hCD137KI mice (as described in reference example 6-3) were used. LLC1/OVA/GPC3 clone C5 cell line was implanted subcutaneously into the abdomen of mice until the tumor volume reached about 200-300mm ³ Mice were grouped at time. Grouping was performed 9 days after transplantation. As shown in Table 15 below, A551-MB110/B379-ml of 0r antibody was administered to the tail vein of mice 10 days and 14 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control. In addition, as shown in table 16 below, agents for depleting various immune cells were administered.

[ Table 15]

[ Table 16]

Tumor volumes were measured at 9, 14, 17 and 20 days post-transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, the efficacy of the A551-MB110/B379-ml0r antibody against LLC1/OVA/GPC3 clone C5 cells was significantly impaired by depletion of CD8T cells (FIG. 9). It is inferred that the pharmacological effects in this model are exerted mainly by CD8T cells. On the other hand, depletion of CD4T cells, B cells, NK cells and macrophages tended to enhance drug efficacy (fig. 9). Thus, it was shown that the combination of the A551-MB110/B379-ml0r antibody and the agent for depleting these immune cells is effective in improving the drug efficacy.

Example 6 evaluation of anti-tumor Activity by Using a syngeneic tumor cell transplantation model (MC 38 cells) of hCD137KI mice Combination therapy with CD137 antibody single agent or with anti-PD-L1 antibody

(6-1) preparation of cell line and syngeneic tumor transplantation mouse model, and A551-MB110/B379-ml0r (Single dose) Or a combination) of anti-tumor activity and rate of change of body weight

In this study, mouse colon cancer cell line MC38 cells and hCD137KI mice (described in reference example 6-3), which were approved by the national cancer institute, were used. The MC38 cell line was implanted subcutaneously into the abdomen of mice when the tumor volume reached about 100-240mm ³ The model is considered to be established. After model establishment, mice transplanted with the MC38 cell line were grouped and then administered with A551-MB110/B379-ml0r and/or anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) at the doses shown in Table 17. A551-MB110/B379-ml0r was administered twice by the tail vein route 15 days and 18 days after transplantation. The anti-mouse PD-L1 antibody was administered twice by intraperitoneal route 15 days and 18 days after transplantation. PBS containing 0.05% tween 20 was used for vehicle control.

[ Table 17]

Tumor volume and body weight were measured at 14, 18, 21 and 25 days post-transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

TV variation (mm) ³ ) = tumor volume 25 days after transplantation-tumor volume at time of grouping

The weight change rate was calculated according to the following formula.

Weight change rate (%) =100 × measurement daily weight/weight of 14 days after transplantation

As a result, for MC38 cells, an increase in tumor growth inhibition was observed in the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination group, as compared with the A551-MB110/B379-ml0r single dose group or the anti-mouse PD-L1 antibody single dose group. Specifically, the tumor growth inhibition rate of the single-dose group of A551-MB110/B379-ml0r was 82%, while the tumor growth inhibition rate of the combined group of A551-MB110/B379-ml0r with the anti-mouse PD-L1 antibody was 117% (FIG. 10, table 18). In all administration groups, a transient weight loss within 6.5% was observed compared to the beginning of administration, but there was a tendency to recover thereafter (fig. 11).

[ Table 18]

Tumor growth inhibition 25 days after transplantation

(6-2) construction of an immune-related Gene Table before and after drug administration based on RNA-seq data of syngeneic mouse model Heatmap of changes (especially anti-PD-L1 antibody combination group)

The expression value FPKM was calculated by the RSEM method (version 1.2.31) by using the RNA-seq data of hCD137KI mouse (described in reference example 6-3) from a syngeneic tumor transplantation mouse model. The reference mRNA list (gene list) is GRCm38.MC38 was used as cell line. There are 4 groups: vehicle control, A551-MB110/B379-ml0r administration group, anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) administration group, A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination group.

The unit was converted from [ FPKM ] to [ FPKM +1] by adding 1 to the expression level per unit FPKM. This is because log conversion cannot be performed when an expression value exists as 0, and thus a minute (minute) value is added. Thereafter, log2 transformation is performed.

Next, human orthologs (orthologs) were assigned to the mouse genes. In principle, they are assigned according to the NCBI homoGene dataset. However, although NCBI homogene has not been orthologously assigned, human CXCL9 and mouse CXCL9 and human GZMB and mouse GZMB, which have been orthologously assigned on NCBI gene web pages by 9 months in 2019, are considered orthologs, respectively. Next, a panel of tumor immune-related genes (all 54 genes including CD3D, CD8A, etc.) was collected from the tumor immune-related literature. Then, 51 genes (which did not include genes without mouse orthologues) were selected from the 54 genes and examined.

Next, from the GRCm38 gene list (mRNA list) obtained from RNA-seq, only the genes in the above tumor immunity-related genome were selected. Since RNA-seq is mouse data and tumor immune-related genome is human genome, mouse genome was obtained by ortholog transformation. Next, the heatmap is drawn using a programming language R program. Here, the heatmaps were normalized so that the mean of all samples was 0 and the variance per gene was 1. The change in the average expression level of each gene was shown by gradation (fig. 12).

(6-3) evaluation of the intratumoral Effect of anti-CD 137 antibody as a Single drug or in combination with anti-PD-L1 antibody (in the tumor group) CD8 expression and PD-L1 expression in tissues)

(6-3-1) preparation of cell line and syngeneic tumor transplantation mouse model, and method for evaluating intratumoral effects

In this study, mouse colon cancer cell line MC38 cells and hCD137KI mice (described in reference example 6-3), which were approved by the national cancer institute, were used. The MC38 cell line was implanted subcutaneously into the abdomen of mice when the tumor volume reached about 100mm ³ The model is considered to be established. After model establishment, mice transplanted with the MC38 cell line were grouped and then administered with A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) at the doses shown in Table 19. A551-MB110/B379-ml0r through the tail vein 14 days after transplantation The route is administered once. Anti-mouse PD-L1 antibody administration was performed once 14 days after transplantation by intraperitoneal route. PBS containing 0.05% tween 20 was used for vehicle control.

Evaluation of intratumoral effects was analyzed using the increase in CD8 expression and PD-L1 expression in tumor tissues as indicators. CD8 expression and PD-L1 expression were assessed by immunohistochemical staining. In these evaluations, when the expression level of CD8 or PD-L1 in the tumor tissue is higher in the A551-MB110/B379-ml0r single drug group, the anti-mouse PD-L1 antibody single drug group, or the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combined group than in the excipient control group, or in the combined group than in each single drug administration group, it is judged that CD8 is present ⁺ The number of cells or PD-L1 expressing cells increases.

[ Table 19]

(6-3-2) tumor tissue was excised from mice transplanted with the MC38 cell line and a tumor tissue sample was prepared

The excised tumor tissue was immersed in a 10% neutral buffered formalin solution for 1 day. The paraffin embedded tumor tissue was then sectioned and immunohistochemically stained.

⁺ (6-3-3) immunohistochemical staining of tumor tissue samples from mice transplanted with the MC38 cell line and CD8 Fine staining ⁺ Assessment of cellular and PD-L1 cell infiltration into tumor tissue

anti-CD 8 α antibodies (Cell Signaling Technology, clone D4W 2Z) and the OptiView DAB kit (Ventana Medical Systems) were used for immunohistochemical staining of CD 8. Anti-rabbit HQ antibody and HRP-labeled anti-HQ antibody were used as secondary and tertiary detection antibodies. Polyclonal anti-PD-L1 antibodies (R & D) and the UltraView DAB kit (Ventana Medical Systems) were used for immunohistochemical staining of PD-L1. HRP-labeled anti-goat antibody was used as the detection secondary antibody. BenchMark XT automated stainers (Ventana Medical Systems) were used for staining.

The immunohistochemical staining results for CD8 are shown in FIG. 13, and for PD-L1 are shown in FIG. 14. In the combined group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody, increased CD8 expression and PD-L1 expression in tumor tissues were observed as compared with the vehicle control group and each single-drug administration group. Furthermore, in the group administered with a single drug of A551-MB110/B379-ml0r and the group administered with a single drug of anti-mouse PD-L1 antibody, CD8 expression and PD-L1 expression were increased in tumor tissues as compared with the group administered with vehicle control.

In addition, high resolution digital images of all tissues of immunohistochemically stained specimens were captured using the pathology scanner system for brightfield microscopy (Aperio ScanScope CS2 slide scanner, leica Biosystems) and a 20-fold objective. For the measurement of immunohistochemically stained positive cells for CD8 or PD-L1 in tumors, the analysis algorithm "Immune Cell v1.3" or "Membrane v1.7" in HALO image analysis software (Indica Labs, v 2.3), respectively, was used. The tumor area in each tissue was manually annotated and evaluated by a pathologist. The number of positive cells per tumor area is determined by the number of cells per tumor area (mm) ² ) And (4) showing. The digital image analysis was visually inspected for positive threshold quality (fig. 82, 83 and 84).

The above results show that single drug administration of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody increases CD8 in tumor tissue ⁺ Cells and PD-L1 ⁺ A cell. Furthermore, it was shown that the combined administration of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody further increased CD8 in tumor tissues compared to the administration of each single drug ⁺ Cells and PD-L1 ⁺ The number of cells. Due to CD8 ⁺ The cells have cytotoxic activity against cancer cells, and thus CD8 in tumor tissues is considered ⁺ When the number of cells is increased, stronger antitumor effect is shown. Here, PD-L1 expression is induced by a tumor immunity-related gene such as IFN-. Gamma. (see FIG. 12), and PD-L1 ⁺ The cells have the effect of suppressing abnormally activated immune responses. As described above, the tumor immunity-related gene was shown to be elevated in the A551-MB110/B379-ml0r administration group (example 6-2, FIG. 12). Therefore, it is considered that in the combination group of A551-MB110/B379-ml0r and the anti-mouse PD-L1 antibody, the anti-mouse PD-L antibodyThe 1 antibody inhibits the action of PD-L1, thereby obtaining a stronger antitumor effect. These events are believed to contribute to the strong efficacy of the combined administration shown in example 6-1.

(6-4) evaluation of liver and intratumoral effects of anti-CD 137 antibodies as a single agent or in combination with anti-PD-L1 antibodies

(6-4-1) preparation of cell line and syngeneic tumor transplantation mouse model, and evaluation of liver action and intratumoral action By means of

In this study, mouse colon cancer cell line MC38 cells and hCD137KI mice (described in reference example 6-3), which were approved by the national cancer institute, were used. The MC38 cell line was transplanted subcutaneously into the abdomen of mice when the tumor volume reached about 111-200mm ³ The model is considered to be established. After model establishment, mice transplanted with the MC38 cell line were grouped and then administered with A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) at the doses shown in Table 20. A551-MB110/B379-ml0r was administered twice by the tail vein route 11 days and 14 days after transplantation. The anti-mouse PD-L1 antibody was administered twice by intraperitoneal route 11 days and 14 days after transplantation. PBS containing 0.05% tween 20 was used for vehicle control.

Liver effects were assessed using blood collected 4 days after the first administration. The resulting blood was centrifuged and plasma was separated. Measurements of various markers in plasma were evaluated by the methods detailed below. In the evaluation of each of these indices, when the index showing liver function disorder was higher in the a551-MB110/B379-ml0r single drug group, the anti-mouse PD-L1 antibody single drug group, or the a551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combined group than in the excipient control group, impaired liver function was evaluated.

To evaluate the intratumoral response, tumor tissues were removed 4 days after the initial administration by the methods detailed below and the number of cells in the lymphocyte fraction was determined. In this evaluation, CD8 in tumor tissue ⁺ The cell number is higher than that of the excipient control group in A551-MB110/B379-ml0r single drug group, anti-mouse PD-L1 antibody single drug group or A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination groupThen the evaluation promoted CD8 in tumor tissue ⁺ Proliferation of T cells.

[ Table 20]

(6-4-2) evaluation of liver function marker value by testing blood of MC38 cell line-transplanted mice

Liver function markers in blood samples were measured by the sample testing system TBA-120FR (canon medical system).

As a result of liver function marker measurement by blood test, no change in AST and ALT values was observed in both the a551-MB110/B379-ml0r single drug administration group and the anti-mouse PD-L1 antibody single drug administration group and the a551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination group, compared to the excipient administration group (fig. 15).

As described above, neither A551-MB110/B379-ml0r nor anti-mouse PD-L1 antibody impaired liver function.

(6-4-3) removal of tumor tissue from MC38 cell line-transplanted mice and preparation of lymphocyte fraction

(6-4-4) evaluation of lymphocyte fraction derived from MC38 cell line-transplanted mouse

As a result of measuring the number of cells in the lymphocyte fraction using FCM, CD8 in tumor tissue was observed in the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination group, compared to the vehicle-administered group ⁺ The number of T cells increased (fig. 16).

In summary, the combination of A551-MB110/B379-ml0r with anti-mouse PD-L1 antibody promotes intratumoral CD8 ⁺ Proliferation of T cells.

Example 7: model of syngeneic tumor cell transplantation by using hCD137KI mouse (L)LC1/OVA/GPC3 clones C5 cells) as a single agent or in combination with an anti-PD-L1 antibody

In this study, the internally established mouse lung cancer cell lines LLC1/OVA/GPC3 cloned C5 cells (as described in reference example 9-4-1) and hCD137KI mice (as described in reference example 6-3) were used. LLC1/OVA/GPC3 clone C5 cell line was subcutaneously transplanted into mice when tumor volume reached 148-495mm ³ Mice were grouped at time. Grouping was performed 11 days after transplantation. As shown in Table 21 below, vehicle control and A551-MB110/B379-ml0r were administered to the tail vein of mice 11 days and 14 days after transplantation. In addition, as shown in Table 21 below, vehicle control and an anti-mouse PD-L1 antibody (BioXcell, clone: 10F.9G2) were intraperitoneally administered to mice 11 days and 14 days after the transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 21]

Tumor volumes were measured at 11, 14, 17, 20, 25, 28, 31 and 35 days post-transplantation. Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, the average tumor volume of the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody at 35 days after transplantation was 782mm ³ However, the mean tumor volume of the A551-MB110/B379-ml0r single drug group was 1812mm ³ (Table 22). Therefore, the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody showed stronger tumor growth inhibition than the single drug group of A551-MB110/B379-ml0r or the single drug group of anti-mouse PD-L1 antibody. In particular, although the anti-mouse PD-L1 antibody single drug group had almost no tumor growth inhibitory effect compared to the vehicle control, the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody had a high tumor growth inhibitory effect (FIG. 17). Thus, this indicates that the combined use of A551-MB110/B379-ml0r and an immune checkpoint inhibitor is useful for using immune checkpoint inhibitors The formulations (e.g., PD1 and/or PD-L1 inhibitors) are effective in treating poorly effective cancer types.

[ Table 22]

Example 8-1: anti-tumor by using syngeneic tumor cell transplantation model (C1498 cells) of hCD137KI mouse Evaluation of anti-tumor Activity of CD137 antibody as a Single drug or in combination with anti-PD-L1 antibody

In this study, mouse AML (acute myelogenous leukemia) cell line C1498 cells (ATCC, ITEM No.: TIB-49) and hCD137KI mice (described in reference example 6-3) were used. C1498 cell line was subcutaneously transplanted into mice when the tumor volume reached 173-381mm ³ Mice were grouped together. Grouping was performed 8 days after transplantation. As shown in Table 23 below, vehicle control, A375-mIgG1/B167-ml0r antibody and anti-mouse PD-L1 antibody (BioXcell, clone 10 F.9G2) were administered into the tail vein of mice at 8 and 12 days post-transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 23]

Tumor Volume (TV) was measured at 8, 10, 12 and 15 days post-transplantation.

Tumor volume was calculated by the following formula.

Tumor volume (mm) ³ ) = Length (mm) x Width (mm)/2

TV Change (mm) ³ ) = tumor volume at measurement-tumor volume at grouping

As a result, an enhanced tumor growth inhibition was observed for the group combining A375-mIgG1/B167-ml0r and anti-mouse PD-L1 antibody against C1498 cells, compared with the group combining A375-mIgG1/B167-ml0r or anti-mouse PD-L1 antibody. Specifically, the tumor growth inhibition rate of the single drug group of A375-mIgG1/B167-ml0r was 62%, while the tumor growth inhibition rate of the combined group of A375-mIgG1/B167-ml0r and anti-mouse PD-L1 antibody was 82% (FIG. 18, table 24).

[ Table 24]

Example 8-2: evaluation of the singles by using a syngeneic tumor cell transplantation model (C1498 cells) of hCD137KI mice Antitumor Activity of drug-converting anti-CD 137 antibody or combination of converting anti-CD 137 antibody and anti-TIGIT antibody

In this study, mouse AML (acute myelogenous leukemia) cell line C1498 cells (ATCC ITEM No.: TIB-49) and hCD137KI mice (described in reference example 6-3) were used. The C1498 cell line was subcutaneously transplanted into the right abdomen of mice, and divided into groups 7 days after transplantation. The tumor volume is about 270 to 546mm ³ For grouping. 7 days after transplantation, the vehicle and A551-MB110/B379-ml0r antibody were administered to the tail vein of mice. The vehicle and anti-TIGIT antibody (clone: 1G9; catalog number: BE0274; bioXcell) were administered intraperitoneally to mice 7 days, 9 days, 11 days, 13 days, 15 days, and 18 days after transplantation, as shown in the following table. PBS containing 0.05% Tween-20 was used as excipient.

[ Table 86]

Tumor volumes were measured 7 days, 9 days, 11 days, 13 days, 15 days, 17 days, 19 days, 22 days, 25 days, 27 days, 29 days, and 32 days after transplantation. When the tumor volume exceeded 10% of body weight, the individual was euthanized according to internal euthanization criteria, 1000mm ³ The tumor volume of (2) was converted to 1g. For each group, the calculation was done while all individuals were aliveMean tumor volume.

Tumor volume was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, tumor growth inhibition was observed for the combination of the single-drug conversion anti-CD 137 antibody A551-MB110/B379-ml0r and the anti-TIGIT antibody.

22 days after transplantation, the mean tumor volume of the single-drug administration group of the anti-CD 137 antibody A551-MB110/B379-ml0r was converted to 1588mm ³ And the mean tumor volume of the group administered with the combination of anti-CD 137 antibody A551-MB110/B379-ml0r and anti-TIGIT antibody was switched to 734mm ³ . The tumor growth inhibitory effect tended to be stronger in the group administered with the combination of the conversion anti-CD 137 antibody and the anti-TIGIT antibody than in the group administered with the conversion anti-CD 137 antibody alone (fig. 105).

Example 9 anti-CD 137 by Using a syngeneic tumor cell transplantation model (AE 17 cells) of hCD137KI mice Evaluation of anti-tumor Activity of antibodies as a Single drug or in combination with anti-PD-L1 antibodies

In this study, mouse malignant mesothelioma AE17 cells (DS Pharma biomedicalal) and hCD137 KI mice (described in reference example 6-3) were used. In vivo passaged AE17 tumor was subcutaneously transplanted into mice until the tumor volume reached 70-306mm ³ Grouping is performed. Grouping was performed 9 days after transplantation. As shown in Table 25 below, vehicle control, A375-mIgG1/B167-ml0r and anti-mouse PD-L1 antibody (BioXcell, clone 10 F.9G2) were administered into the tail vein of mice at 9 and 12 days post-transplantation. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 25]

Tumor volumes were measured at 9, 12, 15 and 19 days post-transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = Length (mm) x Width (mm)/2

TV Change (mm) ³ ) = tumor volume at measurement-tumor volume at grouping

As a result, with respect to AE17 cells, an increase in tumor growth inhibition was observed in the a375-mIgG1/B167-ml0r and anti-mouse PD-L1 antibody combination group, as compared with the a375-mIgG1/B167-ml0r single drug group or anti-mouse PD-L1 antibody single drug group. Specifically, the tumor growth inhibition ratio of the single drug group of A375-mIgG1/B167-ml0r was 39%, while the tumor growth inhibition ratio of the combined group of A375-mIgG1/B167-ml0r and anti-mouse PD-L1 antibody was 60% (FIG. 19, table 26).

[ Table 26]

Example 10 evaluation by Using the syngeneic tumor cell transplantation model (LLC 1/hGPC3 cells) of hCD137KI mice The anti-CD 137 antibody is estimated as a single agent or in combination with an anti-hGPC 3-mCD antibody

(10-1) preparation of cell line and syngeneic tumor transplantation mouse model, and A551-MB110/B379-ml0r (Mono-tumor transplantation) Drug or combination) of the drugs

In the present study, the mouse lung cancer cell lines LLC1/hGPC3 cells (Science relative Medicine 2017, 10/4, volume 9, stage 410, eaal 4291) and hCD137KI mice (described in reference example 6-3) established internally were used. The LLC1/hGPC3 cell line was transplanted subcutaneously into the abdomen of mice when the tumor volume reached about 287mm ³ Mice were grouped at time. Grouping was performed 11 days after transplantation. As shown in Table 27 below, 11 days after transplantation, vehicle control, A551-MB110/B379-ml0r antibody and anti-hGPC 3-mCD3 antibody (bispecific antibody binding to human GPC3 and mouse CD 3) were administeredInto the tail vein of the mouse. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 27]

Tumor Volume (TV) was measured 11, 14, 17 and 21 days after transplantation.

Tumor volume was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

The results are shown in FIG. 20. The mean tumor volume of the vehicle control group was 2393mm 21 days after the transplantation ³ . The average tumor volumes of the single drug group of the anti-hGPC 3-mCD3 antibody, the single drug group of A551-MB110/B379-ml0r, and the combined group of the anti-hGPC 3-mCD3 antibody and A551-MB110/B379-ml0r were 1592mm ³ ，1340mm ³ And 35mm ³ . Both the A551-MB110/B379-ml0r unit drug group and the anti-hGPC 3-mCD3 antibody unit drug group showed lower mean tumor volumes compared to the vehicle control group. Furthermore, the mean tumor volume was significantly reduced for the anti-hppc 3-mCD3 antibody and a551-MB110/B379-ml0r combination group compared to the a551-MB110/B379-ml0r and anti-hppc 3-mCD3 antibody monotherapy groups.

From the above, the combination of anti-hGPC 3-mCD3 antibody and A551-MB110/B379-ml0r synergistically enhanced the anti-tumor effect on LLC1/hGPC3 cancer-bearing human CD137 knock-in mice.

(10-2) preparation of cell line and syngeneic tumor transplantation mouse model, and method for evaluating intratumoral reaction

In the present study, the mouse lung cancer cell lines LLC1/hGPC3 cells (Science relative Medicine 2017, 10/4, volume 9, stage 410, eaal 4291) and hCD137KI mice (described in reference example 6-3) established internally were used. The LLC1/hGPC3 cell line is transplanted to the abdomen of the mouse subcutaneously when the tumor volume reaches about 230mm ³ Mice were grouped at time. Grouping was performed 10 days after transplantation. As shown in Table 28 below, vehicle controls, A551-MB110/B379-ml0r antibody and anti-hGPC 3-mCD were administered 10 days after transplantation3 antibody (bispecific antibody binding to human GPC3 and mouse CD 3) was administered into the tail vein of mice. PBS containing 0.05% tween 20 was used as vehicle control.

[ Table 28]

Tumor volumes for the above groupings were calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

Tumors were harvested 13 days after transplantation. The collected tumors were immersed in RNA-later (QIAGEN) and stored. Total RNA was extracted from tumor samples using RNeasy Mini kit (QIAGEN). Gene expression levels of total RNA were measured using nCounter punch Mouse Profiling Panel (NanoString Technologies) and nCounter Digital analyzer (NanoString Technologies).

The measured gene expression levels were analyzed using nSolver (NanoString Technologies) and Microsoft Excel 2013 (Microsoft corporation). Among the genes whose expression levels were measured, the expression levels of Cd3e, cd8b1, gzmb, prf1, ifng, which correlated with the cytotoxic activity of T cells, were plotted (fig. 21). The combined group of a551-MB110/B379-ml0r and anti-hpgc 3-mCD3 antibody showed increased expression levels of Cd3e, cd8B1, gzmb and Prf1 genes compared to the vehicle control group (Dunnett test p <0.05, p <0.01, p < 0.001)). The expression level of the combination of A551-MB110/B379-ml0r and anti-hGPC 3-mCD3 antibody group tended to increase even for Ifng, compared with the vehicle group. Compared with the A551-MB110/B379-ml0r single drug group and the anti-hGPC 3-mCD3 antibody single drug group, the expression level of the A551-MB110/B379-ml0r and anti-hGPC 3-mCD3 antibody combined group tends to be increased for all genes. In addition, the expression level of the cytotoxic activity-associated gene tends to increase in each single drug group as compared with the excipient.

As can be seen from the above, under the conditions of this study, the combination of A551-MB110/B379-ml0r and anti-hGPC 3-mCD3 antibody induced a strong intratumoral response in LLC1/hGPC3 cancer-bearing human CD137 knock-in mice.

Example 11 anti-tumor by Using syngeneic tumor cell transplantation model (breast cancer cell line) of hCD137KI mouse Evaluation of anti-tumor Activity of CD137 antibody

In this study, commonly available or established breast cancer cell lines and hCD137KI (knock-in) mice were used. The breast cancer cell lines were transplanted into mice and grouped when the tumor volume reached a certain level. anti-CD 137 antibodies and excipients were then administered to mice.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate compared to the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against breast cancer cell lines.

Example 12 evaluation by Using a syngeneic tumor cell transplantation model (breast cancer cell line) of hCD137KI mouse Combination therapy with anti-CD 137 antibodies, either alone or in combination with anti-PD-L1 antibodies

In this study, a commonly available or established breast cancer cell line and hCD137KI mouse were used. The breast cancer cell lines were transplanted into mice and grouped when the tumor volume reached a certain level. Then, an anti-CD 137 antibody single agent, an anti-PD-L1 antibody single agent, an anti-CD 137 antibody, and an anti-PD-L1 antibody combination or excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate compared to the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against breast cancer cell lines. Furthermore, when the anti-CD 137 antibody and anti-PD-L1 antibody combined administration group showed a higher tumor growth inhibition rate than any single agent administration group, synergistic or additive enhancement by the presence of tumor growth inhibition by combined use can be evaluated.

Example 13 evaluation by Using a syngeneic tumor cell transplantation model (breast cancer cell line) of hCD137KI mouse Combination therapy of anti-CD 137 antibodies, either alone or with anti-PD-L1 antibodies and/or anti-TIGIT antibodies

In this study, commonly available or established breast cancer cell lines and hCD137KI mice were used. Breast cancer cell lines were transplanted into mice and grouped when tumor volume reached a certain level. Thereafter, (1) an anti-CD 137 antibody single agent, (2) an anti-PD-L1 antibody single agent, (3) an anti-CD 137 antibody and anti-PD-L1 antibody two agent combination, (4) an anti-CD 137 antibody and anti-TIGIT antibody two agent combination, (5) an anti-CD 137 antibody, anti-PD-L1 antibody and anti-TIGIT antibody three agent combination, or (6) an excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate than the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against breast cancer cell lines. Further, when the two-drug combination administration group of the anti-CD 137 antibody and the anti-PD-L1 antibody, or the two-drug combination administration group of the anti-CD 137 antibody and the anti-TIGIT antibody has a higher tumor growth inhibition rate than that of their single-drug administration group, then the synergistic or additive enhancement of the tumor growth inhibition effect by the combined use can be evaluated. Furthermore, if the three-drug combination group of the anti-CD 137 antibody, the anti-PD-L1 antibody, and the anti-TIGIT antibody shows a higher tumor growth inhibition rate than the above-mentioned single-drug or two-drug combination group, the synergistic or additive enhancement of the tumor growth inhibition by the three-drug combination can be evaluated.

Example 14 evaluation of anti-tumor Activity by Using a syngeneic tumor cell transplantation model (Lung cancer cell line) of hCD137KI mouse Combination therapy of CD137 antibodies, either alone or with anti-PD-L1 antibodies and/or anti-TIGIT antibodies

In this study, commonly available or established lung cancer cell lines and hCD137KI mice were used. The lung cancer cell lines were transplanted into mice and grouped when the tumor volume reached a certain level. Thereafter, (1) an anti-CD 137 antibody single agent, (2) an anti-PD-L1 antibody single agent, (3) an anti-CD 137 antibody and anti-PD-L1 antibody two agent combination, (4) an anti-CD 137 antibody and anti-TIGIT antibody two agent combination, (5) an anti-CD 137 antibody, anti-PD-L1 antibody and anti-TIGIT antibody three agent combination, or (6) an excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate compared to the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against lung cancer cell lines. Furthermore, when the two-drug combination administration group of the anti-CD 137 antibody and the anti-PD-L1 antibody, or the two-drug combination administration group of the anti-CD 137 antibody and the anti-TIGIT antibody has a higher tumor growth inhibition rate than those of the single-drug administration group, then the synergistic or additive enhancement of the tumor growth inhibition effect by the combined use can be evaluated. Furthermore, if the three-drug combination group of the anti-CD 137 antibody, the anti-PD-L1 antibody, and the anti-TIGIT antibody shows a higher tumor growth inhibition rate than the above-mentioned single-drug or two-drug combination group, the synergistic or additive enhancement of the tumor growth inhibition by the three-drug combination can be evaluated.

Example 15 evaluation by Using a syngeneic tumor cell transplantation model (melanoma cell line) of hCD137KI mice Evaluation of combination therapy of anti-CD 137 antibodies alone or with anti-PD-L1 antibodies and/or anti-TIGIT antibodies

In this study, a widely available or established melanoma cell line and hCD137KI mice were used. Melanoma cell lines were transplanted into mice and grouped when tumor volume reached a certain level. Thereafter, (1) an anti-CD 137 antibody single agent, (2) an anti-PD-L1 antibody single agent, (3) an anti-CD 137 antibody and anti-PD-L1 antibody two agent combination, (4) an anti-CD 137 antibody and anti-TIGIT antibody two agent combination, (5) an anti-CD 137 antibody, anti-PD-L1 antibody and anti-TIGIT antibody three agent combination, or (6) an excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate than the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against melanoma cell lines. Furthermore, when the tumor growth inhibition rate is higher in the group administered with the combination of the anti-CD 137 antibody and the anti-PD-L1 antibody, or the group administered with the combination of the anti-CD 137 antibody and the anti-TIGIT antibody than in these single drug administration groups, it is possible to evaluate the synergistic or additive enhancement of the tumor growth inhibition effect by the combined use. Furthermore, if the three-drug combination group of the anti-CD 137 antibody, the anti-PD-L1 antibody, and the anti-TIGIT antibody shows a higher tumor growth inhibition rate than the above-mentioned single-drug or two-drug combination group, the synergistic or additive enhancement of the tumor growth inhibition effect by the three-drug combination can be evaluated.

Example 16 syngeneic tumor cell transplantation model by Using hCD137KI mouse (colorectal cancer cell line) Evaluating combination therapy of anti-CD 137 antibodies, either alone or with anti-TIGIT antibodies

In this study, commonly available or established colorectal cancer cell lines and hCD137KI mice were used. Colorectal cancer cell lines were transplanted into mice and grouped when tumor volume reached a certain level. Then, any one of the anti-CD 137 antibody single agent, anti-TIGIT antibody single agent, anti-CD 137 antibody and anti-TIGIT antibody combination, and the excipient were administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate than the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against colorectal cancer cell lines. Furthermore, when the anti-CD 137 antibody and anti-TIGIT antibody combined administration group showed a higher tumor growth inhibition rate than any single-drug administration group, synergistic or additive enhancement by the presence of tumor growth inhibition by the combined use can be evaluated.

Example 17 homologous tumor cell transplantation model by Using hCD137KI mice (JAK 1 knockout cancer cell line) Evaluating combination therapy of anti-CD 137 antibodies, either alone or with anti-PD-L1 antibodies and/or anti-TIGIT antibodies

In this study, a universally available or established JAK1 knockout cancer cell line and hCD137KI mice were used. JAK1 knockout cancer cell lines were transplanted into mice and grouped when tumor volume reached a certain level. Thereafter, (1) an anti-CD 137 antibody single agent, (2) an anti-PD-L1 antibody single agent, (3) an anti-CD 137 antibody and anti-PD-L1 antibody two agent combination, (4) an anti-CD 137 antibody and anti-TIGIT antibody two agent combination, (5) an anti-CD 137 antibody, anti-PD-L1 antibody and anti-TIGIT antibody three agent combination, or (6) an excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate than the vehicle-administered group, it can be evaluated that the anti-CD 137 antibody has an antitumor activity against the JAK1 knockout cancer cell line. Further, when the anti-CD 137 antibody and anti-PD-L1 antibody two-drug combination administration group, or the anti-CD 137 antibody and anti-TIGIT antibody two-drug combination administration group showed a higher tumor growth inhibition rate than those of the single-drug administration group, then the synergistic or additive enhancement of the tumor growth inhibition effect by the combined use can be evaluated. Furthermore, if the three-drug combination group of the anti-CD 137 antibody, the anti-PD-L1 antibody, and the anti-TIGIT antibody shows a higher tumor growth inhibition rate than the above-mentioned single-drug combination group or two-drug combination group, the synergistic or additive enhancement of the tumor growth inhibition effect by the three-drug combination can be evaluated.

Example 18-1: syngeneic tumor cell transplantation model by using hCD137KI mouse (B2M knockout cancer cell) Line) evaluation of anti-CD 137 antibodies monotherapy or combination therapy with anti-PD-L1 antibodies and/or anti-TIGIT antibodies

In this study, a widely available or established B2M knockout cancer cell line and hCD137KI mice were used. B2M knockout cancer cell lines were transplanted into mice and grouped when tumor volume reached a certain level. Thereafter, (1) an anti-CD 137 antibody single agent, (2) an anti-PD-L1 antibody single agent, (3) an anti-CD 137 antibody and anti-PD-L1 antibody two agent combination, (4) an anti-CD 137 antibody and anti-TIGIT antibody two agent combination, (5) an anti-CD 137 antibody, anti-PD-L1 antibody and anti-TIGIT antibody three agent combination, or (6) an excipient is administered to the mouse.

Tumor volume was measured multiple times after transplantation. When the anti-CD 137 antibody-administered group showed a higher tumor growth inhibition rate compared to the vehicle-administered group, the anti-CD 137 antibody can be evaluated to have anti-tumor activity against B2M knockout cancer cell lines. In addition, when the anti-CD 137 antibody and anti-PD-L1 antibody combined administration group, or the anti-CD 137 antibody and anti-TIGIT antibody combined administration group showed a higher tumor growth inhibition rate than those of the single administration group, it was possible to evaluate the synergistic or additive enhancement of the tumor growth inhibition effect by the combined use. Furthermore, if the three-drug combination group of the anti-CD 137 antibody, the anti-PD-L1 antibody, and the anti-TIGIT antibody shows a higher tumor growth inhibition rate than the above-mentioned single-drug combination group or two-drug combination group, the synergistic or additive enhancement of the tumor growth inhibition effect by the three-drug combination can be evaluated.

Example 18-2: anti-CD 137 anti-human tumor cells transformed by using syngeneic tumor cell transplantation model of hCD137KI mouse Evaluation of antitumor Activity of body

(18-2-1) preparation of tumor cell line and syngeneic tumor transplantation mouse model, and method for evaluating antitumor Activity Method of

Tumors having a mutation in the β 2 microglobulin (B2M) gene (B2M mutation) are known to be resistant to treatment with an anti-PD-1 antibody, an anti-PD-L1 antibody, or the like. (Sharma P., et al, cell.168, (9), 707-232017; and Zaretsky JM. Et al, N Engl J Med.375 (9): 819-29.2016).

In this study, an internally established mouse colon cancer cell line MC38-hGPC3# G64B2M KO clone 5 cells with a β 2 microglobulin (B2M) knock-out and hCD137 KI mice (described in reference to examples 6-3) were used. The MC38-hGPC3# G64B2M KO clone 5 cell line was subcutaneously transplanted into the right abdomen of mice, and was grouped 14 days after transplantation. Having a thickness of 111 to 228mm ³ Individuals of tumor volume were used for grouping. The conversion anti-CD 137 antibody A551-MB110/B379-ml of 0r was administered into the tail vein of mice at 14 days and 21 days after transplantation. PBS containing 0.05% Tween-20 was used as excipient.

[ Table 87]

Tumor volumes were measured at 14, 17, 21, 24, 27 and 31 days post-transplantation.

Tumor volume was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

TV Change (mm) ³ ) = tumor volume at measurement-tumor volume at grouping

TGI (%) = (1- (mean value of TV change per group/mean value of TV change of excipient group)) × 100

As a result, the conversion anti-CD 137 antibody A551-MB110/B379-ml0r showed tumor growth inhibition on MC38-hGPC3# G64B2M KO clone 5 cells throughout the period after tumor transplantation compared to the control, and showed 29% tumor growth inhibition at 31 days after tumor transplantation (FIG. 106).

The CD137 converting antibody of the present invention shows extremely significant antitumor activity against tumors with b2m mutation known to be resistant to treatment with an anti-PD-1 antibody, an anti-PD-L1 antibody, or the like.

Example 19 anti-syngeneic tumor cell transplantation model (MC 38 cells) by Using hCD137KI mice Evaluation of anti-tumor Activity of CD137 antibodies as a Single drug or in combination with anti-PD-L1 antibodies

(19-1) preparation of tumor cell line and syngeneic tumor transplantation mouse model, and A551-MB110/B379-ml0r And evaluation of anti-tumor Activity of UreH-MB110/UreL-mk1 (alone or in combination with anti-mouse PD-L1 antibody)

In this study, MC38 cells, a mouse colon cancer cell line licensed by the national cancer institute, and hCD137KI mice (described in reference example 6-3) were used. The MC38 cell line is transplanted to the abdomen of the mouse subcutaneously when the tumor volume reaches 106-200mm ³ The model is considered to be established. After model establishment, mice transplanted with the MC38 cell line were grouped and then administered with the transforming antibody A551-MB110/B379-ml0r or the non-transforming antibody UreH-MB110/UreL-mk1 (positive control), and/or the anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) at the doses shown in Table 77. The transducible antibody A551-MB110/B379-ml0r or the non-transducible antibody UreH-MB110/UreL-mk1 was administered twice by the tail vein route at 11 days and 14 days after transplantation. The anti-mouse PD-L1 antibody was administered twice by intraperitoneal route 11 days and 14 days after transplantation. Contains 0 PBS 05% tween 20 was used for vehicle control.

The non-switching antibody UreH-MB110/UreL-mk1 used as a positive control was a non-switching CD137 antibody having the variable region of the known anti-CD 137 antibody Urelumab (CAS registry No.: 934823-49-1) as the variable region and MB110 (SEQ ID NO: 145) as the heavy chain constant region and mouse kappa chain mk1 (the same amino acid sequence as mouse kappa chain mk0 (SEQ ID NO: 147)) as the light chain constant region, which are described in WO 2014030750.

[ Table 77]

Tumor volume and body weight were measured 10, 14, 18, 22, 25, 29 and 32 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

TV Change (mm) ³ ) = tumor volume 32 days after transplantation-tumor volume at time of grouping

TGI (%) = (1- (mean value of TV change per group/mean value of TV change of vehicle control group)) × 100

As a result, the single-drug administration group of the crossover antibody a551-MB110/B379-ml0r, the combination group of the crossover antibody a551-MB110/B379-ml0r and the anti-mouse PD-L1 antibody, and the combination group of the non-crossover antibody urea h-MB 110/urea L-mk1 and the anti-mouse PD-L1 antibody showed significant tumor growth inhibition compared to the vehicle group for MC38 cells, and the tumor growth inhibition rates were 78%, 109%, and 81%, respectively (fig. 85, table 78). Thus, it was shown that the combination group of the converting antibody A551-MB110/B379-ml0r and the anti-mouse PD-L1 antibody had the same or higher anti-tumor effect as that of the combination group of the non-converting antibody UreH-MB110/UreL-mk1 and the anti-mouse PD-L1 antibody.

[ Table 78]

Tumor growth inhibition 32 days after transplantation

Example 20 anti-CD 137 antibody by Using a syngeneic tumor cell transplantation model of hCD137KI mouse Assessment of systemic Effect of a Single drug or in combination with an anti-PD-L1 antibody

(20-1) preparation of tumor cell line and syngeneic tumor transplantation mouse model, and method for evaluating intratumoral effects

In this study, mouse colon cancer cell line MC38 cells and hCD137KI mice (described in reference example 6-3), which were approved by the national cancer institute, were used. The MC38 cell line was implanted subcutaneously into the abdomen of mice when the tumor volume reached about 200mm ³ The model is considered to be established. After model establishment, mice transplanted with the MC38 cell line were grouped, and then the transfer antibody A551-MB110/B379-ml0r and the anti-mouse PD-L1 antibody (BioXcell, clone 10F.9G2) were administered at the doses shown in Table 79. As positive control substances, non-transformed anti-CD 137 antibody UreH-MB110/UreL-mk1 (top) and anti-mouse PD-L1 antibody were administered.

On days 15 and 18 after the transplantation, the crossover antibody A551-MB110/B379-ml0r or the non-crossover antibody UreH-MB110/UreL-mk1 was administered from the caudal vein route twice. The anti-mouse PD-L1 antibody was administered twice by intraperitoneal route 15 days and 18 days after transplantation. PBS containing 0.05% tween 20 was used for vehicle control.

(20-2) removal of spleen, tumor regional lymph node (DLN) and liver, lymphocytes from MC38 cell line-transplanted mice Fraction preparation and peripheral blood sampling

Peripheral blood was collected from the inferior vena cava for blood collection. Lymphocyte fraction isolation was performed using excised spleens, tumor regional lymph nodes and liver. For the excised spleen and tumor regional lymph nodes, organ weights were measured before lymphocyte fraction isolation was performed. For spleen and tumor regional lymph nodes, lymphocyte fractions obtained by using a cell filter after tissue destruction were used. For the liver, a lymphocyte fraction obtained by performing tissue destruction using a liver dissociation kit, a mouse (Miltenyi Biotec) and then using a cell filter was used. The obtained peripheral blood was analyzed by a hemocytometer, and the obtained spleen, tumor regional lymph node and liver-derived lymphocyte fraction were analyzed by a Flow Cytometer (FCM).

[ Table 79]

(20-3) evaluation of blood parameter values by blood test of MC38 cell line-transplanted mice

For sample blood, blood parameters were measured using sample testing system XT-2000iV (Sysmex corp.).

Blood parameter measurements of blood tests showed no change in the leukocyte, platelet and lymphocyte concentrations of the A551-MB110/B379-ml0r single drug group. Slight fluctuations in leukocyte concentration and lymphocyte concentration were observed in the anti-mouse PD-L1 antibody single-agent group, whereas slight fluctuations in leukocyte concentration and lymphocyte concentration similar to those in the anti-mouse PD-L1 antibody single-agent group were observed in the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combined group (FIG. 86).

On the other hand, in the UreH-MB110/UreL-mk1 single drug group, significant decrease in the leukocyte concentration and lymphocyte concentration was observed (FIG. 86). In addition, in the combination group of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody, a decrease in platelet concentration was observed in addition to a significant decrease in leukocyte concentration and lymphocyte concentration (FIG. 86).

In conclusion, compared to UreH-MB110/UreL-mk1, A551-MB110/B379-ml0r does not cause fluctuation of blood parameters even when used in combination with an anti-mouse PD-L1 antibody, and its effect on peripheral blood is extremely limited.

(20-4) evaluation of transplanted mice by organ weight measurement and Flow Cytometry (FCM) analysis Using MC38 cell line Immune cell activation

Organ weight assessment of spleen and lymph node in tumor region (DLN) showed no increase in each organ weight in the A551-MB110/B379-ml0r monotherapy group, the anti-mouse PD-L1 antibody monotherapy group, and the A551-MB110/B379-ml0r and anti-mouse PD-L1 combination group (FIG. 87).

On the other hand, in the UreH-MB110/UreL-mk1 single drug group, an increase in organ weight of each organ was observed. Furthermore, in the combination group of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody, a more significant increase in organ weight per organ was observed compared to the single drug group of UreH-MB110/UreL-mk1 (FIG. 87).

Next, FCM was used to analyze the activation of T cells derived from spleen, lymph nodes in tumor area and liver. In FCM to assess immune cell activation, CD8 was used ⁺ Expression Rate of activation marker, CD4, in T cells ⁺ Foxp3 in T cells ⁺ Increased rate of regulatory T cells and CD45 ⁺ Foxp3 in cells ⁺ The rate of increase of regulatory T cells. KLRG-1, ICOS, PD-1 and LAG-3 were used as CD8 ⁺ Activation marker in T cells. In the FCM assay, anti-CD 45 antibodies (BD Biosciences), anti-CD 3 antibodies (BioLegent), anti-CD 8 antibodies (BioLegent), anti-CD 4 antibodies (BioLegent), anti-KLRG-1 antibodies (BD Biosciences), anti-ICOS antibodies (BioLegent), anti-PD-1 antibodies (BioLegent), anti-LAG-3 antibodies (BD Biosciences), and anti-Foxp 3 antibodies (BD Biosciences) were used. BD LSR Fortessa X-20 (BD Biosciences) was used for measurement.

Spleen evaluation results showed that CD8 was present in the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody in comparison with the combination group of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody ⁺ Expression rates of KLRG-1, ICOS, PD-1, LAG-3, CD45 in T cells ⁺ Activation marker positive CD8 for each cell ⁺ Ratio of T cells, and CD4 ⁺ Foxp3 in T cells ⁺ Ratio of regulatory T cells and CD45 ⁺ Foxp3 in cells ⁺ There was no significant increase in the ratio of regulatory T cells (figures 88 and 89).

The evaluation results of lymph nodes in tumor regions showed that CD8 was present in the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody, as compared with the combination group of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody ⁺ In T cellsExpression rates and absolute amounts of KLRG-1, ICOS, PD-1, LAG-3, and CD4 ⁺ Foxp3 in T cells ⁺ The ratio and absolute number of regulatory T cells did not increase significantly (fig. 90 and 91).

The liver evaluation results showed that CD8 was present in the combination group of A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody, as compared with the combination group of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody ⁺ Expression rates of KLRG-1, ICOS, PD-1, LAG-3, CD45 in T cells ⁺ Activation marker positive CD8 for each cell ⁺ Ratio of T cells, and CD4 ⁺ Foxp3 in T cells ⁺ Ratio of regulatory T cells and CD45 ⁺ Foxp3 in cells ⁺ There was no significant increase in the ratio of regulatory T cells (figures 92 and 93).

Based on the above, even though the transition antibody A551-MB110/B379-ml0r showed a tumor growth inhibitory effect comparable to that of the non-transition antibody UreH-MB110/UreL-mk1, its effect on immune cells in normal organs such as peripheral blood, spleen, tumor regional lymph nodes, and liver was extremely limited even when used in combination with the anti-mouse PD-L1 antibody.

Example 21 tissue distribution evaluation of anti-CD 137 antibody

(21-1) preparation of tumor cell line and syngeneic tumor transplanted mouse model, and method for evaluating tissue distribution of antibody Method of

In this study, MC38 cells, a mouse colon cancer cell line licensed by the national cancer institute, and hCD137KI mice (described in reference example 6-3) were used. The MC38 cell line was implanted subcutaneously into the abdomen of mice when the tumor volume reached about 200mm ³ The model is considered to be established. After model establishment, MC38 cell line transplanted mice were grouped and Alexa488 labeled A551-MB110/B379-ml0r was administered at the dose shown in Table 80. As a negative control substance, alexa 488-labeled IC17HdK-MB110/IC17L-mk was administered. As a positive control substance, non-transformed anti-CD 137 antibody Alexa 488-labeled UreH-MB110/UreL-mk1 was administered. For Alexa488 labeling of each antibody, alexa Fluor 488 antibody labeling kit (Thermo Fisher Scientific) was used.Alexa 488-labeled IC17HdK-MB110/IC17L-mk or Alexa 488-labeled A551-MB110/B379-ml0r or Alexa 488-labeled UreH-MB110/UreL-mk1 was administered once 14 days after transplantation.

(21-2) tumor, spleen and liver were excised from MC38 cell line-transplanted mice and lymphocyte fractions were prepared

Lymphocyte fraction isolation was performed on the excised tumors, spleen and liver. For the spleen, a lymphocyte fraction obtained by a cell filter after tissue destruction was used. For the tumor or liver, a lymphocyte fraction obtained by performing tissue destruction using a tumor dissociation kit, a mouse (Miltenyi Biotec) or a liver dissociation kit, a mouse (Miltenyi Biotec) and then using a cell filter was used. The obtained tumor, liver and spleen derived lymphocyte fractions were subjected to Flow Cytometry (FCM) analysis.

[ Table 80]

(21-3) evaluation of antibody tissue distribution by Flow Cytometry (FCM) analysis of MC38 cell line-transplanted mice

Each Alexa 488-labeled antibody that binds to tumor, spleen and liver-derived lymphocyte fractions was analyzed using FCM. Alexa488 in CD8 ⁺ Expression rate in T cells was used for assessment by FCM. anti-CD 45 antibodies (BD Biosciences), anti-CD 3 antibodies (BioLegend), anti-CD 8 antibodies (BioLegend), anti-CD 19 antibodies (BioLegend) and anti-CD 11b antibodies (BioLegend) were used for FCM analysis. BD LSR Fortessa X-20 (BD Biosciences) was used for measurement.

CD8 as an assessment and infiltration tumor ⁺ Results for T cell binding of each Alexa 488-labeled antibody, high binding rates were observed in the Alexa 488-labeled A551-MB110/B379-ml0r administration group or the Alexa 488-labeled UreH-MB110/UreL-mk1 administration group compared to the Alexa 488-labeled IC17HdK-MB110/IC17L-mk administration group (FIG. 94). In spleen and liver, the transducin antibody Alexa 488-labeled CD8 from A551-MB110/B379-ml0r administration group ⁺ The T cell binding rate was almost comparable to that of the negative control Alexa 488-labeled IC17HdK-MB110/IC17L-mk administration group (FIG. 94).

On the other hand, in the Alexa 488-labeled UreH-MB110/UreL-mk1 administration group (which is a non-switching antibody), CD8 was compared with the Alexa 488-labeled IC17HdK-MB110/IC17L-mk administration group and the Alexa 488-labeled A551-MB110/B379-ml0r administration group ⁺ The T cell binding rate was very high (fig. 94).

From the above, it was found that the converting antibody A551-MB110/B379-ml0r binds only to lymphocytes present in tumor tissues, and that the binding to lymphocytes present in normal organs, spleen and liver is limited.

Example 22 validation of a set of genes showing expression Change associated with extracellular ATP in tumor tissue

(22-1) mapping of extracellular ATP-related expression based on RNA-seq data of syngeneic tumor-transplanted mouse model Gene heatmap of expression changes

From the RNA-seq data obtained from each of the syngeneic tumor-transplanted mouse models prepared by transplanting the MC38 cell line, the LLC/OVA/hGPC3 cell line, the LLC/OVA cell line, or the Hepa1-6/hGPC3 cell line into the C57BL/6 mice, the expression value (unit: FPKM) was calculated by the RSEM method (version 1.2.31). The reference mRNA list (gene list) is GRCm38. In addition, expression values were similarly calculated from RNA-seq data obtained from each normal tissue of C57BL/6 mice by the RSEM method. For RNA-seq raw Data (. Fastq files) of 12 normal organs, data published in literature (Sollner, sci Data,2017, 4.

The unit was converted from [ FPKM ] to [ FPKM +1] by adding 1 to the gene expression level in FPKM unit. This is because log conversion cannot be performed when an expression value exists as 0, and thus a minute (minute) value is added. Thereafter, a log2 conversion is performed.

Next, human orthologs (orthologs) were assigned to the mouse genes. In principle, they were assigned according to the NCBI HomoloGene dataset.

Next, genes associated with extracellular ATP were collected from the literature (Hu, clin Cancer Res,2019,25 (4), 1318-1330, li, cancer discov,2019,9 (12), 1754-1773 yang, cancer Sci,2019,110 (8), 2456-2470, gobbault, front immunol,2013, 414.

Furthermore, EMT-associated genomes whose expression was increased by addition of ATP were collected from the literature (Yang, cancer Sci,2019,110 (8), 2456-2470) (EMT: epithelial mesenchymal transition).

Next, from the GRCm38 gene list (mRNA list) obtained from RNA-seq, only genes in the above ATP-related genome and EMT-related genome were selected. Since RNA-seq is mouse data and ATP-related and EMT-related genomes are human genomes, they were converted by ortholog to obtain mouse genomes. Next, genes whose expression was significantly higher than that of the normal tissue group (p < 0.05) in each cancer cell line were selected by t-test.

Next, the heatmap is drawn using a programming language R program. Here, the heatmaps were normalized so that the mean of all samples was 0 and the variance per gene was 1. The change in the average expression level of each gene is shown by gradation (fig. 95).

The results indicate that there are a number of significantly highly expressed genes in the ATP-related genome and EMT-related genome in tumor tissues compared to 12 normal organs. For this reason, it is considered that A551-MB110/B379-ml0r binds only to immune cells in tumor tissues, but not to immune cells in normal tissues such as liver and spleen (example 21). Furthermore, it is considered that the transition antibody bound to the antigen in the presence of ATP induces a strong immune response only in tumor tissues (example 20), while it does not elicit an immune response in normal organs such as liver, spleen, tumor regional lymph nodes and peripheral blood (example 20).

Example 23 assessment of CD73 and CD39 expression in various cancer cells by Flow Cytometry (FCM) analysis

(23-1) evaluation of CD73 expression in various cancer cell lines

In this study, the mouse acute myelogenous leukemia cell line C1498 cells (described in example 3) and the mouse leukemia cell line e.g7-OVA cells (described in example 2) were used, which were purchased from ATCC; mouse colon cancer cell line MC38 cells (described in example 6) approved by the national cancer institute; and the mouse hepatoma cell line Hepa1-6/hGPC3 cells (as described in example 4), the mouse hepatoma cell line LLC1/OVA/GPC3 clone C5 cells (as described in reference example 9-4-1), and the internally established mouse hepatoma cell line LLC1/OVA clone 17 cells.

Mouse lung cancer cell line LLC1/OVA clone 17 cells were prepared by introducing an Ovalbumin (OVA) expression plasmid into the mouse lung cancer-derived cell line LLC1 (LL/2 (also referred to as LLC 1), distributor: ATCC, catalog number: CRL-1642). anti-CD 73 antibody (Bio Legend) was used for FCM analysis in each cell line. FACS Lyric (BD Biosciences) was used for the measurement.

As a result of evaluating the membrane surface expression of CD73 in each cell line, almost no expression of CD73 was observed on the cell membranes of C1498 cells and e.g. g7-OVA cells, while expression of CD73 was observed on the cell membranes of MC38 cells, hepa 1-6/hpgc 3 cells, LLC1/OVA/GPC3 clone C5 cells and LLC1/OVA clone 17 cells (fig. 96). CD73 is known to be a membrane-type enzyme that breaks AMP down into adenosine. Thus, even though MC38 cells, hepa 1-6/hpgc 3 cells, LLC1/OVA/GPC3 clone C5 cells, and LLC1/OVA clone 17 cells had the property of breaking down AMPs (which are required for the conversion antibody to bind to the antigen) to adenosine, the conversion antibody was able to exhibit an anti-tumor effect (examples 1 to 10), indicating that the conversion antibody acted on tumor tissue without being affected by CD73 expression.

(23-2) expression of CD39 and CD73 in tumor tissues from mice transplanted with LLC1/OVA/GPC3 cloned C5 cell line Evaluation of (2)

(23-2-1) preparation of cell line and syngeneic tumor transplantation mouse model, and evaluation of infiltration of immune cells in tumor And methods for tumor cell CD39 and CD73 expression

In the course of this study, the internally established mouse lung cancer cell lines LLC1/OVA/GPC3 clone C5 cells and hCD137 KI mice were used (see above for further discussion of examples of cancer)As described in reference example 6-3). LLC1/OVA/GPC3 clone C5 cell line was implanted subcutaneously into the abdomen of mice until the tumor volume reached about 500mm ₃ Or greater. Samples were taken 14 days after transplantation. After sampling, tumor tissue was removed and a tumor cell fraction or lymphocyte fraction was prepared by the method described in detail below, further, CD39 or CD73 was present in tumor cells, CD4 ⁺ T cell, CD8 ⁺ Expression on T cells, and non-T cell fractions, was assessed using Flow Cytometry (FCM).

(23-2-2) tumor tissue was excised from LLC1/OVA/GPC3 clone C5 cell line-transplanted mice and tumor cells were prepared Cellular or lymphocyte fractions

For the excised tumor tissue, cell preparation of the tumor cell fraction or lymphocyte fraction was performed. A tumor dissociation kit, mouse (Miltenyi Biotec), was used for cell preparation, and the obtained tumor cell fraction or lymphocyte fraction was used for FCM analysis.

(23-2-3) evaluation of tumor cell grade by FCM from mice transplanted with LLC1/OVA/GPC3 clone C5 cell line CD39 and CD73 expression in fractions or lymphocyte fractions

For evaluation by FCM, CD39 and CD73 on tumor cells, CD4, were used ⁺ T cell, CD8 ⁺ Positive rate on T cell and non-T cell fractions. For this purpose, anti-CD 45 antibodies (BD Biosciences), anti-CD 3 antibodies (BioLegend), anti-CD 8 antibodies (BioLegend), anti-CD 4 antibodies (BioLegend), anti-CD 39 antibodies (BioLegend) and anti-CD 73 antibodies (BioLegend) were used in the FCM analysis. BD LSR Fortessa X-20 (BD Biosciences) was used for the measurements.

Tumor cells, CD4, as a result of analysis of CD39 expression in each fraction ⁺ T cell, CD8 ⁺ CD39 expression on T cells and non-T cells is such that in some tumor cells and in almost all CD4 cells ⁺ T cell, CD8 ⁺ Expression was observed in both T cells and non-T cells (fig. 97). Next, as a result of analyzing CD73 expression in each fraction, some tumor cells, some CD8 ⁺ T thinExpression was observed in cells and some non-T cells, while in almost all CD4 cells ⁺ Expression was observed in both T cells (fig. 97). A representative example of each data is shown in fig. 98.

(23-3) evaluation of expression of CD39 and CD73 in tumor tissues from MC38 cell line-transplanted mice

(23-3-1) preparation of cell line and syngeneic tumor transplantation mouse model, and evaluation of infiltration of immune cells in tumor And methods for tumor cell CD39 and CD73 expression

In this study, MC38 cells, a mouse colon cancer cell line licensed by the national cancer institute, and hCD137KI mice (described in reference example 6-3) were used. The MC38 cell line was transplanted subcutaneously to the abdomen of mice when the tumor volume reached 163-204mm ³ Mice were grouped at time. Grouping was performed 14 days after transplantation, and sampling was performed 16 days after transplantation. Tumor tissue was removed by the method described in detail below to prepare a tumor cell fraction or lymphocyte fraction, and further using Flow Cytometry (FCM), CD39 or CD73 was evaluated at tumor cells, CD4 ⁺ T cell, CD8 ⁺ Expression on T cells and on non-T cell fractions.

(23-3-2) tumor tissue was excised from MC38 cell line-transplanted mice and tumor cell fractions or lymphocytes were prepared Cellular fraction

(23-3-3) evaluation of tumor cell fraction or lymphocyte fraction from MC38 cell line-transplanted mice by FCM CD39 and CD73 expression of

For evaluation by FCM, CD39 and CD73 on tumor cells, CD4, were used ⁺ T cell, CD8 ⁺ Positive rate on T cell and non-T cell fractions. For this purpose, in FCM analysisanti-CD 45 antibodies (BD Biosciences), anti-CD 3 antibodies (BioLegend), anti-CD 8 antibodies (BioLegend), anti-CD 4 antibodies (BioLegend), anti-CD 39 antibodies (BioLegend) and anti-CD 73 antibodies (BioLegend) were used. BD LSR Fortessa X-20 (BD Biosciences) was used for measurement.

Tumor cells, CD4, as a result of analysis of CD39 expression in each fraction ⁺ T cell, CD8 ⁺ CD39 expression on T cells and non-T cells is such that on some CD4 s ⁺ T cells and some CD8 ⁺ Expression was observed in T cells as well as in most non-T cells (figure 99). Subsequently, as a result of analyzing CD73 expression in each fraction, in some tumor cells and in almost all CD4 cells ⁺ T cells and CD8 ⁺ Expression was observed in both T cells (fig. 99). Little expression was observed in non-T cells. A representative example of each datum is shown in graph 100.

Based on the above results, it was revealed that in tumor tissues from mice transplanted with the mouse colon cancer cell line MC38 cells and the mouse lung cancer cell line LLC1/OVA/GPC3 clone C5 cells, CD39 and CD73 were widely expressed in the intratumoral infiltrating immune cells, in addition to the tumor cells. The membrane-type enzymes CD39 and CD73 are known as enzymes that decompose ATP into adenosine. The conversion antibody showed antigen binding ability in the presence of ATP, ADP and AMP, but not in the presence of adenosine (see WO 2020/032230). However, as has been demonstrated, even in tumor tissues in which CD39 and CD73 are widely expressed, for example, tumor tissues from mice transplanted with MC38 cells, a mouse colon cancer cell line, and LLC1/OVA/GPC3, a mouse lung cancer cell line, and C5 cells, the crossover antibody binds to immune cells (example 21) and exhibits an anti-tumor effect (examples 1 to 10), showing that the crossover antibody can act on tumor tissues without being affected by the expression of CD39 and CD 73.

Example 24 homologous tumor transplantation model by Using hCD137KI mice (of LLC1/OVA clone 17 cells) Subcutaneous transplantation), evaluation of anti-tumor activity of anti-CD 137 antibody as a single agent or in combination with anti-PD-L1 antibody

(24-1) tumor cell line and syngeneic tumor transplantation mouse modelPreparation of form A551-MB110/B379-ml0r Evaluation of antitumor Activity (alone or in combination with anti-mouse PD-L1 antibody)

In this study, the internally established mouse lung cancer cell lines LLC1/OVA clone 17 cells (as described in example 23, hereinafter referred to as LLC1/OVA cell line in this example) and hCD137KI mice (as described in reference examples 6-3) were used. The LLC1/OVA cell line is transplanted to the abdomen of the mouse subcutaneously when the tumor volume reaches 79-199mm ³ The model is considered to be established. After modeling, mice transplanted with LLC1/OVA cell line were grouped and then the conversion antibody A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody (BioXcell, inc., clone 10F.9G2) were administered at the doses indicated in Table 81. As positive control substances, non-switching antibody UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody were administered. The transformant antibody A551-MB110/B379-ml0r or the non-transformant antibody UreH-MB110/UreL-mk1 was administered twice by the tail vein route 10 days and 13 days after transplantation. Administration of anti-mouse PD-L1 antibody was performed twice by intraperitoneal route 10 days and 13 days after transplantation. PBS containing 0.05% tween 20 was used for vehicle control.

[ Table 81]

Tumor volumes were measured 10, 13, 17 and 20 days after transplantation.

Tumor Volume (TV) was calculated by the following formula.

Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

TV Change (mm) ³ ) = tumor volume 20 days after transplantation-tumor volume at the time of grouping

As a result, the combined group of the conversion antibody A551-MB110/B379-ml0 and the anti-mouse PD-L1 antibody, the combined group of the non-conversion antibody UreH-MB110/UreL-mk1 and the anti-mouse PD-L1 antibody, and the single drug group of the conversion antibody A551-MB110/B379-ml0r showed almost the same tumor growth inhibition effect against LLC1/OVA tumors.

Specifically, the combined group of the switch antibody a551-MB110/B379-ml0 and the anti-mouse PD-L1 antibody exhibited a tumor growth inhibition rate of 95%, whereas the combined group of the non-switch antibody urea h-MB 110/urea L-mk1 and the anti-mouse PD-L1 antibody exhibited a tumor growth inhibition rate of 93%, and the unit group of the switch antibody a551-MB110/B379-ml0r exhibited a tumor growth inhibition rate of 101% (fig. 101, table 82).

From the above results, it was revealed that the combination of the diabody A551-MB110/B379-ml0 and the anti-mouse PD-L1 antibody had a tumor growth inhibitory effect similar to that of the combination of the non-diabody UreH-MB110/UreL-mk1 and the anti-mouse PD-L1 antibody (FIG. 101, table 82).

[ Table 82]

Tumor growth inhibition (%) 20 days after transplantation

Example 25 anti-syngeneic tumor cell transplantation model (LLC 1/OVA cells) by Using hCD137KI mice Assessment of systemic effects of CD137 antibodies as a single agent or in combination with anti-PD-L1 antibodies

(25-1) preparation of tumor cell line and syngeneic tumor transplanted mouse model, and method for evaluating intratumoral effects

In this study, the internally established mouse lung cancer cell lines LLC1/OVA cells (as described in example 23) and hCD137KI mice (as described in reference examples 6-3) were used. The LLC1/OVA cell line is transplanted to the abdomen of mouse subcutaneously until the tumor volume reaches 91-205mm ³ The model is considered to be established. After modeling, mice transplanted with LLC1/OVA cell line were grouped and then the conversion antibody A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody (BioXcell, inc., clone 10F.9G2) were administered at the doses indicated in Table 83.

As positive control substances, non-transformed anti-CD 137 antibody UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody were administered. On days 14 and 17 after transplantation, the crossover antibody A551-MB110/B379-ml0r or the non-crossover antibody UreH-MB110/UreL-mk1 was administered by the tail vein route twice. Administration of anti-mouse PD-L1 antibody was performed twice by intraperitoneal route 14 days and 17 days after transplantation. PBS containing 0.05% tween 20 was used for vehicle control.

(25-2) excision of lymph nodes from tumor regions from mice transplanted with LLC1/OVA cell line and preparation of lymphocyte fractions

For the excised tumor area lymph nodes, lymphocyte fractions obtained by using a cell filter after tissue destruction were used. The obtained lymphocyte fraction derived from lymph nodes in the tumor area was subjected to Flow Cytometry (FCM) analysis.

[ Table 83]

(25-3) evaluation of immune cells in LLC1/OVA cell line transplanted mice by Flow Cytometry (FCM) analysis Activation of

FCM was used to analyze T cell activation from lymph nodes in the tumor area. CD8 ⁺ The expression rate of activation markers in T cells was used for evaluation by FCM. In the FCM analysis, anti-CD 45 antibodies (BD Biosciences), anti-CD 3 antibodies (BioLegend), anti-CD 8 antibodies (BioLegend), anti-KLRG-1 antibodies (BD Biosciences), anti-ICOS antibodies (BioLegend) and anti-PD-1 antibodies (BioLegend) were used. BD LSR Fortessa X-20 (BD Biosciences) was used for measurement.

CD8 in the A551-MB110/B379-ml0r monotherapy group, the anti-mouse PD-L1 antibody monotherapy group, and the A551-MB110/B379-ml0r and anti-mouse PD-L1 antibody combination group as a result of lymph node evaluation in the tumor region ⁺ There was no increase in all expression rates and absolute numbers of KLRG-1, ICOS, PD-1 in T cells (FIGS. 102 and 103). On the other hand, in the UreH-MB110/UreL-mk1 single drug group and the combination of UreH-MB110/UreL-mk1 and anti-mouse PD-L1 antibody To a significant increase (fig. 102 and 103).

From this result, as shown in example 24, even though both the switch antibody A551-MB110/B379-ml0r and the non-switch antibody UreH-MB110/UreL-mk1 showed similar tumor growth inhibitory effects even when used in combination with the anti-mouse PD-L1 antibody, the effect of the switch antibody A551-MB110/B379-ml0r on T cells in lymph nodes in tumor regions of normal organs was extremely limited when used as a single drug and in combination with the anti-mouse PD-L1 antibody, compared to the non-switch antibody UreH-MB110/UreL-mk 1.

Example 26 model of homologous tumor cell transplantation by Using hCD137KI mice (quiescence of LLC1/OVA cells) Intravenous administration) of anti-CD 137 antibody

(26-1) preparation of tumor cell line and syngeneic tumor transplantation mouse model and evaluation of antitumor activity

In this study, the internally established mouse lung cancer cell lines LLC1/OVA clone 17 cells (as described in example 23) and hCD137KI mice (as described in reference examples 6-3) were used. LLC1/OVA clone 17 cell line was transplanted into the tail vein of mice and divided into groups 7 days after transplantation. As shown in Table 84 below, A551-MB110/B379-ml0r antibody was administered to the tail vein of mice 7 days and 14 days after transplantation. PBS containing 0.05% tween 20 was used as vehicle control. Body weight was measured 18 days after tumor implantation and lung weight was measured after euthanasia under exsanguination and anesthesia. 10 normal mice without cell transplantation were sampled by the same method on the same day. Lung weight was calculated as a lung weight value per 100 grams of body weight.

[ Table 84]

As a result, the group administered with the transition antibody A551-MB110/B379-ml0r had significantly suppressed lung weight compared to the group administered with vehicle (FIG. 104, table 85). Thus, the switch antibody A551-MB110/B379-ml0r was shown to have a pharmacological effect not only on subcutaneously transplanted tumors (as described in example 24) but also on micrometastases of the lung. One mouse in the vehicle-administered group died 11 days after tumor transplantation. Small traces and bumps indicative of tumors were found in the lungs of dead mice.

[ Table 85]

Lung weight 18 days post-transplant

Reference example 1: antigen preparation

(1-1) preparation of extracellular region of human CD137

The extracellular domain of human CD137 (also known as hCD 137) was prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding a histidine tag and a gene fragment encoding a specific sequence to which biotin was added (AviTag sequence, SEQ ID NO: 86) were ligated downstream of the gene fragment encoding the extracellular region of human CD 137. A gene fragment encoding a protein (human CD137 or hCD137-HisBAP, SEQ ID NO: 87) in which the extracellular domain of human CD137, a histidine tag, and Avitag were ligated was incorporated into an animal cell expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At this time, a plasmid vector containing a gene expressing EBNA1 (SEQ ID NO: 88) was transfected at the same time. The cells transfected with the genes according to the above procedure were treated at 37 ℃ 8% ₂ Performing downward culture; the protein of interest (extracellular domain of human CD 137) was then secreted into the culture supernatant. The cell culture medium was filtered through a 0.22 μm filter to obtain a culture supernatant.

The culture supernatant was applied to HisTrap-HP (GE healthcare) and the extracellular domain of human CD137 was bound to the column. Human CD137 extracellular domain was eluted using a solution of 20mM sodium phosphate, 500mM sodium chloride and 500mM imidazole (pH 7.5). Aggregates were then removed by gel filtration chromatography using Superdex 200/600 (GE healthcare) to produce purified human CD137 extracellular domain.

The concentration of human CD137 was calculated based on the amino acid sequence excluding the signal sequence deduced from SEQ ID NO:87 (SEQ ID NO: 183) using the method of Pace et al (Pace, C.N. et al, protein Science (Protein Science), 1995.

(1-2) preparation of the ectodomain of human CD137 (hCD 137 (FXa digested))

The human CD137 ectodomain, also known as hCD137 (FXa digested), was prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding a factor Xa cleavable sequence and a gene fragment encoding a constant region of an antibody are linked downstream of a gene fragment encoding an extracellular region of human CD 137. A gene fragment encoding a protein (hCD 137-F-Fc, SEQ ID NO: 89) in which the extracellular domain of human CD137, the factor Xa cleavable sequence, and the antibody constant region are ligated is incorporated into an animal cell expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At this time, a plasmid vector containing the EBNA1 (SEQ ID NO: 88) -encoding gene was transfected at the same time. The cells transfected with the gene according to the above procedure are treated at 37 ℃,8% ₂ Performing downward culture; hCD137-F-Fc was secreted into the culture supernatant. The cell culture medium was filtered through a 0.22 μm filter and the culture supernatant was collected.

The culture supernatant was applied to a column filled with protein A (MabSelect SuRe, GE medical treatment). hCD137-F-Fc was bound to the column and eluted with 50mM acetic acid solution. After neutralizing the eluate with 1M Tris-HCl, pH 8.0, the solvent for hCD137-F-Fc was replaced with 20mM Tris, 100mM sodium chloride, 2mM calcium chloride (pH 8.0). Next, factor Xa protease (NEW ENGLAND BioLabs, cat. No. P8010L) was added and hCD137-F-Fc was digested. Protease inhibitors (DNS-GGACK, calbiochem, cat # 251700) were then added to stop the reaction. 5M sodium chloride was added to the protease reaction solution, and the total volume of the prepared solution was applied to HiTrap benzamidine (GE medical, catalog No. 17-5143-01). The column filtered solution was further applied to a protein a column (MabSelect SuRe, GE medical care) and the pass fractions were collected. Aggregates that passed through the fractions were removed by gel filtration chromatography using Superdex 200Increate, 10/30 (GE healthcare, catalog No. 28990944) to give a purified extracellular domain of human CD 137.

(1-3) preparation of biotinylated Fc fusion human CD137

Biotinylated Fc fusion human CD137 (also known as "biotinylated hCD137-Fc" or "Bio-hCD137-Fc" or hCD 137-Fc-Bio) was prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding an antibody constant region and a gene fragment encoding a specific sequence (AviTag sequence, SEQ ID NO: 86) to which biotin was added were ligated downstream of the gene fragment encoding the extracellular region of human CD 137. A gene fragment in which a protein encoding the extracellular region of human CD137, an antibody constant region and Avitag (Fc-fused human CD137, SEQ ID NO: 90) had been ligated was incorporated into an animal expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 88) and a gene expressing biotin ligase (BirA, SEQ ID NO: 91) were transfected at the same time, and biotin was added to biotinylate Fc-fused human CD 137. Culturing the cells transfected with the gene according to the above procedure at 37 ℃ under 8% CO2; the protein of interest (biotinylated Fc fusion human CD 137) was then secreted into the culture supernatant. The cell culture was filtered through a 0.22 μm filter to obtain a culture supernatant.

The culture supernatant was applied to a column filled with protein a (MabSelect SuRe, GE medical) and biotinylated Fc-fusion human CD137 was bound to the column. Biotinylated Fc-fusion human CD137 was eluted using 50mM acetic acid solution. Aggregates were then removed by gel filtration chromatography using Superdex 200, 26/600 (GE healthcare) and purified biotinylated Fc fusion human CD137 was obtained.

(1-4) preparation of Fc fusion human CD137

Fc-fused human CD137 (also known as hCD 137-Fc) was prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding an antibody constant region and a gene fragment encoding a specific sequence (AviTag sequence, SEQ ID NO: 86) to which biotin was added were ligated downstream of the gene fragment encoding the extracellular region of human CD137. A gene fragment encoding a protein (Fc-fused human CD137, SEQ ID NO: 90) in which the extracellular region of human CD137, the antibody constant region, and Avitag have been linked was incorporated into an animal expression vector. The constructed plasmid vector was transfected into FreeStyle293F cells (Invitrogen) using 293-fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 88) was simultaneously transfected. The cells transfected with the gene according to the above procedure were cultured at 37 ℃ in 8% CO2, and the target protein (Fc-fused human CD 137) was secreted into the culture supernatant. The cell culture was filtered through a 0.22 μm filter to obtain a culture supernatant.

The culture supernatant was applied to a column filled with protein a (MabSelect SuRe, GE medical) and Fc-fused human CD137 was bound to the column. Fc-fused human CD137 was eluted using 50mM acetic acid solution. Next, aggregates were removed by gel filtration chromatography using Superdex 200, 26/600 (GE healthcare) to obtain purified Fc fusion human CD137.

(1-5) preparation of biotinylated Fc fusion monkey CD137

Biotinylated Fc fusion monkey CD137 (also known as "cyCD 137-Fc-BAP") was prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding an antibody constant region and a gene fragment encoding a specific sequence to which biotin was added (AviTag sequence, SEQ ID NO: 86) were ligated downstream of the gene fragment encoding the extracellular region of monkey CD137. A gene fragment encoding a protein in which the extracellular region of monkey CD137, the antibody constant region, and Avitag have been ligated (Fc fusion monkey CD137, SEQ ID NO: 92) was incorporated into an animal expression vector.

The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 88) and a gene expressing biotin ligase (BirA, SEQ ID NO: 91) were transfected at the same time; and biotin was added to label Fc-fused monkey CD137 with biotin. Culturing the cells transfected with the gene according to the above procedure at 37 ℃,8% in CO2; the protein of interest (biotinylated Fc fusion monkey CD 137) was then secreted into the culture supernatant. The cell culture was filtered through a 0.22 μm filter to obtain a culture supernatant.

The culture supernatant was applied to a column filled with protein a (MabSelect SuRe, GE medical care) and biotinylated Fc fusion monkey CD137 was bound to the column. Biotinylated Fc fusion monkey CD137 was eluted using 50mM acetic acid solution. Aggregates were then removed by gel filtration chromatography using Superdex 200 increments 10/300 (GE healthcare) and purified biotinylated Fc fusion monkey CD137 was obtained.

Reference example 2: obtaining ATP-dependent CD137 antibodies

(2-1) obtaining of antibody having Small molecule-dependent antigen binding Activity Using ATP through rationally designed library (Small molecule converting antibody) (1)

(2-1-1) panning

Antibodies exhibiting binding activity to an antigen in the presence of adenosine triphosphate (adenosine 5' -triphosphate; ATP) were obtained from rationally designed antibody phage display libraries constructed in the prior patent WO 2015/083764. Note that an antibody having a small molecule-dependent antigen (e.g., CD 137) binding activity may be referred to as a "switch antibody" or a "small molecule switch antibody", and an antibody having an ATP-dependent antigen (e.g., CD 137) binding activity is referred to as a "switch antibody" or an "ATP switch antibody". To obtain, phage presenting antibodies that exhibit binding activity in the presence of ATP to the antigen captured on the beads are harvested. Subsequently, in the absence of ATP, the phage was collected from the eluate eluted from the beads.

Phage were generated from E.coli harboring the constructed phage-display phagemid in a conventional manner. Specifically, E.coli carrying the constructed phagemid vector was infected with M13KO 7. Delta. PIII (referred to as "Superphage") (PROGEN Biotechnik), and the phage were collected from the supernatant cultured overnight at 30 ℃. Obtaining a phage library solution by diluting a phage population with Tris-buffered saline solution (TBS), wherein the phage population is precipitated by adding 2.5M NaCl/10% PEG to an E.coli culture in which phage production is performed. BSA was then added to the phage library solution at a final concentration of 4%. Panning was performed using the antigen immobilized on the magnetic beads. For the magnetic beads, sera-Mag NeutrAvidin beads (Sammer Feishila technology) or Dynabeads M-280 StreptAvidin (Life technologies, USA) were used. As the antigen, biotinylated adenosine triphosphate (5' -adenosine triphosphate; ATP) or hCD137-Fc-Bio (SEQ ID NO: 90) or Bio-hCD137 (SEQ ID NO: 89) purchased from Jena bioscience was used, wherein the hCD137-Fc-Bio (SEQ ID NO: 90) or Bio-hCD137 (SEQ ID NO: 89) was biotinylated from hCD137 (FXa digested) using previously determined weightless NHS-PEO 4-biotin (PIERCE) prepared in reference examples 1-2 and 1-3.

Panning was performed to efficiently obtain small molecule dependent switch antibodies that could exert a switching effect in cancer tissues. In particular, with reference to the method shown in the previous patent WO2015/083764, panning is performed to enrich for antibodies that bind to the antigen in the presence of small molecule ATP and do not bind to the antigen in the absence of ATP. In round 1, panning was performed for all biotinylated hCD137 (Bio-hCD 137), hCD137-Fc-Bio and biotinylated ATP (Bio-ATP) using this method: the biotinylated antigen is immobilized on magnetic beads (referred to as the "bead immobilization method"), and then a prepared phage library solution is added. For Bio-ATP panning was performed to enrich for antibodies that can bind antigen (Bio-ATP) in the absence of ATP as a small molecule compound, with reference to the method described in the above-mentioned prior patent WO 2015/083764. For hCD137-Fc-Bio, 4nmol of non-biotinylated human IgG1Fc region was added to remove antibody bound to the Fc region. The harvested phage was added to E.coli strain ER2738 to infect E.coli with phage, then the harvested E.coli was infected with super phage, and phage were harvested from the supernatant that was cultured overnight at 30 ℃.

Starting from round 2, only biotinylated hCD137 (Bio-hCD 137) and hCD137-Fc-Bio were subjected to panning to enrich for antibodies that bind to the antigen in the presence of ATP and do not bind to the antigen in the absence of ATP in the bead solid phase method, see the method shown in prior patent WO 2015/083764. For hCD137-Fc-Bio in both cases, 4nmol of non-biotinylated human IgG1Fc region was added to remove antibody bound to the Fc region. Similar panning was repeated until round 5 to enrich for the antibody sequence of interest.

(2-1-2) evaluation of binding Activity in the Presence and absence of ATP by phage ELISA

Phage-containing culture supernatants were harvested from individual colonies of E.coli obtained by the above-described Methods using conventional Methods (Methods mol. Biol.), (2002) 178, 133-145). NucleoFast 96 (MACHEREY-NAGEL) was used to ultrafilter the harvested culture supernatant. The flow-through was removed by centrifugation (4,500g, 45 min) of NucleoFast 96 (100. Mu.L of culture supernatant had been added to each well). By adding 100. Mu.L of H to each well ₂ O and the NucleoFast 96 is washed again by centrifugation (4,500g, 30 minutes). Finally, 100. Mu.L of TBS was added and the phage solution contained in the supernatant in the wells of NucleoFast 96, which had been left to stand at room temperature for 5 minutes, was recovered.

ELISA was performed on the purified phage with TBS or ATP/TBS added by the following procedure. 384-well streptavidin-coated microplates (Greiner) were coated overnight with 10. Mu.L TBS containing biotinylated antigens (Bio-hCD 137, hCD137-Fc-Bio, and Bio-Fc) generated in reference example 1. After removing biotinylated antigen not bound to the plate by washing each well of the plate with Tris buffered saline solution (TBST) with tween 20, the wells were blocked with 80 μ Ι _ of 2% skim milk TBS for 1 hour or longer. 2% of skim milk-TBS was removed by TBST washing, and then antibody-presenting phage were bound to biotinylated antigen present in each well in the absence and presence of ATP by allowing the plate to sit at room temperature for 1 hour with the prepared purified phage added to each well. To each well washed with TBST or ATP/TBST, HRP-conjugated anti-M13 antibody (GE medical 27-9421-01) diluted with TBS or ATP/TBS was added and the plate was incubated for 1 hour. After washing with TBST or ATP/TBST, the color development of the solution in each well to which the TMB single solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm.

As a result, several antibodies with altered binding activity to Bio-hCD137 or hCD137-Fc-Bio in the presence and absence of ATP were identified.

The phage ELISA results using the clones after 4 th and 5 th panning are shown in table 29.

Here, a clone having an absorbance in the presence of ATP of 0.2 or more and an S/N ratio of absorbance in the presence/absence of antigen of more than 2 was determined as a positive clone. In addition, among the positive clones, the clone having an absorbance S/N higher than 2 in the presence/absence of ATP was judged as a clone having ATP-dependent antigen binding activity (switch clone).

[ Table 29]

(2-1-3) sequence analysis of transition antibody whose antigen-binding activity is changed in the Presence/absence of ATP

From the phage ELISA results, the nucleotide sequence of the gene amplified from the clone having ATP-dependent antigen-binding activity (switch clone) using the specific primer pBAD-F, G1seq-R was analyzed. As a result of the analysis, the nucleotide sequence of the clone judged to bind to human CD137 in the presence of ATP and not to bind to human CD137 in the absence of ATP was obtained.

(2-2) obtaining antibodies binding to antigens in the presence of small molecules from rationally designed libraries using ATP (2)

(2-2-1) panning

Antibodies exhibiting binding activity to antigen in the presence of ATP are obtained from rationally designed antibody phage display libraries constructed in prior patent WO 2015/083764. To obtain, phage presenting antibodies showing binding activity to an antigen in the presence of ATP are recovered, and then phage are recovered from the eluate eluted from the beads in the absence of ATP.

Phage were generated in a conventional manner from E.coli harboring the constructed phage-display phagemid. Specifically, E.coli carrying the constructed phagemid vector was infected with M13KO7TC (WO 2015046554A 1) or M13KO 7. Delta. PIII (super phage) (PROGEN Biotechnik), and the phage was recovered from the supernatant cultured overnight at 30 ℃. Obtaining a phage library solution by diluting a phage population with TBS, wherein said phage population is precipitated by adding 2.5M NaCl/10% PEG to an E.coli culture in which phage production is performed. BSA was then added to the phage library solution at a final concentration of 4%. Panning was performed using the antigen immobilized on the magnetic beads. Neutravidin-coated beads (Sera-Mag express beads (SpeedBead) neutravidin-coated), neutravidin beads (TAMAGAGAWA SEIKI) or Dynabeads MyOne Streptavidin T1 (Sammer Feishal technology) were used as the magnetic beads. As the antigen, hCD137-Fc-Bio or Bio-hCD137 produced in reference example 1 was used.

Panning is performed to efficiently obtain small molecule switch antibodies that are dependent on small molecules that can switch in cancer tissues. In particular, panning is performed with reference to the method described in the previous patent WO2015/083764 to enrich for antibodies that bind to antigen in the presence of small molecules of adenosine triphosphate (adenosine 5' -triphosphate; ATP) and do not bind to antigen in the absence of ATP. For bio-hCD137, the following procedure was performed: a method of immobilizing a biotinylated antigen on a magnetic bead in advance and then adding the prepared phage library solution (referred to as a "bead solid phase method") and a method of mixing the prepared phage library solution and a biotinylated antigen in advance and then adding a magnetic bead (referred to as a "liquid phase method"). For hCD137-Fc-Bio, 4nmol of non-biotinylated human IgG1Fc region was added to remove antibody bound to the Fc region, and panning was performed only in the liquid phase method. The collected phages were added to the E.coli strain ER2738 and allowed to infect E.coli, which was then infected with M13KO7TC (WO 2015046554A 1) or M13KO 7. Delta. PIII (super phage) (PROGEN Biotechnik), and the phages were collected from the supernatant after overnight culture at 30 ℃. Similar panning was repeated until round 5.

(2-2-2) evaluation of binding in the Presence and absence of ATP by phage ELISAActivity of

Phage-containing culture supernatants were harvested from individual colonies of E.coli obtained by the method described above after each round using conventional Methods (Methods mol. Biol.), (2002) 178, 133-145). NucleoFast 96 (MACHEREY-NAGEL) was used to ultrafilter the harvested culture supernatant. The flow-through was removed by centrifugation (4,500g, 45 min) of NucleoFast 96 (100. Mu.L of culture supernatant had been added to each well). Washed again by centrifugation (4,500g, 30 min) where 100. Mu.L H was added to each well ₂ NucleoFast 96 of O. Finally, 100. Mu.L of TBS was added and the phage solution contained in the supernatant of the NucleoFast 96 well which had been left to stand at room temperature for 5 minutes was recovered.

ELISA was performed on the purified phage with TBS or ATP/TBS added by the following procedure. StreptaWell 96 microtiter plates (Roche) were fixed overnight with 100. Mu.L TBS containing biotinylated antigen (hCD 137-Fc-Bio or Bio-hCD 137) generated in reference example 1. After removing biotinylated antigen not bound to the plate by washing each well of the plate with TBST, the wells were blocked with 250 μ L of 2% skim milk-TBS for 1 hour or more. 2% of skim milk-TBS was removed, and then antibody-presenting phage were bound to biotinylated antigen present in each well, in the presence and absence of ATP, by allowing the plate with the prepared purified phage added to each well to stand at 37 ℃ for 1 hour. To each well washed with TBST or ATP/TBST, HRP-conjugated anti-M13 antibody (GE medical 27-9421-01) diluted in TBS or ATP/TBS was added and the plate was incubated for 1 hour. After washing with TBST or ATP/TBST, the solution color development in each well to which TMB single solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm.

For example, phage ELISA results using clones after panning round 5 are shown in table 30.

Here, a clone having an absorbance of 0.2 or more in the presence of ATP and an S/N ratio of greater than 2 in the presence/absence of antigen was determined as a positive clone. In addition, among the positive clones, those having an S/N ratio of absorbance in the presence/absence of ATP of more than 2 were determined as clones having ATP-dependent antigen-binding activity (switch clones).

[ Table 30]

(2-2-3) sequence analysis of transition antibody whose antigen-binding activity is changed in the Presence/absence of ATP

The nucleotide sequence of the gene amplified from the clone having ATP-dependent antigen-binding activity (switch clone) based on the phage ELISA result was analyzed using the specific primers pBAD-F, G1 seq-R. As a result of the analysis, the nucleotide sequence of the clone judged to bind to human CD137 in the presence of ATP and not to bind to human CD137 in the absence of ATP was obtained.

(2-3) selection of the conversion antibody

From the analysis results of reference examples 2-1-3 and 2-2-3, 17 samples were selected from the clones judged to have ATP-dependent antigen-binding activity, and the clone names were reassigned as described in Table 31.

[ Table 31]

(2-4) expression and purification of a Trans-antibody having altered antigen-binding Activity in the Presence/absence of ATP

Genes encoding the antibody variable regions described in Table 31 obtained from rationally designed phage libraries were inserted into human IgG 1/lambda plasmids for expression in animals. Expressed using the following methodAn antibody. The prepared plasmid was introduced into human fetal kidney cell-derived FreeStyle 293-F (Invitrogen) by lipofection at 1.33x10 ⁶ Cell density of cells/mL was suspended in FreeStyle293 expression medium (Invitrogen), with 3mL seeded in each well of a 6-well plate. The rProtein A Sepharose was used in a manner known to those skilled in the art ^TM Fast Flow (Amersham biosciences) purified antibodies from culture supernatant in CO ₂ Incubator (37 ℃,8% CO) ₂ 90 rpm) for four days. The absorbance of the purified antibody solution at 280nm was measured using a spectrophotometer. From the obtained measurement values, the concentration of the purified antibody was calculated using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

(2-5) evaluation of anti-CD 137 antibodies identified by phage display

(2-5-1) expression and binding of antigen-binding switching antibody depending on the presence or absence of ATP and its metabolites Purification of

Genes encoding the variable regions of antibodies obtained from human rationally designed phage libraries were inserted into animal expression plasmids with a heavy chain constant region (SEQ ID NO: 93) fused to genes encoding Gly and Lys downstream of modified human IgG1 (P253) and a light chain constant region Lamlib chain (SEQ ID NO: 63). Clone names and sequence numbers are listed in table 32.

Clones evaluated

[ Table 32]

	Clone name	Heavy chain (full-Length) SEQ ID NO	Light chain (full length) SEQ ID NO
				1	dBBAT007-P253	94	113
2	dBBAT013-P253	95	114
				3	dBBAT015-P253	96	115
4	dBBAT017-P253	97	116
				5	dBBAT019-P253	98	117
6	dBBAT025-P253	99	118
				7	dBBAT029-P253	100	119
8	dBBAT031-P253	100	119
				9	dBBAT037-P253	101	120
10	dBBAT042-P253	102	121
				11	dBBAT053-P253	103	122
12	dBBAT056-P253	104	123
				13	dBBAT021-P253	105	124
14	dBBAT091-P253	106	125
				15	dBBAT112-P253	107	126
16	dBBAT118-P253	108	127
				17	dBBAT119-P253	109	128
18	dBBAT121-P253	110	129
				19	dBBAT122-P253	111	130
20	dBBAT134-P253	112	131

Antibodies are expressed and purified using methods known to those skilled in the art. The absorbance of the purified antibody solution at 280nm was measured using a spectrophotometer. From the obtained measurement values, the concentration of the purified antibody was calculated using the extinction coefficient calculated by the PACE method (Protein Science, 1995) 4, 2411-2423).

(2-5-2) evaluation of ATP, ADP and AMP binding to human CD137 by surface plasmon resonanceInfluence of

Biacore T200 (GE medical) was used to analyze the interaction of the antigen-antibody reaction between anti-CD 137 antibodies and hCD137 (FXa digestion). The anti-CD 137 antibody was captured on a sensor chip CM3 (GE medical treatment) on which an appropriate amount of protein G (CALBIOCHEM) was immobilized by an amine coupling method, and the hCD137 (FXa digestion) prepared in reference example 1-2 was allowed to interact. For the running buffer, 20mM ACES, 150mM NaCl, 2mM MgCl were used ₂ 0.05% tween 20 (pH 7.4), and 10mM glycine-HCl (pH 1.5) was used as a regeneration solution.

After capture of anti-CD 137 antibody suspended in TBS, 500nM hcd137 (FXa digest) was injected into each flow cell at a flow rate of 10 μ L/min for 3 minutes. This three minute period served as the binding phase for hCD137 (FXa digestion) and a two minute period of switching to running buffer after the binding phase was over served as the dissociation phase for hCD137 (FXa digestion). After completion of the dissociation phase, the regeneration solution was injected at a flow rate of 30. Mu.l/min for 30 seconds. The above is the cycle for measuring the binding activity of the anti-CD 137 antibody. The amount of binding of hCD137 (FXa digestion) interacting with the anti-CD 137 antibody during the binding phase is regulated by the amount of captured antibody. The binding amount (RU) of 1RU per capture ligand was shown using Biacore T200 evaluation software version 2.0, and values of the antibody capture amount (captured antibody amount) and the antigen binding amount were obtained. The amount of antigen bound is shown in table 33. Since the amount of antigen binding reflects the binding activity, it can be said that the dependence on small molecules is recognized when the value in the presence of small molecules (ATP, ADP or AMP) is higher than the value in the absence of small molecules. In particular, the larger the difference, the higher the dependence on small molecules.

[ Table 33]

(2-5-3) evaluation of binding Activity to monkey CD137 antigen

The binding of the obtained antibody to monkey CD137 was assessed by ELISA. The cyCD137-Fc-BAP prepared in reference example 1 was immobilized on Streptawell microtiter plates. After removing unbound antigen from the plate by washing each well of the plate with washing buffer, the wells were blocked with 150 μ L blocking buffer (2% BSA in TBS) for 1 hour or more. Blocking buffer was removed from each well and 100 μ L of purified antibody diluted in TBS at a final concentration of 1mM ADP or diluted in TBS at a final concentration of 1mM ADP was added to each well. The plates with added antibody were shaken at 600rpm for 1 hour. After washing with washing buffer containing 1mM ADP at final concentration (TBS containing 0.1% tween 20), AP-conjugated anti-human lambda antibody (BETHYL) diluted in TBS at 1mM ADP final concentration was added to each well. After one hour incubation and washing with washing buffer containing 1mM ATP final concentration, bluePhos phosphate substrate (KPL) was added. Color development was measured by absorbance at 600 nm. The increase rate of absorbance at an antibody concentration of 0. Mu.g/mL is shown in Table 34. Samples with concentration-dependent increases in absorbance ratios can be considered to bind monkey CD 137.

Absorbance ratio

[ Table 34]

(2-5-4) evaluation of CD137 agonist Activity Using Jurkat cells

GloResponse ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line (Promega) was used to measure antibody activity in vitro. To each well of 384 well plates, 10 μ L at 2X10 was added ⁶ concentration/mL Fc γ RIIB CHO-K1 cells (Promega) prepared in assay medium (99% RPMI,1% FBS). Subsequently, 10 μ L of antibody solution containing ADP, antibody solution containing ATP, or antibody solution without ATP or ADP was added to each well. Then, 10 μ L was applied at 2 × 10 ⁶ GloResponse/mL prepared with assay medium (99% RPMI,1% FBS) ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell lines were added to each well. The final concentration of ADP is 50 μ M; and the final concentration of ATP was 50. Mu.M. The plate was 5% CO ₂ The resulting mixture was left at 37 ℃ for 6 hours in an incubator, then left at room temperature for 15 minutes,then 30. Mu.L of Bio-Glo reagent was added to each well. The Bio-Glo luciferase assay system (buffer and substrate) was used for the Bio-Glo reagent. Subsequently, luminescence of each well was measured with a microplate reader. The luminescence value of each well was divided by the luminescence value of a well to which no antibody was added to obtain a luminescence magnification, which was used as an index for evaluating the activity of each antibody.

The results obtained are shown in FIGS. 22 and 23. From fig. 22 and 23, it was confirmed that all antibodies except NS1-P253 (non-switch antibody) showed human CD137 agonist activity in an ATP and ADP dependent manner.

(2-6) evaluation of in vitro CD137 agonist Activity of the transformed antibody obtained by human T cell panning

(2-6-1) expansion of human T cell culture

Human peripheral blood mononuclear cells isolated from blood samples of healthy volunteers were used. 50mL of blood was mixed with 0.5mL of heparin and further diluted with 50mL of PBS. Human peripheral blood mononuclear cells were isolated by the following two steps. In the first step, leucosep (greiner bio-one) supplemented with Ficoll-Paque PLUS (GE medical) was centrifuged at 1000 Xg for one minute at room temperature, then PBS-diluted blood was added and centrifuged at 400 Xg for 30 minutes at room temperature. In the second step, the buffy coat was collected from the tube after centrifugation and then washed with 60ml PBS (Wako). The culture of T cells was then expanded using a T cell activation/expansion kit/human (MACS Miltenyi biotec).

(2-6-2) evaluation of CD137 agonist Activity in vitro Using human T cells

2x10 ⁴ Human peripheral blood monocyte-derived T cells and 2x10 ⁴ REC-1 cells were suspended in 100 μ L of RPMI1640 medium containing 30U/mL human IL-2 (SIGMA), 10ng/mL PMA (SIGMA), 0.5 μ g/mL ionomycin, 500 μ M ADPbetaS (Sigma) and 10% FBS (Sigma) and plated in a 96-well flat-bottom plate (Corning) with control antibodies IC17HdK-hIgG1/IC17L-k0, NS1-P253, or any of the clones described in Table 32, wherein the control antibodies were antibodies with ATP-independent human CD137 binding activity (referred to as ATP-independent antibodies in the entire examples and reference examples) (referred to as "Corning" throughout the examples and reference examples) A "non-switching antibody" or a "non-switching CD137 antibody"). NS1-P253, IC17HdK-hIgG1/IC17L-k0 and the clones described in Table 32 were evaluated at 10. Mu.g/mL. IFN- γ production in ADPbetaS minimal medium was also assessed. Shaking the plate, then at 5% CO ₂ The incubator was left at 37 ℃ for 72 hours. Subsequently, the culture supernatant was harvested, and the amount of IFN-. Gamma.contained in the culture supernatant was quantified using a human IFN-. Gamma.ELISA development kit (PeproTech). The ELISA was performed according to the instructions of the kit manufacturer (PeproTech). The absorbance measurements were performed with EnVision (PerkinElmer).

The results are shown in FIG. 24.

dBAT 007-P253, dBAT 013-P253, dBAT 015-P253, dBAT 019-P253, dBAT 021-P253, dBAT 025-P253, dBAT 031-P253, dBAT 042-P253, dBAT 056-P253, dBAT 118-P253, dBAT 121-P253, dBAT 122-P253, and dBAT 119-P253 have been identified to show human CD137 agonist activity in an ADPbaSet dependent manner. ADPbetaS is an analogue of ADP, which is less susceptible to hydrolysis than ADP. This suggests that these human CD137 conversion antibodies may exhibit human CD137 agonist activity in a manner that is dependent on small molecules (e.g., ATP, ADP, or AMP).

(2-6-3) evaluation of CD137 agonist Activity in vitro Using human T cells (2)

2x10 ⁴ Human peripheral blood monocyte-derived T cells and 2x10 ⁴ REC-1 cells were suspended in 100. Mu.L RPMI1640 medium containing 30U/mL human IL-2 (SIGMA), 10ng/mL PMA (SIGMA), 0.5. Mu.g/mL ionomycin, 500. Mu.M ADPbetaS (Sigma) and 10% FBS (Sigma) and plated into 96-well flat-bottom plates (Corning) with NS1-P253 (non-converting antibody) or dBAT 119-P253. NS1-P253 and dBAT 119-P253 were evaluated at 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 μ g/mL. Shaking the plate, then at 5% CO ₂ The incubator was left at 37 ℃ for 72 hours. Subsequently, the culture supernatant was harvested, and the amount of IFN-. Gamma.contained in the culture supernatant was quantified using a human IFN-. Gamma.ELISA development kit (PeproTech). The ELISA was performed according to the instructions of the kit manufacturer (PeproTech). The absorbance measurements were performed with EnVision (PerkinElmer).

The results are shown in FIG. 25.

It was demonstrated that dBBAT119-P253 exhibits human CD137 agonist activity in the presence of ADP β S. This suggests that dBBAT119-P253 may exhibit human CD137 agonist activity in a manner that is dependent on small molecules (e.g., ATP, ADP, or AMP).

Reference example 3: enhanced antigen binding in the presence of small molecules using rationally designed light and heavy chain libraries Binding Activity of antibodies

(3-1) construction of a library for enhancing binding Activity Using rationally designed light chain library

For an antibody library containing a large number of antibodies having ATP-dependent antigen binding activity harvested in reference example 2-2-1, the binding activity was enhanced by reconstituting an antibody light chain library.

The light and heavy chain regions of rationally designed antibody phage display libraries constructed in prior patent WO2015/083764 were used to construct antibody light chain libraries and antibody heavy chain libraries to enhance binding activity. They were introduced into the light chain or heavy chain region of the light chain library described above or the phagemid vector library harvested in reference example 2-2-1 and introduced into E.coli strain ER2738 by electroporation.

(3-2) enhancing the binding Activity of an antibody having ATP-dependent antigen-binding Activity using a rationally designed library

Phage were generated from E.coli harboring the constructed phage-display phagemid in a conventional manner. Specifically, E.coli carrying the constructed phagemid vector was infected with M13KO7TC (WO 2015/046554) or M13KO 7. Delta. PIII (super phage) (PROGEN Biotechnik), and the phage was collected from the supernatant cultured overnight at 30 ℃. Obtaining a phage library solution by diluting a phage population with TBS, wherein said phage population is precipitated by adding 2.5M NaCl/10% PEG to an E.coli culture in which phage production is performed. BSA was then added to the phage library solution at a final concentration of 4%. Panning was performed using the antigen immobilized on the magnetic beads. Neutravidin-coated beads (neutravidin-coated Sera-Mag SpeedBeads), neutravidin beads (TAMAGAWA SEIKI), or Dynabeads MyOne Streptavidin T1 (Semmerfell technology) were used as the magnetic beads. As antigen, biotinylated hCD137-Fc was used.

Panning is performed to efficiently obtain small molecule switch antibodies that are dependent on small molecules that can exert a switching effect in cancer tissues. In particular, panning is performed to enrich for antigen-bound antibodies that bind to the antigen in the presence of small molecules of adenosine triphosphate (adenosine 5' -triphosphate; ATP) and do not bind to the antigen in the absence of ATP, with reference to the method described in the previous patent WO 2015/083764.

(3-3) evaluation of binding Activity in the Presence and absence of ATP by phage ELISA

The culture supernatants containing the phages were harvested from individual colonies of E.coli obtained in each round by the above-described Methods using conventional Methods (Methods mol. Biol.), (2002) 178, 133-145). The binding activity to human CD137 in the presence and absence of ATP was then confirmed by phage ELISA using the method described in reference example 2-2-2.

The results are shown in FIG. 5.

Many clones judged to have an ATP-dependent antigen-binding activity were obtained, which had an absorbance S/N ratio of more than 2 in the presence/absence of an antigen and an absorbance S/N ratio of more than 2 in the presence/absence of ATP (switch clones).

Reference example 4: production of modified CD137 antibodies and evaluation of their Activity

(4-1) increase in binding Activity due to changes in dBAT 119H-P253/dBAT 119L-LamLib

The variants of the heavy chain variable region dBAT 119H and the light chain variable region dBAT 119L of the anti-CD 137 antibody (clone name: dBAT 119H-P253/dBAT 119L-LamLib) obtained in reference examples 2-4 were prepared by methods known to those skilled in the art (e.g., PCR). For the heavy chain variable region, variants were made in which D10 (referring to aspartic acid (Asp) at position 10 (Kabat numbering)) and G17 (referring to glycine (Gly) at position 17 (Kabat numbering)) of dBdB119H were replaced with glycine (Gly) and serine (Ser), respectively, to yield dBBAT119H010; and N99 (referring to asparagine (Asn) at position 99 (Kabat numbering)), M100a (referring to methionine (Met) at position 100a (Kabat numbering)) and N100b (referring to asparagine (Asn) at position 100b (Kabat numbering)) of dBBAT119H010 are replaced with other amino acids for the light chain variable region, variants are produced in which F87 of dBBAT119L (referring to phenylalanine (Phe) at position 87 (Kabat numbering)) is replaced with tyrosine (Tyr) to produce dBBAT119L010, and D27b (aspartic acid (Asp) at position 27b (Kabat numbering)), N31 (asparagine (Asn) at position 31 (Kabat numbering)) and D94 (aspartic acid (Asp) at position 94 (Kabat numbering)) of dBBAT119L010 are replaced with other amino acids.

For the variants of the heavy chain variable region, the binding activity to human CD137 was measured by the surface plasmon resonance analyzer biacore t200 (GE medical). The antibody was captured by interacting the purified variant with protein G (CALBIOCHEM) immobilized S series sensor chip CM3 (GE medical). Then, human CD137 (FXa digested) solution supplemented with ATP or human CD137 (FXa digested) solution without ATP were interacted in the presence and absence of ATP to assess the binding activity of the variants to human CD137 (FXa digested). 20mM ACES, 150mM NaCl, 0.05% Tween 20 and 2mM MgCl were used ₂ (pH 7.4) as running buffer, measurements were carried out at 25 ℃. The measurement results showed that the binding activity of the L100a variant (variant in which methionine (Met) at position 100a (Kabat numbering) was replaced by leucine (Leu)) to human CD137 was enhanced only in the presence of ATP (fig. 27). The heavy chain variable region of the L100a variant was dBBATk119H024 (SEQ ID NO: 132). The amount of human CD137 bound was regulated by the amount captured (1000 RU) of each variant.

For the light chain variable region variants, binding activity to human CD137 (FXa digested) was determined by BiacoreT200 under conditions similar to those described above. The measurements showed that the binding activity of the E94 variant (variant wherein aspartic acid (Asp) at position 94 (Kabat numbering) is replaced by glutamic acid (Glu)) to human CD137 was enhanced only in the presence of ATP (fig. 27). The light chain variable region of the E94 variant was dBBATk119L020 (SEQ ID NO: 133).

Such variants, which combine these heavy and light chain variants together, are abbreviated as dBATk 119H 024-P253/dBATk 119L020-LamLib (heavy chain variable region SEQ ID NO:132; light chain variable region SEQ ID NO:133; heavy chain constant region SEQ ID NO:93; and light chain constant region SEQ ID NO: 63).

Antibodies in the present specification are specified according to the following rules: (heavy chain variable region) - (heavy chain constant region)/(light chain variable region) - (light chain constant region).

For example, the antibody designation dBAT 119H-P253/dBAT 119L-LamLib indicates that the heavy chain variable region of the antibody is dBAT 119H, the heavy chain constant region is P253, the light chain variable region is dBAT 119L, and the light chain constant region is LamLib.

(4-2) evaluation of in vitro CD137 agonist Activity of modified anti-human CD137 antibody Using human T cells

(4-2-1) expansion of human T cell culture

Human T cells were expanded and cultured as described in reference example 2-6-1.

(4-2-2) evaluation of in vitro CD137 agonist Activity Using human T cells

IFN- γ production was evaluated in the presence of NS1-P253 (non-switching antibody), dBAT 119H-P253/dBAT 119L-LamLib, dBATk 119H 024-P253/dBATk 119L020-LamLib, or control antibody IC17HdK-hIgG1/IC17L-k0 at 10, 2, 0.4, 0.08, and 0.016. Mu.g/mL, respectively, as described in reference to examples 2-6-2. IFN- γ production in ADPbetaS minimal medium was also assessed.

These two results are shown in fig. 28.

The modified dBATk 119H 024-P253/dBATk 119L020-LamLib showed a stronger ADPbetaS dependent agonist activity than dBAT 119H-P253/dBAT 119L-LamLib. This suggests that dBATk 119H 024-P253/dBATk 119L020-LamLib may exhibit greater ATP, ADP and AMP-dependent human CD137 agonist activity than dBAT 119H-P253/dBAT 119L-LamLib.

Reference example 5: further modification of CD137 antibodies

(5-1) search for by introducing comprehensive modificationsModifications to increase binding activity

In order to produce an excellent anti-CD 137 antibody, amino acid modifications were fully introduced into the heavy chain variable region dBBATk119H024 and the light chain variable region dBBATk119L020 of the anti-CD 137 antibody produced in reference example 4-1. Variants were each prepared by methods known to those skilled in the art, such as PCR, in which each of all amino acid residues constituting the CDRs of dBBATk119H024 and dBBATk119L020 was replaced with all 18 amino acids except cysteine. Approximately 1200 variants of the generated bound human CD137 were measured using Biacore 4000. The antibody was captured by interacting the culture supernatant of the variant with the S series sensor chip CM3 (GE medical) immobilized with protein G (CALBIOCHEM). Then, the human CD137 solution supplemented with a small molecule (ATP) or the human CD137 solution without the small molecule added was allowed to interact in the presence or absence of the small molecule to evaluate the binding activity of the antibody to human CD 137. Use and supplement CaCl ₂ Was measured at 25 ℃ in a running buffer of 20mM ACES, 150mM NaCl, 0.02% Tween 20 and 2mM MgCl2 (pH 7.4).

(5-2) increased ATP binding

Antibody heavy chain gene A002-P253 (SEQ ID NO: 134) was created in dBATk 119H024 (SEQ ID NO: 132) having dBATk 119H024 (SEQ ID NO: 93) as the heavy chain variable region and P253 (SEQ ID NO: 93) as the heavy chain constant region created by introducing S267E/L328F modifications into human IgG1 and deleting the C-terminal Gly and Lys from human IgG1 by combining the modifications found in reference example 5-1 that increase binding activity to human CD137 in the presence of small molecules. Antibody light chain gene B040-Lamlib (SEQ ID NO: 135) was also created in antibody light chain dBATk 119L020-Lamlib with dBATk 119L020 (SEQ ID NO: 133) as the light chain variable region and human lambda chain Lamlib (SEQ ID NO: 63) as the light chain constant region by combining the modifications found in reference example 5-1 that increase binding activity to human CD137 in the presence of small molecules. These genes are combined by methods known to those skilled in the art to express and purify the antibody to produce the desired anti-CD 137 antibody A002-P253/B040-Lamlib. The heavy chain variable region of A002-P253/B040-Lamlib is A002 (SEQ ID NO: 136), the light chain variable region is B040 (SEQ ID NO: 137), the heavy chain constant region is P253 (SEQ ID NO: 93), and the light chain constant region is human lambda chain Lamlib (SEQ ID NO: 63).

In the heavy chain variable region of A002-P253/B040-Lamlib produced in this section, various modifications were made in which the amino acid at position 53, 54 or 55 of Kabat numbering was replaced with another amino acid in order to improve ATP-binding activity. Table 35 shows the amino acid modifications (Kabat numbering) of a002 in the heavy chain variable region of the resulting antibodies.

[ Table 35]

Heavy chain variable region	Amino acid change of A002 (Kabat numbering)
		A002	―
A146	S54A/N55S
		A159	R53S/N55S
A160	R53T/N55S
		A161	R53Q/N55S
A162	R53K/N55S
		A163	R53H/N55S
A164	R53S/N55T
		A165	R53T/N55T
A166	R53Q/N55T
		A167	R53K/N55T
A168	R53H/N55T
		A169	R53S/N55H
A170	R53T/N55H
		A171	R53Q/N55H

The resulting variants were assessed for binding activity to ATP and human CD137 by Biacore T200.

20mM ACES (pH 7.4), 150mM NaCl, 2mM MgCl were used ₂ And 0.05% Tween 20 as running buffer, ATP-binding activity was measured at 37 ℃. First, the antibody was captured by interacting an antibody solution prepared in a running buffer with the Sure Protein a (GE medical) immobilized S-series sensor chip CM3 (GE medical). The binding activity of the antibody was then assessed by interaction with ATP solutions prepared in running buffer. 25mM NaOH and 10mM glycine-HCl (pH 1.5) were usedThe chip was green and measured by repeated capture of the antibody. The binding amount of ATP for each antibody was calculated by adjusting the binding amount of ATP when injected at a concentration of 100nM with the amount of antibody captured on the chip surface as the amount of ATP per unit amount of antibody.

20mM ACES (pH 7.4), 150mM NaCl, 2mM MgCl were used ₂ And 0.05% tween 20 as running buffer, the binding activity to human CD137 was measured at 37 ℃. First, an antibody solution prepared in a running buffer was interacted with the S-series sensor chip CM3 (GE medical) on which Sure protein a (GE medical) was immobilized to capture the antibody. Then, the binding activity to human CD137 was assessed by interaction with a solution of human CD137 supplemented with 100 μ M ATP as small molecule. For the human CD137 antigen, hCD137-HisBAP prepared in reference example (1-1) was used and measurement was performed at an antigen concentration of 0, 15.625, 62.5, 250, or 1000 nM. The chip was regenerated using 25mM NaOH and 10mM glycine-HCl (pH 1.5) and measured by repeated capture of antibody. Dissociation constants (KD) of various antibodies to human CD137 were calculated using Biacore T200 evaluation software 2.0. Specifically, by using 1: the association rate constant ka (L/mol/s) and dissociation rate constant KD (1/s) were calculated by overall fitting of the sensorgram obtained by measurement to the 1Langmuir binding model, and the dissociation constant KD (mol/L) was calculated from these values.

Table 36 shows the results of these measurements.

[ Table 36]

As shown in Table 36, variants in which amino acids 53, 54, or 55 (Kabat numbering) of the heavy chain variable region were replaced with other amino acids in addition to A146-P253/B040-Lamlib and A160-P253/B040-Lamlib had enhanced ATP-binding activity compared to A002-P253/B040-Lamlib before modification. Comparison of the association rate constants ka (L/mol/s) also shows that, among the antibodies evaluated above, the association rate constants of A146-P253/B040-Lamlib, A159-P253/B040-Lamlib, A162-P253/B040-Lamlib, A163-P253/B040-Lamlib, A164-P253/B040-Lamlib, A167-P253/B040-Lamlib, and A168-P253/B040-Lamlib increased for human CD 137.

(5-3) increasing the binding Activity by introducing comprehensive modifications

To produce better anti-CD 137 antibodies, the modifications found in reference example 5-1 that increase human CD137 binding activity in the presence of small molecules and decrease binding to human CD137 in the absence of small molecules, and the modifications found in reference example 5-2 that increase ATP binding activity and increase the association rate constant for human CD137 were combined to produce anti-human CD137 antibodies that exhibited better characteristics. An antibody heavy chain gene was generated which combined the modifications found in reference examples 5-1 and 5-2 in antibody heavy chain gene A002-G1T3 having heavy chain variable region A002 (SEQ ID NO: 136) and heavy chain constant region G1T3 (SEQ ID NO: 138) produced by introducing K214R/Q419E modification into human IgG1 and deleting C-terminal Gly and Lys from human IgG 1. The antibody light chain gene was generated by combining the modifications found in reference example 5-1 in antibody light chain B040-Lamlib with light chain variable region B040 (SEQ ID NO: 137) and light chain constant region human lambda chain Lamlib.

As a comparative subject, the following genes were generated: antibody heavy chain gene 20H4.9-P253, having the heavy chain variable region 20H4.9 (SEQ ID NO: 139) and heavy chain constant region P253 (SEQ ID NO: 93) of the existing anti-CD 137 antibody described in US 8137667; and an antibody light chain gene combining the light chain variable region 20H4.9LC (SEQ ID NO: 140) and the light chain constant region human kappa chain k0 (SEQ ID NO: 141). As another comparison, an antibody heavy chain gene MOR-7480.1H-P253 was generated having the heavy chain variable region MOR-7480.1H (SEQ ID NO: 142) and the heavy chain constant region P253 (SEQ ID NO: 93), wherein said heavy chain variable region MOR-7480.1H constitutes the existing anti-CD 137 antibody MOR-7480.1 described in US 8337850; and antibody light chain MOR-7480.1L-lam comprising the light chain variable region MOR-7480.1L (SEQ ID NO: 143) and the light chain constant region human lambda chain lam (SEQ ID NO: 63) (note: both human lambda chain Lamlib and lam have the same amino acid sequence (SEQ ID NO: 63)).

These genes are combined to express and purify the antibody using methods known to those skilled in the art to produce the anti-CD 137 antibody of interest. Table 37 is a list of the sequence numbers of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region (hypervariable region; also referred to as HVR or CDR) of the antibodies produced.

Amino acid sequences of heavy and light chains and hypervariable regions thereof (indicated by sequence ID numbers)

[ Table 37]

The resulting variants were evaluated for binding to ATP and to human CD137 in Biacore T200. 20mM ACES (pH 7.4), 150mM NaCl, 2mM MgCl were used ₂ And 0.05% tween 20 as running buffer, measurements of human CD137 binding were performed at 37 ℃. First, 250-400RU of antibody was captured by interacting an antibody solution prepared in a running buffer with Sure Protein a (GE medical) immobilized S series sensor chip CM3 (GE medical). Human CD137 binding activity in the presence or absence of ATP was then assessed by interaction with a human CD137 solution prepared in running buffer supplemented with the required concentration of ATP or a human CD137 solution prepared in running buffer without ATP. For the human CD137 antigen, hCD137-HisBAP prepared in reference example (1-1) was used, and KD value measurement was performed at antigen concentrations of 0, 15.625, 62.5, 250, and 1000 nM. To assess the amount of binding, measurements were made at antigen concentrations of 0 and 1000 nM. The chip was regenerated using 25mM NaOH and 10mM glycine-HCl (pH 1.5) and measured by repeated capture of antibody. Dissociation constants for various antibodies to human CD137 were calculated using Biacore T200 evaluation software 2.0. Specifically, by using 1:1Langmuir binding model the sensorgrams obtained from the measurements were fit to calculate the association rate constant ka (L/mol/s) and dissociation rate constant KD (1/s) and from these values the dissociation constant KD (mol/L) was calculated.

Table 38 shows the results of these measurements.

Binding assay for modified antibodies to human CD137

[ Table 38]

The value of "binding to human CD 137" in Table 38 above represents the amount of binding of human CD137 per unit antibody when human CD137 was allowed to interact at 1000nM under each ATP concentration condition described above, and "K of human CD 137" is used _D (M) "represents the dissociation constant of human CD137 at each ATP concentration. KD values marked in the table are calculated by a steady state model. In the presence of 10. Mu.M ATP, all the variants produced bound more than 1. Mu.M ATP and even more in the presence of 100. Mu.M, indicating that they bound to human CD137 in an ATP concentration-dependent manner. On the other hand, the comparison subjects 20H4.9-P253/20H4.9LC-k0 and MOR-7480.1H-P253/MOR-7480.1L-lam did not show binding to human CD137 in an ATP concentration-dependent manner.

(5-4) use of 4-1BB Jurkat reporter assay to evaluate in vitro ATP dependent of modified anti-human CD137 antibodies Leydig CD137 agonist Activity

GloResponse ^TM The NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line (Promega, CS 196004) was used to measure the in vitro activity of the produced variants. To each well of a 96-well plate, 200. Mu.L of 5X10 concentration was added ⁴ /mL Fc γ RIIB CHO-K1 cells (Promega) prepared in culture medium, and 5% CO ₂ The incubator was left overnight at 37 ℃. CHO medium (90% ham's F12, 10% FBS) was used as the medium. Then, after aspirating all the medium, 25 μ L of 2X10 prepared with assay medium (99% RPMI,1% FBS) ⁶ GloResponse/mL ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell lines were added to each well. Subsequently, 25. Mu.L of each antibody solution diluted with the assay medium was added to give final concentrations of 0, 0.001, 0.01, 0.1, 1 and 10. Mu.g/mL; finally 25. Mu.L of ATP solution diluted with assay medium was added to bring the final concentrations to 0 and 250. Mu.M. Mixing the plate with 5% CO ₂ Standing in incubator at 37 deg.C for 6 hr, standing at room temperature for 15 minA clock. Add 75. Mu.L Bio-Glo reagent to each well. The Bio-Glo luciferase assay system (buffer and substrate) was used for the Bio-Glo reagent. Subsequently, luminescence of each well was measured with a microplate reader. The luminescence value of each well divided by the luminescence value of wells without antibody addition was the relative light unit (fold induction) and used as an index to evaluate the CD137 agonist activity of each antibody.

The results are shown in FIG. 29.

(5-5) evaluation of ATP-dependent CD137 in vitro of modified anti-human CD137 antibody Using human peripheral blood mononuclear cells Agonist activity

(5-5-1) isolation of human peripheral blood mononuclear cells

Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from blood samples of healthy volunteers of the applicant company as described in reference example 2-6-1. Subsequently, the cells were diluted in culture medium (5% human Serum (SIGMA), 95% AIM-V (Saimer Feishal technology)) at a cell density of 5X10 ⁶ /mL。

(5-5-2) evaluation of CD137 agonist Activity Using human peripheral blood mononuclear cells

Adjusting the cell density of human peripheral blood mononuclear cells to 5x10 ⁶ mL, and seeded at 100 μ L into each well of a 96-well flat-bottom plate (Corning). Subsequently, 50. Mu.L of 0.04. Mu.g/mL anti-human CD 3. Epsilon. Antibody (BD, clone SP 34) and 20. Mu.g/mL anti-human CD28 antibody (BD, clone: CD 28.2) diluted in the medium were added. Shaking plate, then 5% of CO at 37 ℃ ₂ The incubator was left for 6 hours. Then, each well was supplemented with 25. Mu.L of 2mM ATP (SIGMA) medium or ATP-free medium, and 25. Mu.L of 40. Mu.g/mL of each antibody, the plate was shaken, and then placed into 5% CO ₂ The resulting mixture was left at 37 ℃ for 18 hours in an incubator. Subsequently, part of the culture supernatant was harvested and used with human IL-2 DuoSet ELISA kit (R)&D System) or human IL-2ELISA Set (BD biosciences) culture supernatants were used to quantify IL-2 content in culture supernatants. After harvesting the culture supernatant, the plates are again placed 5% CO ₂ The culture was carried out in an incubator at 37 ℃ for 24 hours. Subsequently, part of the culture is harvestedCulture supernatants and using human IFN-. Gamma.DuoSet ELISA kit (R)&D system) or human IFN- γ ELISA development kit (PeproTech) to quantify the IFN- γ content in the culture supernatants. The ELISA was performed essentially according to the protocol attached to the kit. For human IL-2 DuoSet ELISA kit (R)&D System) and human IFN-. Gamma.DuoSet ELISA kit (R)&D System), according to the protocol, using a catalyst containing H ₂ O ₂ And tetramethylbenzidine, 1 NH ₂ SO ₄ (Wako) substrate solution (R)&D system) for color development and stopping the color development. For the human IL-2ELISA kit (BD biosciences), 1 NH was used ₂ SO ₄ (Wako) color development was terminated. For IFN-. Gamma.ELISA development kit (PeproTech), TMB chromogen solution (Saimer Feishel technology) and 1N H ₂ SO ₄ (Wako) for color development and termination of color development. The absorbance measurements were then made with EnVision (PerkinElmer).

The results are described in detail with reference to examples 5-5-3 and thereafter.

(5-5-3) evaluation of enhancement of agonist Activity of heavy chain constant region by increasing binding Activity of Fc γ receptor

The effect of using P587, MY518, TT14 and TT16 on CD137 agonist activity was evaluated by in vitro evaluation using human peripheral blood mononuclear cells as described in reference examples 5-5-1 and 5-5-2, wherein the P587, MY518, TT14 and TT16 differentially increased Fc γ RIIb binding activity of the heavy chain constant region.

The antibodies evaluated are shown in table 39.

The production of the antibodies listed in Table 39 is described in reference example 7-1. When the addition of the antibody increased the amount of IL-2 in the culture supernatant by 1.05-fold or more and the amount of IFN- γ by 1.15-fold or more, the antibody was judged to exhibit CD137 agonist activity, as compared to the amounts of IL-2 and IFN- γ in the culture supernatant when the negative control antibodies (IC 17HdK-MY518/IC17L-k0, IC17HdK-G4d/IC17L-k 0) were added in the presence of 250. Mu.M ATP. Measurement of agonist activity using human Peripheral Blood Mononuclear Cells (PBMCs) may vary from donor to donor of the blood sample. In view of this, it was determined that an antibody that does not show CD137 agonist activity in a portion or more than half of human PBMCs isolated from multiple donors can be judged not to show CD137 agonist activity even if it meets the criteria for agonist activity for another portion of human PBMCs.

Table 39 shows antibodies judged to have CD137 agonist activity in the presence of 250 μ MATP. This suggests that CD137 agonist activity was enhanced by combining TT16 with P587, which increases Fc γ RIIb binding activity of the heavy chain constant region (fig. 30 and 31). It is believed that agonistic activity is enhanced by increasing binding activity to Fc γ RIIb-expressing cells, which are the cross-linked scaffolds required for antibodies to exhibit agonistic activity to CD 137-expressing cells (see Protein Eng Des sel.2013.10;26 (10): 589-598. Published online at 2013, 6/month 5. Doi: 10.1093/Protein).

[ Table 39]

Evaluated antibody name	Name of antibody judged to be active in the Presence of ATP
		A375-MY518/B167-LamLib	A375-P587/B167-LamLib
A356-MY518/B040-LamLib	A356-P587/B040-LamLib
		A375-P587/B167-LamLib	A375-TT16/B167-LamLib
A356-P587/B040-LamLib	A356-TT16/B040-LamLib
		A375-TT16/B167-LamLib
A356-TT16/B040-LamLib
		A375-TT14/B167-LamLib

(5-5-4) evaluation of ATP-dependent CD137 agonist Activity of variable region

The ATP-dependent CD137 agonist activity of each variable region by combining the heavy chain constant regions P587 and P253 with various variable regions was assessed by using human peripheral blood mononuclear cell in vitro evaluation as described in reference examples 5-5-1 and 5-5-2.

The antibodies evaluated are shown in table 40. The production of the antibodies listed in Table 40 is described in reference example 7-1. When the addition of the antibody increased the amount of IL-2 in the culture supernatant by 1.05-fold or more and the amount of IFN- γ by 1.15-fold or more, the antibody was judged to have CD137 agonist activity, as compared to the amounts of IL-2 and IFN- γ in the culture supernatant when the negative control antibody (IC 17HdK-P253/IC17L-k0, IC17HdK-P587/IC17L-k 0) was added in the presence of 250. Mu.M ATP. Measurement of agonist activity using human Peripheral Blood Mononuclear Cells (PBMCs) may vary depending on the donor of the blood sample. In view of this, it was determined that an antibody that does not show CD137 agonist activity in some or more than half of human PBMCs isolated from multiple donors can be judged not to show CD137 agonist activity even if it meets the agonist activity criteria for other parts of human PBMCs. Table 40 (fig. 32, 33, 34, 35, and 36) shows antibodies judged to exhibit CD137 agonist activity in the presence of 250 μ M ATP.

Furthermore, of the antibodies judged to show CD137 agonist activity in the presence of 250 μ M ATP, the following antibodies were judged to have a lower CD137 agonist in the absence of ATP: the fold change in the amounts of IL-2 and IFN- γ in the culture supernatants relative to the groups supplemented with negative control antibodies (IC 17HdK-P253/IC17L-k0, IC17HdK-P587/IC17L-k 0) in the absence of ATP was less than the fold change in the presence of 250 μ M ATP relative to the negative control. Table 40 (fig. 32, 33, 34, 35, and 36) shows antibodies that show CD137 agonist activity in the presence of 250 μ Μ ATP and are judged to have lower CD137 agonist activity in the absence of ATP. These antibodies were judged to exhibit ATP-dependent CD137 agonist activity.

From the above, it was confirmed that the combination of the heavy chain variable region/light chain variable region of the variable regions (A375/B167), (A356/B040), (A372/B040), (A486/B167), (A488/B226), (A489/B223), (A551/B256), (A548/B256) and (A551/B379) showed an ATP-dependent manner of CD137 agonist activity.

[ Table 40]

Evaluated antibody name	Name of antibody judged to be active in the Presence of ATP	Name of antibody judged to have lower activity in absence of ATP
			A372-P253/B040-LamLib	A372-P253/B040-LamLib	A372-P253/B040-LamLib
A486-P253/B167-LamLib	A486-P253/B167-LamLib	A486-P253/B167-LamLib
			A488-P253/B226-LamLib	A488-P253/B226-LamLib	A488-P253/B226-LamLib
A489-P253/B223-LamLib	A489-P253/B223-LamLib	A489-P253/B223-LamLib
			A551-P587/B256-LamLib	A551-P587/B256-LamLib	A551-P587/B256-LamLib
A375-P587/B167-LamLib	A375-P587/B167-LamLib	A375-P587/B167-LamLib
			A548-P587/B256-LamLib	A548-P587/B256-LamLib	A548-P587/B256-LamLib
A356-P587/B040-LamLib	A356-P587/B040-LamLib	A356-P587/B040-LamLib
			A551-P587/B379-LamLib	A551-P587/B379-LamLib	A551-P587/B379-LamLib

(5-5-5) evaluation will increase heavy chain constantAlteration of pI of a targeting region and compounds with increased Fc gamma receptor binding activity Enhancement of CD137 agonist activity by combination of chain constant regions

The effect of introducing various amino acid modifications that increase the pI of the heavy chain constant region into heavy chain constant regions with various increased Fc γ RIIb binding activities on CD137 agonist activity was evaluated by in vitro evaluation using human peripheral blood mononuclear cells as described in reference examples 5-5-1 and 5-5-2. Amino acid changes that increase pI were introduced into the heavy chain constant regions TT16, MY518 and TT14, resulting in heavy chain constant regions TT16+ P343R/D413K (SCF 028), TT16+ Q311R/P343R (SCF 033), MY518+ P343R/D413K (SCF 025), MY518+ Q311R/P343R (SCF 030), TT14+ P343R/D413K (SCF 027) and TT14+ Q311R/P343R (SCF 032). The names of the evaluated antibodies to which the amino acid change that increases the pI has been introduced, and the names of the corresponding antibodies before the amino acid change that increases the pI is introduced, are shown in table 41. The production of the antibodies cited in Table 41 is described in reference example 7-2.

With respect to the evaluation of enhancing agonist activity by introducing amino acid modifications that increase the pI, CD137 agonist activity was judged to be enhanced when the addition of the antibody increased the amount of IL-2 in the culture supernatant by 1.04-fold or more and the amount of IFN- γ by 1.1-fold or more, relative to the groups a375-TT16/B167-LamLib, a375-MY518/B167-LamLib, and a375-TT14/B167-LamLib to which the antibody containing a heavy chain constant region that did not contain these amino acid modifications was added. Antibodies comprising a heavy chain constant region into which amino acid modifications showing enhanced CD137 agonist activity in the presence of 250 μ M ATP were introduced are shown in table 42 (fig. 31) compared to antibodies comprising a heavy chain constant region without amino acid modifications increasing the pI.

This indicates that introduction of the modification that increases the heavy chain constant region pI enhances CD137 agonist activity of anti-human CD137 antibodies, regardless of the degree of increase in Fc γ RIIb binding activity of the heavy chain constant region. This shows that combining a modification that increases the pI of the heavy chain constant region with a modification that increases Fc γ RIIb binding of the heavy chain constant region into the heavy chain constant region enhances interaction with the negatively charged cell surface expressing Fc γ RIIb; the antibody or immune complex of the antigen-binding antibody is closer to the surface of the Fc γ RIIb-expressing cell; and this further increases binding to Fc γ RIIb-expressing cells, which are the cross-linked scaffold required for the antibody to exhibit agonistic activity against CD 137-expressing cells; therefore, the agonistic activity is further enhanced.

[ Table 41]

[ Table 42]

Names of antibodies judged to exhibit increased agonistic activity
	A375-SCF028/B167-LamLib
A375-SCF033/B167-LamLib
	A375-SCF025/B167-LamLib
A375-SCF030/B167-LamLib
	A375-SCF027/B167-LamLib
A375-SCF032/B167-LamLib

(5-5-6) evaluation of the Effect of introducing a modification to increase the pI of the heavy chain constant region on agonist Activity

The effect of the introduction of various amino acid modifications that increase the pI of the heavy chain constant regions MY201aPh and MY518 on CD137 agonist activity was assessed by using the in vitro assessment of human peripheral blood mononuclear cells described in reference examples 5-5-1 and 5-5-2. Introducing amino acid modifications with increasing pI to heavy chain constant regions MY518, MY518a, and MY201 apph; thus, MY518+ P343R/D413K (SCF 025), MY518+ Q311R/P343R (SCF 030), MY518+ P343R (SCF 039), MY518+ D413K (SCF 040), MY518a + Q311R (SCF 060 a), MY201aPh + P343R (SCF 041 aPh), MY201aPh + P343R/D413K (SCF 043 aPh), MY201aPh + Q311R (SCF 056 aPh), MY201aPh + Q311R/P343Rb (SCF 057 aPh), and MY201aPh + Q311R/D413K (SCF 059 aPh) were generated. The evaluated antibody names in which amino acid changes that increase the pI have been introduced, and the antibody names before introducing amino acid changes that increase the corresponding pI are shown in table 43. The production of the antibodies cited in Table 43 is described in reference example 7-2.

With respect to the evaluation of the enhancement of agonist activity by introducing amino acid modifications that increase the pI, CD137 agonist activity was judged to be enhanced when the addition of the antibody increased the amount of IL-2 in the culture supernatant by 1.04-fold or more and the amount of IFN- γ by 1.1-fold or more, relative to the group to which the antibody comprising the heavy chain constant region that did not contain these amino acid modifications was added, i.e., a375-MY518a/B167-LamLib and a375-MY201 apph/B167-LamLib. Antibodies comprising a heavy chain constant region into which amino acid changes have been introduced that show enhanced CD137 agonist activity in the presence of 250 μ M ATP are shown in table 44 (fig. 37 and 38) compared to antibodies comprising a heavy chain constant region without amino acid modifications that increase pI.

[ Table 43]

[ Table 44]

Names of antibodies judged to exhibit increased agonistic activity
	A375-SCF025/B167-LamLib
A375-SCF030/B167-LamLib
	A375-SCF039/B167-LamLib
A375-SCF040/B167-LamLib
	A375-SCF060a/B167-LamLib
A375-SCF041aPh/B167-LamLib
	A375-SCF043aPh/B167-LamLib
A375-SCF056aPh/B167-LamLib
	A375-SCF057aPh/B167-LamLib
A375-SCF059aPh/B167-LamLib

(5-5-7) evaluation of ATP-dependent CD137 agonist Activity of anti-human CD137 antibody, wherein the anti-human CD137 antibody Combining the variable region and constant region generated by the antibody

The ATP-dependent CD137 agonist activity of an anti-human CD137 antibody prepared by combining the above-described variable region with a heavy chain constant region that increases the pI was evaluated by in vitro evaluation using the human peripheral blood mononuclear cells in reference examples 5-5-1 and 5-5-2. The antibodies evaluated are shown in table 45.

The antibody was judged to exhibit CD137 agonist activity when the addition of the antibody increased the amount of IL-2 in the culture supernatant by more than 1.05 fold and the amount of IFN- γ by more than 1.15 fold, compared to the amount of IL-2 and IFN- γ in the culture supernatant when a negative control antibody (IC 17HdK-MY518a/IC17L-k0, IC17HdK-MY201aPh/IC17L-k0, IC17HdK-MY201/IC17L-k0, IC17HdK-G4d/IC17L-k0, IC17HdK-MY518/IC17L-k0, or IC17HdK-TT16/IC17L-k 0) was added in the presence of 250 μ M ATP. Measurement of agonist activity using human Peripheral Blood Mononuclear Cells (PBMCs) may vary from donor to donor of the blood sample. In view of this, it was determined that an antibody that does not show CD137 agonist activity in a portion or more than half of human PBMCs isolated from multiple donors can be judged not to show CD137 agonist activity even if it meets the agonist activity criterion for another portion of human PBMCs. Antibodies judged to have CD137 agonist activity in the presence of 250 μ M ATP are shown in table 45 (fig. 37, fig. 38, fig. 39, fig. 40, fig. 41, fig. 42, fig. 43, and fig. 44).

In addition, among the antibodies judged to show CD137 agonist activity in the presence of 250 μ M ATP, the following antibodies were judged to have lower CD137 agonist activity in the absence of ATP: regarding the amount of IL-2 and IFN- γ in the culture supernatants, the fold change in the absence of ATP relative to the group to which the negative control antibodies (IC 17HdK-MY518a/IC17L-k0, IC17HdK-MY201aPh/IC17L-k0, IC17HdK-MY201/IC17L-k0, IC17HdK-G4d/IC17L-k0, IC17HdK-MY518/IC17L-k0, or IC17HdK-TT16/IC17L-k 0) were added was less than the fold change in the presence of 250 μ M ATP relative to the negative control. Antibodies that were judged to have lower CD137 agonist activity in the absence of ATP are shown in table 45 (fig. 37, fig. 38, fig. 39, fig. 40, fig. 41, fig. 42, fig. 43, and fig. 44). These antibodies show ATP-dependent CD137 agonist activity.

[ Table 45]

Reference example 6: human CD137 knock-in mouse administration test of anti-human CD137 conversion antibody

(6-1) production of antibody for human CD137 knock-in mouse administration study

Anti-human CD 137-converted and non-converted antibodies with mouse constant regions were generated for human CD137 knock-in mouse administration studies. Specifically, an anti-human CD137 non-converting antibody (20H4.9-mIgG 1/20H4.9LC-mk0, abbreviation: NS1-mIgG1, 20H4.9-MB110/20H4.9LC-mk0, abbreviated as NS1-MB110, 20H4.9-MB492/20H4.9LC-mk0, abbreviated as NS1-MB492, abbreviated as MOR-7480.1H-MB110/MOR-7480.1L-ml0r, abbreviated as NS2-MB110, MOR-7480.1H-MB492/MOR-7480.1L-ml0r, abbreviated as NS2-MB 492), and anti-human CD137 converting antibody (A375-mIgG 1/B167-ml0r, A372-mIgG1/B040-ml0r, A372-MB110/B040-ml0r, A372-MB 492/B492-0 r, A356-MB 110/B-ml 0r, A486/B167-MB 0r, A488-MB 70/B120 r, A551-MB 70/B24-40 ml, A1-MB 1/223-MB 1/40-ml, A223-MB 1/32-40-ml).

For the heavy chains of NS1-mIgG1, NS1-MB110, and NS1-MB492 antibodies, the genes of the antibody heavy chains were generated by combining the heavy chain variable region 20H4.9 (SEQ ID NO: 139) with any of the following as a heavy chain constant region: (i) mIgG1, the heavy chain constant region of murine IgG1 (SEQ ID NO: 144),

(ii) MB110 (SEQ ID NO: 145) as described in WO2014030750, and

(iii) MB492 (SEQ ID NO: 146) as described in WO2014030750, i.e., the genes of 20H4.9-mIgG1, 20H4.9-MB110, and 20H4.9-MB492 were produced.

For the light chain of NS1-mIgG1, NS1-MB110, and NS1-MB492 antibodies, an antibody light chain gene 20H4.9LC (SEQ ID NO: 140) was generated by combining the light chain variable region 20H4.9LC (SEQ ID NO: 140) with the light chain constant region murine kappa chain mk0 (SEQ ID NO: 147). By combining these heavy and light chain genes, each antibody was expressed and purified by methods known to those skilled in the art.

For the heavy chains of NS2-MB110 and NS2-MB492 antibodies, the genes for the antibody heavy chains were generated by combining the heavy chain variable region MOR-7480.1H (SEQ ID NO: 142) with either:

(i) MB110 (SEQ ID NO: 145), or

(ii)MB492(SEQ ID NO:146)，

That is, the MOR-7480.1H-MB110 and MOR-7480.1H-MB492 genes were generated.

For the light chains of NS2-MB110 and NS2-MB492 antibodies, the antibody light chain gene MOR-7480.1L-ml0r was generated by combining the light chain variable region MOR-7480.1L (SEQ ID NO: 143) with the murine lambda chain ml0r (SEQ ID NO: 148) as the light chain constant region. By combining these heavy and light chain genes, each antibody was expressed and purified by methods known to those skilled in the art.

For the anti-CD 137 conversion antibody, the genes for the antibody heavy chain and antibody light chain in tables 46 and 47 below were generated; and each antibody is expressed and purified by combining these genes by methods known to those skilled in the art.

Antibody heavy chain of anti-CD 137 switch antibody with murine constant region

[ Table 46]

Antibody light chain of anti-CD 137 transformant antibody with murine constant region

[ Table 47]

Full length light chain	B040-ml0r	Variable region	B040(SEQ ID NO:55)
				Constant region	ml0r(SEQ ID NO:148)
Full length light chain	B167-ml0r	Variable region	B167(SEQ ID NO:54)
				Constant region	ml0r(SEQ ID NO:148)
Full length light chain	B226-ml0r	Variable region	B226(SEQ ID NO:56)
				Constant region	ml0r(SEQ ID NO:148)
Full length light chain	B223-ml0r	Variable region	B223(SEQ ID NO:57)
				Constant region	ml0r(SEQ ID NO:148)
Full length light chain	B256-ml0r	Variable region	B256(SEQ ID NO:59)
				Constant region	ml0r(SEQ ID NO:148)
Full length light chain	B379-ml0r	Variable region	B379(SEQ ID NO:60)
				Constant region	ml0r(SEQ ID NO:148)

(6-2) evaluation of human CD 137-binding Activity of antibodies for human CD137 knock-in mouse administration study

The binding activity of the anti-human CD137 non-converting antibody and the anti-CD 137 converting antibody produced in reference example 6-1 to human CD137 was evaluated. 20mM ACES (pH 7.4), 150mM NaCl,2mM MgCl were used ₂ And 0.05% tween 20 as running buffer, binding to human CD137 was measured at 37 ℃. First, antibodies were captured by interacting an antibody solution prepared in a running buffer with an S-series sensor chip CM5 (GE medical) on which rabbit anti-mouse IgG (seemer science) was immobilized. Human CD137 binding activity in the presence and absence of ATP was then assessed by interaction with a human CD137 solution prepared in a running buffer supplemented with the required concentration of ATP or with a human CD137 solution prepared in a running buffer without ATP . For the human CD137 antigen, hCD137 (FXa-digested) prepared in reference example (1-2) was used, and measurements were made at antigen concentrations of 0, 15.625, 62.5, 250, and 1000 nM. The chip was regenerated using 25mM NaOH and 10mM glycine-HCl (pH 1.5) and measured by repeated capture of antibody. Dissociation constants (KD) of various antibodies to human CD137 were calculated using Biacore T200 evaluation software 2.0. Specifically, by using 1: the association rate constant ka (L/mol/s) and dissociation rate constant KD (1/s) were calculated by overall fitting of the sensorgram obtained by measurement to the 1 Langmuir (Langmuir) binding model, and the dissociation constant KD (mol/L) was calculated from these values.

Table 48 shows the results of these measurements.

Human CD137 binding assay for anti-CD 137 antibodies with murine constant regions

[ Table 48]

N.A.: too weak to determine the KD value.

N.D.: it is not determined.

It was confirmed that both the anti-human CD137 non-converted antibody containing a murine constant region and the anti-human CD137 converted antibody generated as described above bind to human CD137. All anti-human CD137 converting antibodies were also shown to bind human CD137 in an ATP concentration dependent manner. On the other hand, for the non-converted antibody, no such ATP concentration-dependent binding to human CD137 was observed, and binding was almost the same at any ATP concentration.

(6-3) evaluation of plasma kinetics of modified antibodies in human CD137 knock-in mice

(6-3-1) Generation of human CD137 knock-in mice

Human CD137 knock-in mice were generated by introducing the human CD137 gene replacement vector into mouse embryonic stem cells (ES cells), in which the mouse CD137 gene was replaced with the human CD137 gene. This mouse was described as an hCD137KI mouse.

(6-3-2) plasma anti-human CD137 antibody in hCD137KI mouse modelMeasurement of concentration

After the hCD137KI mice were produced in reference example 6-3-1, the respective antibodies were intravenously administered to the hCD137KI mice in a single dose, as shown in table 49. In Table 49, NS1-mIgG1, NS1-MB110, and NS1-MB492 are all non-adapter antibodies, others are adapter antibodies. Blood was collected several times over time from 5 minutes to 28 days after administration. The obtained blood was centrifuged to separate plasma. The plasma was kept in a refrigerator at below-20 ℃ until measured.

Group details of the plasma dynamics assessment study

[ Table 49]

Group of	Number of animals	Medicament	Dosage [ mg/kg]
				1	3	NS1-mIgG1	7.5 Single administration
2	3	NS1-MB110	7.5 Single administration
				3	3	NS1-MB492	7.5 Single administration
4	3	A375-mIgG1/B167-ml0r	7.5 Single administration
				5	3	A356-MB110/B040-ml0r	7.5 Single administration
6	3	A372-mIgG1/B040-ml0r	7.5 Single administration
				7	3	A372-MB110/B040-ml0r	7.5 Single administration
8	3	A372-MB492/B040-ml0r	7.5 Single administration
				9	3	A486-MB492/B167-ml0r	7.5 Single administration
10	3	A488-MB492/B226-ml0r	7.5 Single administration
				11	3	A489-MB492/B223-ml0r	7.5 Single administration
12	3	A548-mIgG1/B256-ml0r	7.5 Single administration
				13	3	A551-mIgG1/B256-ml0r	7.5 Single administration
14	3	A551-MB110/B379-ml0r	7.5 Single administration

The concentration of each of the conversion antibodies in plasma was measured by an Electrochemiluminescence (ECL) method. Specifically, hCD137 (nano Biological inc.) was diluted in PBS (-) and then added to MULTI-ARRAY 96-well plates (Meso Scale Diagnostics, LLC). hCD137 was added to the plate and shaken at room temperature for 1 hour, and hCD137 was fixed on the plate. Next, for blocking, a PBS solution containing 1% BSA and 0.05% Tween 20 was added, and shaken at room temperature for 1 hour. Calibration curves for individual switch antibodies were prepared at plasma concentrations of 64, 32, 16, 8, 4, 2 and 1 ng/mL. After adding a PBS solution containing 3mM ADP, 1% BSA and 0.05% Tween 20 to the hCD 137-fixed plate, plasma samples diluted two times in a PBS solution containing 1% BSA and 0.05% Tween 20 and calibration curve samples were added. After shaking the plate for one hour at room temperature, biotinylated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, inc.). In addition, after the plate was shaken at room temperature for one hour, SULFO-TAG-labeled streptavidin (Meso Scale Diagnostics, LLCs) was added. Further, after the plate was shaken at room temperature for one hour, read buffer T (Read buffer T) (Meso Scale Diagnostics, LLCs) diluted twice with a PBS solution containing 2mM ADP, 1% BSA and 0.05% Tween 20 was added. Measurement of antibody concentration in hCD137KI mouse plasma was performed by using the SULFO-TAG detection on a SECTOR imager (Meso Scale Diagnostics, LLC). Calculations of individual antibody concentrations in mouse plasma were performed using SOFTmax PRO (Molecular Devices).

The concentration of non-converted antibodies in plasma was measured by the Electrochemiluminescence (ECL) method. Specifically, hCD137 (nano Biological inc.) was diluted in PBS (-) and then added to MULTI-ARRAY 96-well plates (Meso Scale Diagnostics, LLC). hCD137 was added to the plate and shaken at room temperature for 1 hour, and hCD137 was fixed on the plate. Next, for blocking, a PBS solution containing 1% BSA and 0.05% Tween 20 was added, and shaken at room temperature for 1 hour. Calibration curves for each non-converted antibody were prepared at plasma concentrations of 32, 16, 8, 4, 2, 1 and 0.5 ng/mL. Plasma samples diluted in PBS solution containing 1% bsa, 0.05% tween 20, and calibration curve samples were added to hCD 137-fixed plates. After shaking the plate for one hour at room temperature, biotinylated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, inc.). In addition, after the plate was shaken at room temperature for one hour, SULFO-TAG-labeled streptavidin (Meso Scale Diagnostics, LLCs) was added. In addition, after the plate was shaken at room temperature for one hour, a diluted two-fold reading buffer T (Meso Scale Diagnostics, LLCs) was added. The measurement of antibody concentration in plasma of hCD137KI mice was similar to the above-described converted antibody.

The results are shown in fig. 45, 46 and 47.

FIG. 45 shows the course of the mean plasma concentration over time of the antibodies with mIgG1 as Fc in the antibodies shown in Table 49 in hCD137 KI mice (NS 1-mIgG1 is a non-shifting antibody, A375-mIgG1/B167-ml0r, A372-mIgG1/B040-ml0r, A548-mIgG1/B256-ml0r or A551-mIgG1/B256-ml0r are shifting antibodies). FIG. 46 shows the course of the mean plasma concentration over time of the antibody with MB110 as Fc in hCD137 KI mice (NS 1-MB110 as non-switching antibody, A356-MB110/B040-ml0r, A372-MB110/B040-ml0r and A551-MB110/B379-ml0r as switching antibody). FIG. 47 shows the course of the mean plasma concentration of antibody with MB492 as Fc over time in CD137 KI mice (NS 1-MB492 as non-shifting antibody, and A372-MB492/B040-ml0r, A486-MB492/B167-ml0r, A488-MB492/B226-ml0r, or A489-MB492/B223-ml0r as shifting antibody). In FIG. 47, for A488-MB492/B226-ml0r, a sharp decline in the time course of plasma concentration suspected of being caused by ADA was confirmed in one subject, and the mean was taken from two subjects.

In hCD137 KI mice with either Fc, the converted antibody showed slower clearance from plasma than the non-converted antibody. This is thought to be because non-switching antibodies bind to CD137 expressed in vivo and eliminate from plasma more rapidly than switching antibodies. Furthermore, this suggests that the extracellular ATP concentration in normal tissues is low enough that the switch antibody used in this study does not bind human CD 137. These results indicate that the binding to systemically expressed antigens can be reduced by using the switch antibody, and thus antibodies with enhanced hemodynamics can be prepared.

(6-4) pharmacological effects of anti-CD 137 antibody by using syngeneic tumor cell transplantation model of hCD137KI mouse Movement of systemic response markers (movement)

(6-4-1) Generation of cell line and syngeneic tumor transplantation mouse model and evaluation of antitumor Effect and systemic reaction Price method

Cells of the murine colorectal cancer cell line MC38, licensed by the national cancer institute, and the hCD137KI mouse described in reference example 6-3-1 were used for various studies. By implanting the MC38 cell line subcutaneously in the abdomen of mice when the tumor volume reaches about 50-300mm ³ When the mold is formed. After modeling, mice transplanted with the MC38 cell line were grouped and then administered with various anti-CD 137 antibodies.

To evaluate the antitumor effect, tumor volume measurements were performed at a frequency of 1-2 times per week. Tumor volume was calculated according to the following formula:

tumor volume (mm) ³ ) = length (mm) x width (mm)/2

By removing the liver and spleen or lymph nodes at the appropriate time after administration of the antibody, systemic responses can be assessed by organ weight, cell count of lymphocyte fraction, or T cell analysis using Flow Cytometry (FCM). In the evaluation of these indices, when the systemic response index is low compared to the same amount of non-transformed antibody (control anti-human CD137 antibody without ATP-dependent human CD137 binding activity), the subject switch antibody is evaluated as having suppressed systemic response and/or T cell activation in non-tumor tissues (e.g., lymph nodes, spleen, and liver).

The antibody produced in reference example 6-1 (production of antibody for human CD137 knock-in mouse administration test) was used in various tests.

(6-4-2) Collection of various organs from MC38 cell line-transplanted mice and preparation of lymphocyte fractions

Weight measurements of collected spleens and lymph nodes were performed, as well as cell counts of lymphocyte fractions. Lymphocyte fractions obtained from mice (Milteny Biotec) using the liver dissociation kit were also used to assess liver activation. The lymphocyte fraction after erythrocyte lysis was used to evaluate activation in the spleen, and the lymphocyte fraction obtained by trituration was used to evaluate activation in the lymph node.

(6-4-3) FCM analysis Using lymphocyte fractions from various organs

To assess activation markers by FCM, granzyme B or PD-1 expression or ICOS expression on CD8 α positive T cells, or the percentage of CD8 α positive T cells in CD45 positive cells was used. Thus, fluorescently labeled anti-granzyme B antibodies, anti-PD-1 antibodies, anti-ICOS antibodies, anti-CD 8 α antibodies, and anti-CD 45 antibodies were used in the FCM assay. Using BD LSRFortessa ^TM X-20 (BD Biosciences).

(6-4-4) study of drug efficacy (anti-tumor Effect) of A375-mIgG1/B167-ml0r antibody

After establishing the MC38 transplantation mouse model in reference example 6-4-1, the vehicle and A375-mIgG1/B167-ml0r were administered at the doses shown in Table 50. Two antibody administrations were performed by tail vein route on the day of grouping and four days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of group for A375-mIgG1/B167-ml0r pharmacodynamic study

[ Table 50]

The antitumor effect in each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. As a result, the dose-dependent antitumor effect of the A375-mIgG1/B167-ml0r antibody was confirmed (FIG. 48).

(6-4-5) antibody administration and sampling and FCM for assessment of systemic Effect of A375-mIgG1/B167-ml0r Analysis of

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS1-mIgG1 (non-switching antibody), and A375-mIgG1/B167-ml0r were administered as shown in Table 51. Two antibody administrations were performed by tail vein route on the day of grouping and three days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Five days after the initial administration, liver, lymph node and spleen were collected as shown in reference example 6-4-2 (various organs were collected from mice transplanted with MC38 cell line and lymphocyte fractions were prepared). In addition, for spleen and lymph node, organs from three mice were pooled and FCM analysis was performed as shown in reference example 6-4-3 (FCM analysis was performed using lymphocyte fractions from various organs).

Group details for assessment of systemic response of A375-mIgG1/B167-ml0r antibody

[ Table 51]

The systemic effect of the administered antibody was evaluated. As a result of evaluating the organ weights of the lymph node and spleen, when NS1-mIgG1 was administered at 0.3mg/kg, an increase in organ weight was observed, and more intense excessive enlargement of organs was observed at 1.5mg/kg and 7.5 mg/kg. On the other hand, when A375-mIgG1/B167-mL0r was administered, no organ enlargement was observed at any of the doses of 1.5mg/kg, 7.5mg/kg, or 37.5mg/kg (FIG. 49).

In addition, as a result of the evaluation of T cell activation by FCM evaluation, increased expression of all markers PD-1, ICOS and granzyme B was observed at 0.3mg/kg after administration of NS1-mIgG 1. On the other hand, when a375-mIgG1/B167-ml0r was administered, the expression of all markers PD-1, ICOS and granzyme B was significantly inhibited at doses of 1.5mg/kg, 7.5mg/kg and 37.5mg/kg (fig. 50 and 51). Since the A375-mIgG1/B167-ml0r results indicate that organ weight correlates with a marker of T cell activation in lymph nodes and spleen, organ weight was used primarily as an indicator of lymph node and spleen-derived immune cell activation in subsequent antibody assessments.

As a result of evaluating T cell activation in the liver, increased expression of PD-1 and granzyme B was observed when NS1-mIgG1 was administered at 0.3 mg/kg. On the other hand, when A375-mIgG1/B167-mL0r was administered, expression was inhibited at all doses of 1.5mg/kg, 7.5mg/kg, and 37.5mg/kg (FIG. 52).

Thus, the switch antibody inhibits activation of T cells in lymph nodes, spleen and liver compared to the same amount of non-switch antibody.

(6-4-6) study of drug efficacy (anti-tumor Effect) of A356-MB110/B040-ml0r antibody

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle and A356-MB110/B040-ml0r were administered at the doses shown in Table 52. Two antibody administrations were performed by tail vein route on the day of grouping and three days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of A356-MB110/B040-ml0r group pharmacodynamic study

[ Table 52]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. The results confirmed the dose-dependent antitumor effect of A356-MB110/B040-ml0r (FIG. 53).

(6-4-7) antibody administration and sampling and FCM for assessment of systemic Effect of A356-MB110/B040-ml0r Analysis of

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS2-MB110 (non-switching antibody), and A356-MB110/B040-ml0r were administered as shown in Table 53. Two antibody administrations were performed by tail vein route on the day of grouping and three days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

7 days after the first administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with MC38 cell line and lymphocyte fractions were prepared). For spleen and lymph nodes, only organ weight measurements were made.

Group details to assess systemic response of A356-MB110/B040-ml0r antibody

[ Table 53]

The systemic effect of the administered antibody was evaluated. Evaluation of lymph node and spleen organ weights when NS2-MB110 and A356-MB110/B040-ml were administered at 0r indicated that excessive enlargement was also observed when NS2-MB110 was administered at 2.5 mg/kg. On the other hand, when A356-MB110/B040-mL0r was administered, lymphadenopathy was inhibited at 7.5mg/kg, and splenomegaly was inhibited at 2.5mg/kg (FIG. 54).

In addition, the results of evaluating T cell activation in the liver showed that an increase in PD-1 expression in CD8 α -positive cells was also observed when NS2-MB110 was administered at 0.3 mg/kg. Meanwhile, when A356-MB110/B040-mL0r was administered, PD-1 expression was inhibited at 7.5mg/kg, and significantly inhibited at 2.5 mg/kg. A slight increase in ICOS expression was also observed in CD8 α positive cells following administration of NS2-MB110 antibody at 0.3 mg/kg. On the other hand, when A356-MB110/B040-ml0r was administered, suppression of excipient expression at 2.5mg/kg was observed (FIG. 55).

Thus, the switch antibody inhibits activation of T cells in lymph nodes, spleen and liver, as compared to a non-switch antibody.

(6-4-8) study of drug efficacy (anti-tumor Effect) of A372-mIgG1/B040-ml0r antibody

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle and A372-mIgG1/B040-ml0r were administered at the doses shown in Table 54. Four antibody administrations were performed by tail vein route on the day of grouping and 4, 7 and 10 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of group for A372-mIgG1/B040-ml0r pharmacodynamic study

[ Table 54]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. As a result, the anti-tumor effect of the A372-mIgG1/B040-mL0r antibody was observed when 7.5mg/kg was administered (FIG. 56).

(6-4-9) antibody administration and sampling and FCM for assessment of systemic Effect of A372-mIgG1/B040-ml0r Analysis of

After establishing the MC38 transplantation mouse model in reference example 6-4-1, the vehicle and A372-mIgG1/B040-ml0r were administered as shown in Table 55. Antibody administration was performed 3 times by tail vein route on the day of grouping and 3 days and 6 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

7 days after the initial administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with the MC38 cell line and lymphocyte fractions were prepared). For the spleen, only organ weights were measured. For lymph nodes, cell counts were made only on lymphocyte fractions obtained from lymphocytes pooled from three mice.

Group details for assessment of systemic response of A372-mIgG1/B040-ml0r antibody

[ Table 55]

The systemic effect of the administered antibody was evaluated. As a result of measuring the number of cells in the lymph node and evaluating the organ weight of the spleen, it was confirmed that the number of cells in the lymph node was not increased and the weight of the spleen was not increased and the organ weight was the same as that of the excipient group when A372-mIgG1/B040-ml0r was administered (FIG. 57).

As a result of assessing T cell activation in the liver, the level of granzyme B expression was similar to that of the vehicle group when A372-mIgG1/B040-ml0r was administered (FIG. 58).

Thus, it was demonstrated that the switch antibody inhibits activation of T cells in lymph nodes, spleen and liver.

(6-4-10) study of drug efficacy (anti-tumor Effect) of A372-MB110/B040-ml0r antibody

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle and A372-MB110/B040-ml0r were administered at the doses shown in Table 56. Two antibody administrations were performed by tail vein route on the day of grouping and four days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of group of A372-MB110/B040-ml0r pharmacodynamic study

[ Table 56]

The antitumor effect of each group was evaluated by tumor volume calculated as described in reference example 6-4-1. As a result, the antitumor effect of A372-MB110/B040-ml0r was confirmed (FIG. 59).

(6-4-11) antibody administration and sampling and FCM for assessment of systemic Effect of A372-MB110/B040-ml0r Analysis of

After establishing the MC38 transplantation mouse model in reference example 6-4-1, the vehicle, NS2-MB110 (non-switching antibody), and A372-MB110/B040-ml0r were administered as shown in Table 57. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

7 days after the initial administration, liver, lymph node and spleen were collected as shown in reference example 6-4-2 (various organs were collected from mice transplanted with MC38 cell line and lymphocyte fractions were prepared). For spleen and lymph nodes, only organ weight measurements were made.

Group details to assess systemic response of A372-MB110/B040-ml0r antibody

[ Table 57]

The systemic effect of the administered antibody was evaluated. As a result of evaluating the organ weights of lymph node and spleen, when NS2-MB110 was administered at 2.5mg/kg and 7.5mg/kg, an increase in organ weight was observed. On the other hand, when A372-MB110/B040-mL0r was administered, inhibition of organ enlargement was observed at both doses of 2.5mg/kg and 7.5mg/kg (FIG. 60).

As a result of evaluating T cell activation in the liver, an increase in PD-1 expression was observed when NS2-MB110 was administered. An increase in PD-1 expression was also seen when A372-MB110/B040-mL0r was administered (FIG. 61).

In summary, the switch antibody inhibited the activation of T cells in lymph nodes and spleen compared to the non-switch antibody.

(6-4-12) study of drug efficacy (anti-tumor Effect) of A372-MB492/B040-ml0r antibody

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle and A372-MB492/B040-ml0r were administered at the doses shown in Table 58. Two antibody administrations by tail vein route were performed on the day of grouping and three days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of A372-MB492/B040-ml0r group for pharmacodynamic study

[ Table 58]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. As a result, the anti-tumor effect of the A372-MB492/B040-mL0r antibody was observed at all doses (FIG. 62).

(6-4-13) antibody administration and sampling to assess systemic effects of A372-MB492/B040-ml0r antibody And FCM analysis

After establishing the MC38 transplant mouse model in reference example 6-4-1, vehicle, NS1-MB492 (non-switch antibody), and A372-MB492/B040-ml0r were administered as shown in Table 59. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Eight days after the initial administration, liver, lymph nodes and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with the MC38 cell line and lymphocyte fractions were prepared). For the spleen, only organ weights were measured. For lymph nodes, cell counts were performed only on the prepared lymphocyte fraction.

Group details for assessment of A372-MB492/B040-ml0r antibody systemic response

[ Table 59]

The systemic effect of the administered antibody was evaluated. When NS1-MB492 was administered, an increase in the weight of the spleen organ was observed, and when NS1-MB492 was administered, an increase in the measurement of the cell count of the lymphocyte fraction was observed. On the other hand, when A372-MB492/B040-mL0r was administered at doses of 1.5mg/kg, 3.0mg/kg and 7.5mg/kg, inhibition of organ enlargement was observed compared with NS1-MB492 (FIG. 63).

As a result of assessing T cell activation in the liver, increased expression of granzyme B was observed when NS1-MB492 and A372-MB492/B040-ml0r were administered compared to vehicle (FIG. 64).

(6-4-14) efficacy of antibodies A486-MB492/B167-ml0r and A488-MB492/B226-ml0r (anti-tumor effect) By) research on

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle, A486-MB492/B167-ml0r and A488-MB492/B226-ml0r were administered at the doses shown in Table 60. Grouping was performed 10 days after cell transplantation, and four antibody administrations were performed by tail vein route on days 11, 15, 18 and 22 after cell transplantation. For the vehicle, PBS containing 0.05% tween 20 was used.

Group details of study of drug efficacy of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r antibodies

[ Table 60]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. The results confirmed the antitumor effects of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r (FIG. 65).

(6-4-15) for assessment of systemic Effect of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r Antibody administration and sampling and FCM analysis of

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS1-MB492 (non-transformant antibody), A486-MB492/B167-ml0r and A488-MB492/B226-ml0r were administered as shown in Table 61. Grouping was performed 10 days after cell transplantation, and a total of four antibody administrations were performed by tail vein route on days 11, 15, 18 and 22 after cell transplantation. For the vehicle, PBS containing 0.05% tween 20 was used.

13 days after the initial administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with MC38 cell line and lymphocyte fractions were prepared). For the spleen, only organ weight measurements were made. For lymph nodes, only the lymphocyte fraction was cell counted.

Group details to assess systemic response of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r antibodies

[ Table 61]

As a result of cell count measurement and spleen weight measurement on lymphocyte fraction of lymph node, when NS1-MB492 was administered, cell count of lymphocytes and increase in spleen weight were observed. On the other hand, it was observed that the increase in lymphocyte count and spleen weight was suppressed when the A486-MB492/B167-ml0r and the A488-MB492/B226-ml0r were administered, as compared with that when the NS1-MB492 was administered (FIG. 66).

As a result of the evaluation of T cell activation in the liver, an increase in the percentage of CD8 α positive cells was observed when NS1-MB492, A486-MB492/B167-ml0r and A488-MB492/B226-ml0r were administered compared to vehicle (FIG. 67).

From the above, it is known that the switch antibody inhibits T cell activation in lymph nodes and spleen, compared to the non-switch antibody.

(6-4-16) study of drug efficacy (anti-tumor Effect) of A489-MB492/B223-ml0r antibody

After establishing the MC38 transplant mouse model in reference example 6-4-1, the vehicle and A489-MB492/B223-ml0r were administered at the doses shown in Table 62. Antibody administration was performed four times by tail vein route the second day after cohort and 4, 7 and 10 days after cohort. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of group for A489-MB492/B223-ml0r pharmacodynamic study

[ Table 62]

The antitumor effect of each group was evaluated by tumor volume calculated as described in reference example 6-4-1. As a result, the antitumor effect was confirmed at 7.5mg/kg administration (FIG. 68).

(6-4-17) antibody administration and sampling for evaluation of systemic Effect of A489-MB492/B223-ml0r antibody And FCM analysis

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS1-MB492 (non-switch antibody), and A489-MB492/B223-ml0r were administered as shown in Table 63. Antibody administration was performed 3 times by tail vein route on the day of grouping, 3 days and 6 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

7 days after the initial administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with the MC38 cell line and lymphocyte fractions were prepared). For spleen and lymph nodes, cell count measurements were performed only on the prepared lymphocyte fraction.

Group details for evaluation of A489-MB492/B223-ml0r antibody systemic response

[ Table 63]

As a result of cell count measurements on lymphocyte fractions of lymph nodes and spleen, an increase in cell count was observed when NS1-MB492 was administered. On the other hand, when A489-MB492/B223-ml0r was administered, inhibition of increase in lymphocyte count was observed, compared to when NS1-MB492 was administered (FIG. 69).

As a result of the evaluation of T cell activation in the liver, an increase in the percentage of CD8 α positive cells was observed when NS1-MB492 and A489-MB492/B223-ml0r were administered compared to vehicle (FIG. 70).

From the above, it was confirmed that the conversion antibody inhibited the activation of T cells in lymph nodes and spleen, compared with the non-conversion antibody.

(6-4-18) study of drug efficacy of A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r antibodies (anti-tumor Tumor action)

After establishing the MC38 transplantation mouse model in reference example 6-4-1, the vehicle, A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r were administered at the doses shown in Table 64. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Group details of the study of the drug Effect of A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r antibodies

[ Table 64]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. The results show that A548-mIgG1/B256-ml0r administered at 37.5mg/kg and 100mg/kg has an anti-tumor effect. A551-mIgG1/B256-ml0r also showed significant anti-tumor effect at 100mg/kg (FIG. 71).

(6-4-19) systemic evaluation of A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r antibodies Antibody administration and sampling of action and FCM analysis

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS1-mIgG1 (non-switching antibody), A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r were administered as shown in Table 65. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Five days after the initial administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from mice transplanted with MC38 cell line and lymphocyte fractions were prepared). For spleen and lymph nodes, only organ weight measurements were made.

Group details for evaluating systemic reactions of A548-mIgG1/B256-ml0r antibody and A551-mIgG1/B256-ml0r antibody

[ Table 65]

As a result of evaluating the organ weights of lymph nodes and spleen, when NS1-mIgG1 was administered at 0.3mg/kg, 1.5mg/kg, and 7.5mg/kg, an increase in organ weight was observed. On the other hand, when A548-mIgG1/B256-ml0r or A551-mIgG1/B256-ml0r was administered at any one of the doses of 1.5mg/kg, 7.5mg/kg, 37.5mg/kg, and 100mg/kg, no excessive organ enlargement was observed (FIG. 72).

As a result of evaluating T cell activation in the liver, increased expression of both granzyme B and PD-1 was observed when NS1-mIgG1 was administered at a dose of 0.3 mg/kg. On the other hand, when A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r were administered at any one of the doses of 1.5mg/kg, 7.5mg/kg, 37.5mg/kg, and 100mg/kg, expression was suppressed (FIG. 73).

Thus, the switch antibody inhibits activation of T cells in lymph nodes, spleen and liver compared to the non-switch antibody.

(6-4-20) study of drug efficacy (anti-tumor Effect) of A551-MB110/B379-ml0r antibody

After establishing the MC38 transplantation mouse model in reference example 6-4-1, the vehicle and A551-MB110/B379-ml0r were administered at the doses shown in Table 66. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

Detailed information of group for A551-MB110/B379-ml0r antibody pharmacodynamic study

[ Table 66]

The antitumor effect of each group was evaluated by the calculated tumor volume as described in reference example 6-4-1. As a result, an antitumor effect was confirmed at any dose (fig. 74).

(6-4-21) antibody administration and sampling and FCM analysis for assessment of systemic Effect

After establishing the MC38 transplantation mouse model in reference example 6-4-1, vehicle, NS1-mIgG1 (non-switching antibody), and A551-MB110/B379-ml0r were administered as shown in Table 67. Two antibody administrations were performed by tail vein route on the day of grouping and 3 days after grouping. For the vehicle, PBS containing 0.05% tween 20 was used.

On day 5 after the initial administration, liver, lymph node and spleen were collected in the manner shown in reference example 6-4-2 (various organs were collected from MC38 cell line-transplanted mice and lymphocyte fractions were prepared), and FCM analysis was performed in the manner shown in reference example 6-4-3 (FCM analysis was performed using lymphocyte fractions of various organs).

Group details for assessment of A551-MB110/B379-ml0r antibody systemic response

[ Table 67]

As a result of evaluating the organ weights of the lymph node and spleen, an increase in organ weight was observed when NS1-mIgG1 was administered at 7.5 mg/kg. On the other hand, when A551-MB110/B379-mL0r was administered at any one of the doses of 0.83mg/kg, 2.5mg/kg, 7.5mg/kg, and 22.5mg/kg, no organ enlargement was observed (FIG. 75). Furthermore, in the evaluation of T cell activation in spleen assessed by FCM, the increase in activation markers (PD-1, ICOS, granzyme B) observed after NS1-mIgG1 administration was not confirmed when A551-MB110/B379-ml0r was administered at any dose (FIG. 76).

In assessing T cell activation in the liver, increased expression of both PD-1 and ICOS was observed when NS1-mIgG1 was administered at a dose of 7.5 mg/kg. On the other hand, when A551-MB110/B379-ml0r was administered at 0.83mg/kg and 2.5mg/kg, the expression was suppressed to the excipient level, and when A551-MB110/B379-ml0r was administered at 7.5mg/kg, the suppression of the expression was even more than NS1-mIgG1 (FIG. 77).

Thus, the switch antibody inhibits the activation of T cells in lymph nodes, spleen and liver compared to a non-switch antibody.

Reference example 7: production of modified Fc capable of enhancing agonist activity

(7-1) production of variants with increased Fc Gamma R binding Activity

The following sequences were combined with the heavy chain variable region of the switch antibody (A375; A372; A356; A486; A488; A489; A548; and A551) or the heavy chain variable region of the negative control antibody (IC 17HdK (SEQ ID NO: 152)): heavy chain constant region TT14 (SEQ ID NO: 149) (heavy chain constant region comprising T250V/T307P as amino acid modification and L234Y/P238D/V264I/a330K as amino acid modification in the Fc region comprising SEQ ID NO: 182) to increase Fc γ RIIb binding activity as described in WO2017104783, TT16 (SEQ ID NO: 150) comprising G237D amino acid mutation introducing heavy chain constant region TT11, which mutation increases Fc γ RIIb binding activity (heavy chain constant region comprising amino acid modifications of T250V/T307P and L234Y/P238D/a330K in the Fc region comprising SEQ ID NO: 182) as described in WO2017104783, which modifications increase Fc γ RIIb binding activity (positions of modifications are in EU numbering), P587 (SEQ ID NO: 151) as described in WO 163101, which increase Fc γ RIIb binding activity, which is a combination of heavy chain constant region with heavy chain constant region NO: 253, which constant region heavy chain constant region No. 93 has increased Fc γ binding activity as described in WO 163101.

[ Table 68]

Full length heavy chain	Heavy chain variable region (SEQ ID NO)	Heavy chain constant region (SEQ ID NO)
			A375-TT14	A375(SEQ ID NO:43)	TT14(SEQ ID NO:149)
A375-TT16	A375(SEQ ID NO:43)	TT16(SEQ ID NO:150)
			A375-P587	A375(SEQ ID NO:43)	P587(SEQ ID NO:151)
A372-P253	A372(SEQ ID NO:44)	P253(SEQ ID NO:93)
			A356-TT16	A356(SEQ ID NO:45)	TT16(SEQ ID NO:150)
A356-P587	A356(SEQ ID NO:45)	P587(SEQ ID NO:151)
			A486-P253	A486(SEQ ID NO:46)	P253(SEQ ID NO:93)
A488-P253	A488(SEQ ID NO:48)	P253(SEQ ID NO:93)
			A489-P253	A489(SEQ ID NO:49)	P253(SEQ ID NO:93)
A548-P587	A548(SEQ ID NO:50)	P587(SEQ ID NO:151)
			A551-TT16	A551(SEQ ID NO:51)	TT16(SEQ ID NO:150)
A551-P587	A551(SEQ ID NO:51)	P587(SEQ ID NO:151)
			IC17HdK-TT16	IC17HdK(SEQ ID NO:152)	TT16(SEQ ID NO:150)
IC17HdK-P587	IC17HdK(SEQ ID NO:152)	P587(SEQ ID NO:151)
			IC17HdK-P253	IC17HdK(SEQ ID NO:152)	P253(SEQ ID NO:93)

The heavy chain variable region of the switch antibody (A375, A356 and A551) or the heavy chain variable region of the negative control antibody (IC 17HdK (SEQ ID NO: 152)) was combined with the following sequences: heavy chain constant region MY201 (SEQ ID NO: 153) (heavy chain constant region containing G236N/H268D/a330K in the Fc region comprising the amino acid sequence of SEQ ID NO:182 as an amino acid modification that increases Fc γ RIIb binding activity (modification positions are all in EU numbering)), or MY518 (SEQ ID NO: 154) (heavy chain constant region containing L235W/G236N/H268D/Q295L/K326T/a330K in the Fc region comprising the amino acid sequence of SEQ ID NO:182 as an amino acid modification that enhances Fc γ RIIb binding activity (modification positions are all in EU numbering), which is described in WO 2017104783 and has increased binding activity with Fc γ R; wherein K214R modification has been introduced into heavy chain constant region MY201a (SEQ ID NO: 155) or MY518a (SEQ ID NO: 156) in MY201 or MY518, or wherein L235W modification has been introduced into heavy chain constant region MY201a (SEQ ID NO: 157) that has the heavy chain constant region combined with the heavy chain constant region genes as follows (ph NO: 157).

[ Table 69]

Full length heavy chain	Heavy chain variable region (SEQ ID NO)	Heavy chain constant region (SEQ ID NO)
			A375-MY201aPh	A375(SEQ ID NO:43)	MY201aPh(SEQ ID NO:157)
A375-MY518	A375(SEQ ID NO:43)	MY518(SEQ ID NO:154)
			A375-MY518a	A375(SEQ ID NO:43)	MY518a(SEQ ID NO:156)
A356-MY518	A356(SEQ ID NO:45)	MY518(SEQ ID NO:154)
			A551-MY201a	A551(SEQ ID NO:51)	MY201a SEQ ID NO:155)
A551-MY201aPh	A551(SEQ ID NO:51)	MY201aPh(SEQ ID NO:157)
			A551-MY518	A551(SEQ ID NO:51)	MY518(SEQ ID NO:154)
A551-MY518a	A551(SEQ ID NO:51)	MY518a(SEQ ID NO:156)
			IC17HdK-MY201	IC17HdK(SEQ ID NO:152)	MY201(SEQ ID NO:153)
IC17HdK-MY201aPh	IC17HdK(SEQ ID NO:152)	MY201aPh(SEQ ID NO:157)
			IC17HdK-MY518	IC17HdK(SEQ ID NO:152)	MY518(SEQ ID NO:154)
IC17HdK-MY518a	IC17HdK(SEQ ID NO:152)	MY518a(SEQ ID NO:156)

As shown in Table 70, the objective antibody was expressed and purified using a method known to those skilled in the art by combining the above-described antibody heavy chain gene with the antibody light chain gene of a conversion antibody (B040-Lamlib, B167-Lamlib, B226-Lamlib, B223-Lamlib, B256-Lamlib, and B379-Lamlib prepared in reference examples 5-2 and 5-3) or with the antibody light chain gene of a negative control antibody (IC 17L-k0 produced by combining the light chain variable region IC17L (SEQ ID NO: 158) with the human kappa chain k0 (SEQ ID NO: 141) as a light chain constant region).

Variants with increased Fc γ R binding activity

[ Table 70]

(7-2) production of variants with increased pI by amino acid modification of constant region

The heavy chain constant region was generated by combining the heavy chain constant region having increased Fc γ R binding activity prepared in reference example 7-1 with Q311R, P343R and D413K, wherein the Q311R, P343R and D413K are amino acid mutations that increase pI without greatly changing Fc γ R binding activity as described in WO 2017046994.

Specifically, genes having heavy chain constant regions with amino acid mutations introduced into the heavy chain constant regions of the respective antibodies were generated as shown below.

Introduction of P343R/D413K into MY518 (SEQ ID NO: 154): SCF025 (SEQ ID NO: 64)

Introduction of P343R/D413K into MY518a (SEQ ID NO: 156): SCF025a (SEQ ID NO: 65)

Introduction of P343R/D413K into TT14 (SEQ ID NO: 149): SCF027 (SEQ ID NO: 66)

Introduction of P343R/D413K into TT16 (SEQ ID NO: 150): SCF028 (SEQ ID NO: 67)

Introduction of Q311R/P343R into MY518 (SEQ ID NO: 154): SCF030 (SEQ ID NO: 68)

Introduction of Q311R/P343R into TT14 (SEQ ID NO: 149): SCF032 (SEQ ID NO: 69)

Introduction of Q311R/P343R into TT16 (SEQ ID NO: 150): SCF033 (SEQ ID NO: 70)

Introduction of P343R into MY518 (SEQ ID NO: 154): SCF039 (SEQ ID NO: 71)

Introduction of P343R into MY518a (SEQ ID NO: 156): SCF039a (SEQ ID NO: 72)

Introduction of D413K into MY518 (SEQ ID NO: 154): SCF040 (SEQ ID NO: 73)

Introduction of P343R into MY201a (SEQ ID NO: 155): SCF041a (SEQ ID NO: 74)

Introduction of P343R into MY201aPh (SEQ ID NO: 157): SCF041aPh (SEQ ID NO: 75)

Introduction of D413K into MY201a (SEQ ID NO: 155): SCF042a (SEQ ID NO: 76)

Introduction of P343R/D413K into MY201a (SEQ ID NO: 155): SCF043a (SEQ ID NO: 77)

Introduction of P343R/D413K into MY201aPh (SEQ ID NO: 157): SCF043aPh (SEQ ID NO: 78)

Introduction of Q311R into MY201a (SEQ ID NO: 155): SCF056a (SEQ ID NO: 79)

Introduction of Q311R into MY201aPh (SEQ ID NO: 157): SCF056aPh (SEQ ID NO: 80)

Introduction of Q311R/P343R into MY201a (SEQ ID NO: 155): SCF057a (SEQ ID NO: 81)

Introduction of Q311R/P343R into MY201aPh (SEQ ID NO: 157): SCF057aPh (SEQ ID NO: 82)

Introduction of Q311R/D413K into MY201a (SEQ ID NO: 155): SCF059a (SEQ ID NO: 83)

Introduction of Q311R/D413K into MY201aPh (SEQ ID NO: 157): SCF059aPh (SEQ ID NO: 84)

Introduction of Q311R into MY518a (SEQ ID NO: 156): SCF060a (SEQ ID NO: 85)

By combining the heavy chain constant region gene produced herein with the gene of the heavy chain variable region A375 (SEQ ID NO: 43) or A551 (SEQ ID NO: 51), antibody heavy chain genes as shown in Table 71 below were produced.

[ Table 71]

Full length heavy chain	Heavy chain variable region (SEQ ID NO)	Heavy chain constant region (SEQ ID NO)
			A375-SCF025	A375(SEQ ID NO:43)	SCF025(SEQ ID NO:64)
A375-SCF027	A375(SEQ ID NO:43)	SCF027(SEQ ID NO:66)
			A375-SCF028	A375(SEQ ID NO:43)	SCF028(SEQ ID NO:67)
A375-SCF030	A375(SEQ ID NO:43)	SCF030(SEQ ID NO:68)
			A375-SCF032	A375(SEQ ID NO:43)	SCF032(SEQ ID NO:69)
A375-SCF033	A375(SEQ ID NO:43)	SCF033(SEQ ID NO:70)
			A375-SCF039	A375(SEQ ID NO:43)	SCF039(SEQ ID NO:71)
A375-SCF040	A375(SEQ ID NO:43)	SCF040(SEQ ID NO:73)
			A375-SCF041aPh	A375(SEQ ID NO:43)	SCF041aPh(SEQ ID NO:75)
A375-SCF043aPh	A375(SEQ ID NO:43)	SCF043aPh(SEQ ID NO:78)
			A375-SCF056aPh	A375(SEQ ID NO:43)	SCF056aPh(SEQ ID NO:80)
A375-SCF057aPh	A375(SEQ ID NO:43)	SCF057aPh(SEQ ID NO:82)
			A375-SCF059aPh	A375(SEQ ID NO:43)	SCF059aPh(SEQ ID NO:84)
A375-SCF060a	A375(SEQ ID NO:43)	SCF060a(SEQ ID NO:85)
			A551-SCF025a	A551(SEQ ID NO:51)	SCF025a(SEQ ID NO:65)
A551-SCF039a	A551(SEQ ID NO:51)	SCF039a(SEQ ID NO:72)
			A551-SCF041a	A551(SEQ ID NO:51)	SCF041a(SEQ ID NO:74)
A551-SCF041aPh	A551(SEQ ID NO:51)	SCF041aPh(SEQ ID NO:75)
			A551-SCF043a	A551(SEQ ID NO:51)	SCF043a(SEQ ID NO:77)
A551-SCF043aPh	A551(SEQ ID NO:51)	SCF043aPh(SEQ ID NO:78)
			A551-SCF056a	A551(SEQ ID NO:51)	SCF056a(SEQ ID NO:79)
A551-SCF056aPh	A551(SEQ ID NO:51)	SCF056aPh(SEQ ID NO:80)
			A551-SCF057a	A551(SEQ ID NO:51)	SCF057a(SEQ ID NO:81)
A551-SCF057aPh	A551(SEQ ID NO:51)	SCF057aPh(SEQ ID NO:82)
			A551-SCF059a	A551(SEQ ID NO:51)	SCF059a(SEQ ID NO:83)
A551-SCF059aPh	A551(SEQ ID NO:51)	SCF059aPh(SEQ ID NO:84)
			A551-SCF060a	A551(SEQ ID NO:51)	SCF060a(SEQ ID NO:85)

In addition, by combining these antibody heavy chain genes with the B167-Lamlib, B256-Lamlib and B379-Lamlib genes produced in reference example 5-3 as antibody light chain genes, the desired antibodies were expressed and purified as shown in Table 72 by methods known to those skilled in the art.

[ Table 72]

(7-3) Generation of control antibodies for comparison of agonist Activity

Antibodies (IC 17HdK-MY201/IC17L-k0; IC17HdK-MY201aPh/IC17L-k0; IC17HdK-MY518/IC17L-k0; IC17HdK-MY518a/IC17L-k0; IC17HdK-P253/IC17L-k0; IC17HdK-P587/IC; IC17HdK-TT16/IC17L-k 0) were generated as control antibodies for comparison of agonist activity, with the heavy chain IC17 HdHK (SEQ ID NO: 152) variable region generated in reference example 7-1. In addition, the objective antibody (IC 17HdK-G4d/IC17L-k 0) was expressed and purified by a method known in the art by combining the antibody light chain IC17L-k0 (SEQ ID NO:158, variable region; SEQ ID NO:141, constant region) gene with the antibody heavy chain gene IC17HdK-G4d gene produced by combining the heavy chain variable region IC17HdK (SEQ ID NO: 152) gene with the G4d gene (SEQ ID NO: 159), in which Gly and Lys at the C-terminal end of the human IgG4 heavy chain constant region have been removed in the G4d gene. These control antibodies were used in reference example 5.

(7-4) evaluation of human Fc γ receptor binding of the variant, wherein the variant is modified by amino acid modification of the constant region With increased pI

Biacore T200 (GE medical) was used to evaluate the binding activity to the respective human Fc γ receptor (hereinafter referred to as Fc γ R). The measurement was performed at 25 ℃ using 50mM phosphate, 150mM NaCl, 0.05w/v% -P20, pH 7.4 as a buffer. Using a fixed CaptureSelect ^TM The sensor chip of human Fab λ kinetic biotin conjugate (seimer femtology) acts as a ligand capture molecule, capturing approximately 1000RU of antibody. In the measurement buffer, the Fc γ RIa of human Fc γ R was diluted to 8nM, and the other Fc γ R was diluted to 1000nM, and then it was interacted with the captured antibody. The binding activity of each antibody to Fc γ R was evaluated by calculating the amount of Fc γ R binding (RU) per unit amount of antibody using Biacore T200 evaluation software 2.0.

The extracellular domain of Fc γ R was prepared by the following method. First, the gene synthesis of the extracellular domain of Fc γ R is performed by methods known to those skilled in the art. At that time, fc γ R sequences were prepared according to NCBI enrollment data. Specifically, sequences that generate Fc γ RI based on NCBI accession No. NM _000566.3, fc γ RIIa based on NCBI accession No. NM _001136219.1, fc γ RIIb based on NCBI accession No. NM _004001.3, and Fc γ RIIIa based on NCBI accession No. NM _ 001127593.1; his tags were then added to their C-termini. Polymorphic sites of Fc γ RIIa were generated by reference to j.exp.med.,1990, 172, 19-25; polymorphic sites of Fc γ RIIIa were generated by reference to j.clin.invest.,1997, 100, 1059-1070. The resulting gene fragment is inserted into an animal cell expression vector to produce an expression vector. The resulting expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cells, and the target protein was expressed. The culture supernatant was collected and then passed through a 0.22 μm filter, which was purified in principle by the following four steps. The first step was performed by cation exchange column chromatography (SP sepharose FF), the second by His-tagged affinity column chromatography (HisTrap HP), the third by gel filtration column chromatography (Superdex 200) and the fourth by sterile filtration. However, for Fc γ RI, anion exchange column chromatography was performed in the first step using Q sepharose FF. The concentration of the purified protein was calculated by measuring the absorbance at 280nm using a spectrophotometer and the absorption coefficient calculated from the obtained values by the method of PACE et al (protein science, 1995,4, 2411-2423).

Table 73 shows the amount of binding per unit amount of antibody, and the amount of binding relative to A375-G1T 3/B167-Lamlib. All antibodies in table 73 show that the amount of binding per unit amount of antibody to human Fc γ RIIb is greater than the amount of binding of a375-G1T3/B167-Lamlib, when the amount of binding of a375-G1T3/B167-Lamlib to human Fc γ RIIb is set to 1.0, the relative value of the amount of binding is 2.85-14.26. The relative values of human Fc γ RIIb binding and binding per unit amount of antibody were similar in the presence or absence of amino acid modifications of the constant region to increase pI. Furthermore, the amount of binding of antibody comprising the L235W modification to hFcgRIIa was reduced compared to A375-TT 14/B167-Lamlib.

Binding Activity of Each human Fc γ R

[ Table 73]

(7-5) evaluation of human FcRn binding of variant having amino acid modification of constant region Increased pI

Binding activity to human FcRn was assessed using Biacore T200 (GE medical). The measurement was carried out at 25 ℃ using 50mM phosphate, 150mM NaCl, 0.05w/v% -P20, pH 6.0 as a buffer. The human FcRn protein used in this assay was prepared by the procedure described in WO 2010107110. Using a fixed CaptureSelect ^TM The sensor chip of human Fab λ kinetic biotin conjugate (seimer femhel technology) as a ligand capture molecule, captured approximately 400RU of antibody, and then bound to human FcRn diluted in measurement buffer. By using Biacore T 200 evaluation software 2.0 the activity of each antibody for binding to FcRn was evaluated by calculating KD (mol/L) by a steady state model. KD values were similar in the presence or absence of amino acid modifications of the constant region to increase pI. Table 74 shows the KD (mol/L) for human FcRn for each antibody.

[ Table 74]

Abbreviation name	K _D (mol/L)
		A375-G1T3/B167-Lamlib	1.27E-06
A375-MY201a/B167-Lamlib	1.19E-06
		A375-MY201aPh/B167-Lamlib	1.18E-06
A375-MY518/B167-Lamlib	1.27E-06
		A375-MY518a/B167-Lamlib	1.32E-06
A375-TT11/B167-Lamlib	1.56E-06
		A375-TT14/B167-Lamlib	1.77E-06
A375-TT16/B167-Lamlib	1.51E-06
		A375-P587/B167-Lamlib	1.70E-06
A375-P253/B167-Lamlib	1.42E-06
		A375-SCF025/B167-Lamlib	1.10E-06
A375-SCF025a/B167-Lamlib	1.13E-06
		A375-SCF027/B167-Lamlib	1.33E-06
A375-SCF028/B167-Lamlib	1.27E-06
		A375-SCF030/B167-Lamlib	8.63E-07
A375-SCF032/B167-Lamlib	9.66E-07
		A375-SCF033/B167-Lamlib	8.74E-07
A375-SCF039/B167-Lamlib	1.09E-06
		A375-SCF039a/B167-Lamlib	1.11E-06
A375-SCF040/B167-Lamlib	1.46E-06
		A375-SCF041a/B167-Lamlib	1.10E-06
A375-SCF042a/B167-Lamlib	1.49E-06
		A375-SCF043a/B167-Lamlib	1.18E-06
A375-SCF056a/B167-Lamlib	9.56E-07
		A375-SCF057a/B167-Lamlib	9.28E-07
A375-SCF059a/B167-Lamlib	1.03E-06
		A375-SCF060a/B167-Lamlib	1.01E-06
A375-SCF041aPh/B167-Lamlib	1.13E-06
		A375-SCF043aPh/B167-Lamlib	1.21E-06
A375-SCF056aPh/B167-Lamlib	1.00E-06
		A375-SCF057aPh/B167-Lamlib	9.09E-07
A375-SCF059aPh/B167-Lamlib	1.05E-06

Reference example 8: evaluation of modified anti-human CD137 antibodies using a 4-1BB Jurkat reporter assay In vitro ATP-dependent and ADP-dependent CD137 agonist activity

GloResponse ^TM NF-. Kappa.B Luc2/4-1BB Jurkat cell line (Promega, CS 196004) was used to measure the in vitro activity of the variants produced in reference example 7-2 (Table 72). To each well of the 384-well plate, 10. Mu.L of the assay medium (99% RPMI,1% FBS) was added at a concentration of 4X 10 ⁵ Fc γ RIIB CHO-K1 cells (Promega) per mL. Subsequently, 10. Mu.L of an antibody solution containing ADP or an antibody solution containing ATP, or an antibody solution containing no ATP or ADP was added to each well. Then, 10. Mu.L of the measurement medium (99% RPMI,1% FBS) was prepared as 2X 10 ⁶ GloResponse/mL ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell lines were added to each well. The final concentration of ADP was 10. Mu.M, and the final concentration of ATP was 10. Mu.M. The plate was 5% CO ₂ Standing in an incubator at 37 ℃ for 6 hours, and standing at room temperature for 15 minutes; 30 μ L of Bio-Glo reagent was added to each well. The Bio-Glo luciferase assay system (buffer and substrate) was used for the Bio-Glo reagent. Subsequently, the luminescence amount of each well was measured with a microplate reader. The luminescence value of each well divided by the luminescence value of wells without antibody addition is defined as "relative light units" (fold induction), which is used as an index to evaluate the CD137 agonist activity of each antibody.

The results are shown in fig. 78. From FIG. 78, it was confirmed that A375-P587/B167-LamLib, A551-P587/B256-LamLib, A551-P587/B379-LamLib, A375-SCF041aPh/B167-LamLib, A551-SCF041aPh/B256-LamLib, A551-SCF041aPh/B379-LamLib, A375-SCF043aPh/B167-LamLib, A551-SCF043aPh/B256-LamLib, A551-SCF043aPh/B379-LamLib, A375-05SCF 7aPh/B167-LamLib, A551-SCF057aPh/B256-LamLib and A551-SCF057aPh/B379-LamLib showed human agonist activities in ATP-and ADP-dependent manner.

Reference example 9: potentiation of agonist activity by modified anti-CD 137 antibodies

(9-1) preparation of antibody for evaluation

With respect to the introduction of various amino acid modifications that increase pI into the heavy chain constant region with increased binding activity to fcyriib, the effect of such amino acid modifications on CD137 agonist activity as well as plasma kinetics and pharmacodynamics in hCD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mice was evaluated. First, A375-MY201aPh/B167-Lamlib, A375-SCF041aPh/B167-Lamlib, A375-SCF057aPh/B167-Lamlib, and IC17HdK-MY201aPh/IC17L-k0 were prepared as described in reference example 7-1, reference example 7-2, and reference example 7-3.

(9-2) evaluation of ATP dependence of modified anti-human CD137 antibodies in vitro Using 4-1BB Jurkat reporter Gene assay Sexual CD137 agonist activity

With respect to the introduction of various amino acid modifications that increase pI into the heavy chain constant region with enhanced binding activity to Fc γ RIIb, to evaluate the effect of such amino acid modifications on CD137 agonist activity, CD137 agonist activity of a375-MY201 ah/B167-Lamlib, a375-SCF041 ah/B167-Lamlib, a375-SCF057 ah/B167-Lamlib, and IC17HdK-MY201 ah/IC 17L-k0 was evaluated.

Use of GloResponse ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell line (Promega, CS 196004) measures the in vitro activity of the produced antibodies. To each well of a 96-well plate, media was added at a concentration of 5 × 10 ⁴ 200. Mu.L of Fc γ RIIB CHO-K1 cells (Promega) per mL, and the plates were scored at 5% CO ₂ The incubator was left overnight at 37 ℃. CHO medium (90% Ham's F12, 10% FBS) was used as the medium. Next, all the medium was removed by aspiration and the assay medium (99% RPMI,1% FBS) was used at 2X10 ⁶ 25 μ L of G prepared per mLloResponse ^TM NF-. Kappa.B-Luc 2/4-1BB Jurkat cell lines were added to each well. Next, 25. Mu.L of each antibody solution diluted with the assay medium was added at final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10. Mu.g/mL. Finally, 25 μ L ATP solution was added, which was diluted with assay medium to a final concentration of 250 μ M. The plate was 5% CO ₂ The incubator was left at 37 ℃ for 6 hours and then at room temperature for 15 minutes, and 75. Mu. LBio-Glo reagent was added to each well. The Bio-Glo luciferase assay system (buffer and substrate) was used for the Bio-Glo reagent. The relative light units of each well were then determined using a microplate reader. The luminescence value of each well divided by the luminescence value of the well to which no antibody was added was defined as "relative light unit" as an index for evaluating the CD137 agonist activity of each antibody.

The results are shown in fig. 79. The results show that CD137 agonist activity is increased by introducing various amino acid modifications that increase pI.

(9-3) pharmacokinetic study of modified anti-human CD137 antibody in mouse

(9-3-1) Generation of hCD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mice

First, a human CD137 knock-in mouse in which a mouse CD137 gene is replaced with a human CD137 gene was generated by introducing a human CD137 gene replacement vector into a mouse embryonic stem cell (ES cell) together with a Zinc Finger Nuclease (ZFN) targeting the mouse CD137 gene. Next, ZFN mRNA targeting the mouse Fcgr2b gene was microinjected into mouse zygotes, and mouse Fcgr2b knockout mice were generated by selecting mice that introduced mutations at the target site. In addition, BAC vectors in which the human FCGR2B gene was cloned were microinjected into mouse fertilized eggs, and human FCGR2B transgenic mice were generated by selecting those mice into which the genomic region of the human FCGR2B gene was introduced from among the obtained mice (Iwayanagi et al, J Immunol,2015, 195, 3198-3205).

By crossing the above three lines of mice, a "human CD137 knock-in Fcgr2B knock-out human Fcgr2B transgenic mouse" was established. This mouse was designated as hCD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mouse.

(9-3-2) measurement of anti-human in plasma in hCD137 KI/mFcyR 2 bKO/hFcyR 2bTg #90 mouse model Concentration of CD137 antibody

As shown in table 75, the respective anti-human CD137 antibodies were administered intravenously to CD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mice in a single dose. Blood was collected multiple times over a period of 5 minutes to 28 days after administration. The obtained blood was centrifuged to separate plasma. The plasma was kept in a refrigerator at below-20 ℃ until measured.

[ Table 75]

The concentration of each anti-human CD137 antibody in plasma was measured by an Electrochemiluminescence (ECL) method. hCD137 (nano Biological inc.) was diluted with PBS (-) and then added to a multi-assay 96-well plate (Meso Scale Diagnostics, LLC). The plate with the added hCD137 was shaken at room temperature for 1 hour, and the hCD137 was fixed on the plate. For blocking, a PBS solution containing 1mM ADP, 1% BSA and 0.05% Tween 20 was then added and the plate was shaken at room temperature for 1 hour. Calibration curves for the respective anti-human CD137 antibodies were prepared at plasma concentrations of 640, 320, 160, 80, 40, 20, and 10 ng/mL. Plasma samples diluted with PBS solution containing 1mM ADP, 1% BSA and 0.05% Tween 20 and calibration curve samples were added to hCD137 fixation plates. The plates were then shaken at room temperature for 1 hour, and then an antibody specifically recognizing the constant region of an anti-human CD137 antibody described in WO 2019112027 was added as a secondary antibody. The plate was further shaken at room temperature for 1 hour, and then a SULFO-TAG-labeled goat anti-rabbit antibody (Meso Scale Diagnostics, LLC) was added thereto. The plate was further shaken at room temperature for 1 hour and a two-fold dilution of read buffer T (Meso Scale Diagnostics, LLC) containing 1mM ADP was added thereto. The concentration of each antibody in the plasma of mice was measured by detecting SULFO-TAG with a SECTOR imager (Meso Scale Diagnostics, LLC). Concentrations of each antibody in mouse plasma were calculated using SOFTmax PRO (Molecular Devices).

The results are shown in fig. 80. A375-SCF041aPh/B167-Lamlib showed faster elimination than A375-MY201 aPh/B167-Lamlib. This is thought to be because the heavy chain constant region of A375-SCF041aPh/B167-Lamlib introduces amino acid modifications that increase the pI.

(9-4) evaluation of drug efficacy of modified anti-human CD137 antibody in mice

(9-4-1) preparation of mouse model for transplantation of cell line and syngeneic tumor line

As the cells, there was used a mouse lung cancer cell-derived cell line LLC1[ LL/2 (alias: LLC 1), dealer: ATCC, catalog No.: CRL-1642]The LLC1/OVA/GPC3 clone C5 (LLC 1/OVA/GPC 3) cell line was produced. hCD137KI/mFc γ R2bKO/hFc γ R2bTg #90 mice (11 weeks old, female) described in (19-3-1) above were used as mice. LLC1/OVA/GPC3 cell lines were maintained and passaged in RPMI1640 medium (Sigma-Aldrich, co.LLC) containing 9.8% fetal bovine serum (Sigma-Aldrich, co.LLC), 0.44mg/mL G418 (Nacalai Tesque, inc.), and 0.88mg/mL Zeocin (Semmer Feishell technologies, ltd.). LLC1/OVA/GPC3 cell line is transplanted subcutaneously into the abdomen of mouse until the tumor volume reaches 250-500mm ³ Then, the model is considered to have been formed. After model formation, LLC1/OVA/GPC3 cell lines were transplanted into groups of mice, which were then administered vehicle and the corresponding anti-human CD137 antibody, respectively.

(9-4-2) preparation and administration of an agent to be administered and tumor measurement

On days 11 and 14 post-tumor transplantation, A375-MY201aPh/B167-Lamlib or A375-SCF041aPh/B167-Lamlib prepared with PBS containing 0.05% Tween 20 to the doses shown in Table 76 was administered to the LLC1/OVA/GPC3 cell line transplantation model. PBS containing 0.05% tween 20 was administered to the vehicle group. The prepared administration solution was administered through the tail vein at a dose of 10 mL/kg.

Measurement of antitumor Effect in LLC1/OVA/GPC3 cell line transplantation model

[ Table 76]

To evaluate the antitumor effect, tumor volumes were measured 1 to 2 times per week. Tumor volume was calculated by the following equation. Tumor volume (mm) ³ ) = length (mm) x width (mm)/2

As a result, the results of reference example (9-3-2) showed that the blood concentration of the mice administered with A375-SCF041aPh/B167-Lamlib was less changed than that of A375-MY201 aPh/B167-Lamlib. However, A375-SCF041aPh/B167-Lamlib showed stronger antitumor effects than A375-MY201aPh/B167-Lamlib (FIG. 81).

Based on the above results in this mouse model, the anti-tumor effect of the anti-human CD137 antibody was increased by introducing an amino acid modification that increases pI into the heavy chain constant region.

The foregoing invention has been described in some detail by way of examples and illustrations for purposes of promoting a clear understanding, and the descriptions and illustrations herein should not be construed as limiting the scope of the invention. All disclosures of the patent and scientific literature cited throughout this document are expressly incorporated herein by reference.

[ Industrial Applicability ]

An anticancer agent comprising the anti-CD 137 antigen-binding molecule of the present disclosure as an active ingredient, a combination therapy using such an anticancer agent comprising an anti-CD 137 antigen-binding domain as an active ingredient in combination with another anticancer agent, a pharmaceutical composition for use in the combination therapy, and the like are suitable for the development, manufacture, provision, and use of a drug having an immune cell activating effect, a cytotoxic effect, or an antitumor effect but having a lower effect or less side effects on non-tumor tissues (e.g., normal tissues).

Sequence listing

<110> China and foreign pharmaceuticals corporation

<120> anti-CD 137 antigen binding molecules for cancer therapy

<130> C1-A2003P

<150> JP 2020-21275

<151> 2020-02-12

<150> JP 2020-140489

<151> 2020-08-21

<160> 183

<170> PatentIn version 3.5

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<213> Intelligent (Homo sapiens)

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Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 2

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<400> 2

Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro Gln

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<213> mouse (Mus musculus)

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Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 4

<211> 165

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<213> mouse (Mus musculus)

<400> 4

Ala Val Gln Asn Ser Cys Asp Asn Cys Gln Pro Gly Thr Phe Cys Arg

1 5 10 15

Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser

20 25 30

Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr

35 40 45

Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu

50 55 60

Cys Ile Glu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu

65 70 75 80

Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr

85 90 95

Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly

115 120 125

Thr Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser Phe Ser

130 135 140

Pro Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His

145 150 155 160

Ser Leu Gln Val Leu

165

<210> 5

<211> 254

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<213> cynomolgus monkey (Macaca fascicularis)

<400> 5

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Lys Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Ile Ser Gly Tyr His Cys Leu Gly

85 90 95

Ala Glu Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Phe Phe Leu Ala

180 185 190

Leu Thr Ser Thr Val Val Leu Phe Leu Leu Phe Phe Leu Val Leu Arg

195 200 205

Phe Ser Val Val Lys Arg Ser Arg Lys Lys Leu Leu Tyr Ile Phe Lys

210 215 220

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

225 230 235 240

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250

<210> 6

<211> 165

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<213> Macaca fascicularis

<400> 6

Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro Gln Ile

165

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Thr Phe Thr Met Asn

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Ser Ile Ser Ser Lys Ser Thr Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

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Val

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Ser Ile Ser Ser His Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

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Val

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Ser Ile Ser Ser Ser Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

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Gly

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Ser Ile Ser Ser Lys Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

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Val

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Ser Ile Ser Ser Ser Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

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Leu

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Ser Ile Ser Ser Lys Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Phe Lys

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Gly

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Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Glu Gln Phe Lys

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Val

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Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Glu Ser Phe Lys

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Val

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Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Asp Ser Phe Lys

1 5 10 15

Val

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Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr

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Tyr Gly Pro Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr

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Tyr Gly Lys Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr

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Tyr Gly Ile Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr

1 5 10

<210> 21

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Thr Gly Thr Arg Tyr Asp Val Gly Tyr Tyr Glu Tyr Val Ser

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Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr Asn Tyr Val Ser

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Thr Gly Thr Arg Thr Asp Val Gly Phe Tyr Glu Tyr Val Ser

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<210> 24

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Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr Glu Tyr Val Ser

1 5 10

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Thr Gly Thr Ser Thr Asp Val Gly Tyr Tyr Glu Tyr Val Ser

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Glu Thr Ser Lys Arg Leu Ser

1 5

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Ser Ser Tyr Arg Tyr Glu His Gln Val Ser

1 5 10

<210> 28

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Ser Ser Tyr Arg Tyr Pro His Ile Val Ser

1 5 10

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Ser Ser Tyr Arg Tyr Glu Ala Gln Val Ser

1 5 10

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<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be Lys, his or Ser

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<221> misc_feature

<222> (6)..(6)

<223> Xaa can be Ser or Gly

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be Thr or Ser

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa can be Glu or Tyr

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa can be Asp or Glu

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa can be Ser or Gln

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa can be Val, gly or Leu

<400> 30

Ser Ile Ser Ser Xaa Xaa Xaa Tyr Ile Xaa Tyr Ala Xaa Xaa Phe Lys

1 5 10 15

Xaa

<210> 31

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<221> misc_feature

<222> (3)..(3)

<223> Xaa can be Ala, pro, lys or Ile

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<222> (6)..(6)

<223> Xaa can be Phe or Glu

<400> 31

Tyr Gly Xaa Lys Asn Xaa Leu Asn Trp Val Phe Asp Tyr

1 5 10

<210> 32

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<221> misc_feature

<222> (4)..(4)

<223> Xaa can be Arg or Ser

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<221> misc_feature

<222> (5)..(5)

<223> Xaa can be Tyr or Thr

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa can be Tyr or Phe

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa can be Glu or Asn

<400> 32

Thr Gly Thr Xaa Xaa Asp Val Gly Xaa Tyr Xaa Tyr Val Ser

1 5 10

<210> 33

<211> 10

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<222> (6)..(6)

<223> Xaa can be Glu or Pro

<220>

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<222> (7)..(7)

<223> Xaa can be His or Ala

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa can be Gln or Ile

<400> 33

Ser Ser Tyr Arg Tyr Xaa Xaa Xaa Val Ser

1 5 10

<210> 34

<211> 17

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Ser Ile Ser Ser Arg Ser Asn Tyr Lys Glu Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 35

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 36

<211> 14

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Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

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Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 38

<211> 11

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 39

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Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys

20

<210> 40

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Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

1 5 10 15

<210> 41

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Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

1 5 10 15

Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

20 25 30

<210> 42

<211> 10

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Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

1 5 10

<210> 43

<211> 122

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<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Ser Thr Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser His Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 45

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<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 122

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Pro Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Ser Thr Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Pro Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Leu Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Pro Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Pro Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Glu Gln Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Glu Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ile Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Asp Ser Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ile Lys Asn Glu Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Lys Gly Ser Tyr Ile Glu Tyr Ala Glu Gln Phe

50 55 60

Lys Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 54

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Arg Tyr Asp Val Gly Tyr Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 55

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 55

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 56

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 56

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Arg Tyr Asp Val Gly Tyr Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Pro

85 90 95

His Ile Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 57

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 57

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Arg Tyr Asp Val Gly Tyr Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

Ala Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 58

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 58

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Arg Thr Asp Val Gly Phe Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 59

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 59

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 60

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 60

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Tyr Tyr

20 25 30

Glu Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 61

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 61

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 62

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 62

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 63

<211> 106

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 63

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 64

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 65

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 65

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 66

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 66

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 67

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 67

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 68

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 68

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 69

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 69

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 70

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 70

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 71

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 71

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 72

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 72

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 73

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 73

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 74

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 74

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 75

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 75

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 76

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 76

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 77

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 77

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 78

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 78

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 79

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 79

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 80

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 80

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 81

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 81

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 82

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 82

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 83

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 83

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 84

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 84

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Lys Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 85

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 85

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 86

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 86

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

1 5 10 15

<210> 87

<211> 212

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 87

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln His His His His His His

180 185 190

Gly Gly Gly Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile

195 200 205

Glu Trp His Glu

210

<210> 88

<211> 641

<212> PRT

<213> Human gamma herpes virus 4 (Human gamma herpesvirus 4)

<400> 88

Met Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu

1 5 10 15

Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln

20 25 30

Arg Arg Gly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly

35 40 45

Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro

50 55 60

Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile

65 70 75 80

Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly

85 90 95

Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly

100 105 110

Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly

115 120 125

Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala

130 135 140

Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly

145 150 155 160

Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly

165 170 175

Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly

180 185 190

Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Ala Gly

195 200 205

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala

210 215 220

Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala

225 230 235 240

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly

245 250 255

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly

260 265 270

Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly

275 280 285

Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly

290 295 300

Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly

305 310 315 320

Gly Ala Gly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly

325 330 335

Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly

340 345 350

Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg

355 360 365

Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro

370 375 380

Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro

385 390 395 400

Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu

405 410 415

Tyr His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly

420 425 430

Ala Ile Glu Gln Gly Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr

435 440 445

Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp

450 455 460

Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn

465 470 475 480

Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg

485 490 495

Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly

500 505 510

Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala Ile

515 520 525

Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala

530 535 540

Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys

545 550 555 560

Tyr Phe Met Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys

565 570 575

Asp Ala Ile Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn

580 585 590

Ile Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro

595 600 605

Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly

610 615 620

Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln

625 630 635 640

Glu

<210> 89

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 89

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Asp Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys

165 170 175

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

180 185 190

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

195 200 205

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

210 215 220

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

225 230 235 240

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

245 250 255

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

260 265 270

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

275 280 285

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

290 295 300

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

305 310 315 320

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

325 330 335

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

340 345 350

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

355 360 365

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

370 375 380

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

385 390 395

<210> 90

<211> 442

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 90

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Asp Ile Glu Gly Arg Met

180 185 190

Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

195 200 205

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

210 215 220

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

225 230 235 240

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

245 250 255

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

260 265 270

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

275 280 285

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

290 295 300

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

305 310 315 320

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

325 330 335

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

340 345 350

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

355 360 365

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

370 375 380

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

385 390 395 400

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

405 410 415

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Leu Asn Asp Ile

420 425 430

Phe Glu Ala Gln Lys Ile Glu Trp His Glu

435 440

<210> 91

<211> 347

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 91

Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu

1 5 10 15

Leu Leu Met Leu Ala Gln Pro Ala Met Ala Met Lys Asp Asn Thr Val

20 25 30

Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn Gly Glu Phe His Ser Gly

35 40 45

Glu Gln Leu Gly Glu Thr Leu Gly Met Ser Arg Ala Ala Ile Asn Lys

50 55 60

His Ile Gln Thr Leu Arg Asp Trp Gly Val Asp Val Phe Thr Val Pro

65 70 75 80

Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile Gln Leu Leu Asn Ala Lys

85 90 95

Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser Val Ala Val Leu Pro Val

100 105 110

Ile Asp Ser Thr Asn Gln Tyr Leu Leu Asp Arg Ile Gly Glu Leu Lys

115 120 125

Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln Gln Ala Gly Arg Gly Arg

130 135 140

Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly Ala Asn Leu Tyr Leu Ser

145 150 155 160

Met Phe Trp Arg Leu Glu Gln Gly Pro Ala Ala Ala Ile Gly Leu Ser

165 170 175

Leu Val Ile Gly Ile Val Met Ala Glu Val Leu Arg Lys Leu Gly Ala

180 185 190

Asp Lys Val Arg Val Lys Trp Pro Asn Asp Leu Tyr Leu Gln Asp Arg

195 200 205

Lys Leu Ala Gly Ile Leu Val Glu Leu Thr Gly Lys Thr Gly Asp Ala

210 215 220

Ala Gln Ile Val Ile Gly Ala Gly Ile Asn Met Ala Met Arg Arg Val

225 230 235 240

Glu Glu Ser Val Val Asn Gln Gly Trp Ile Thr Leu Gln Glu Ala Gly

245 250 255

Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala Met Leu Ile Arg Glu Leu

260 265 270

Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu Gly Leu Ala Pro Tyr Leu

275 280 285

Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile Asn Arg Pro Val Lys Leu

290 295 300

Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile Ser Arg Gly Ile Asp Lys

305 310 315 320

Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly Ile Ile Lys Pro Trp Met

325 330 335

Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu Lys

340 345

<210> 92

<211> 442

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 92

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Lys Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Ile Ser Gly Tyr His Cys Leu Gly

85 90 95

Ala Glu Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Asp Ile Glu Gly Arg Met

180 185 190

Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

195 200 205

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

210 215 220

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

225 230 235 240

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

245 250 255

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

260 265 270

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

275 280 285

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

290 295 300

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

305 310 315 320

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

325 330 335

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

340 345 350

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

355 360 365

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

370 375 380

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

385 390 395 400

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

405 410 415

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Leu Asn Asp Ile

420 425 430

Phe Glu Ala Gln Lys Ile Glu Trp His Glu

435 440

<210> 93

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 93

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 94

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ala His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Leu Lys Asn Phe Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 95

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 95

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Met Lys Asn Lys Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 96

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Thr Tyr Ala Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Leu Lys Asn Ser Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 97

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 97

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Thr Lys Asn Ser Arg Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 98

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Arg Tyr Ile Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Lys Asn Tyr His Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 99

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 99

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Ile Lys Asn His Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 100

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 100

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Thr Tyr Ala Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Lys Lys Asn Met Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 101

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Leu Lys Asn Ser Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 102

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 102

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly His Lys Asn Met His Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 103

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Leu Lys Asn Glu Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 104

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 104

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ile Lys Asn Glu Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 105

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Arg Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Gly Lys Asn Ile Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 106

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 106

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Lys Lys Asn Asp Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 107

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Val Lys Asn Asn Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 108

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ala Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Val Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 109

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 110

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 110

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Leu Lys Asn Glu Arg Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 111

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 111

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Gly Lys Asn Asn Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 112

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 112

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ala Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Glu Met Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 113

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 113

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gly Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ala Tyr Asp

85 90 95

Ala Gln Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 114

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 114

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Asn Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Arg Tyr Asn

85 90 95

Asn Leu Leu Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 115

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 115

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ser Tyr Asp

85 90 95

Glu Ser Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 116

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 116

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ala Tyr Thr

85 90 95

Lys Asn Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 117

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 117

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Val Tyr Arg Tyr Asn

85 90 95

Asp Gln Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 118

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 118

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Ala Tyr Ser

85 90 95

Thr Ile Val Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 119

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 119

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Arg Tyr Arg

85 90 95

Asp Tyr Val Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 120

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 120

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ser Gly Ala

85 90 95

Ser Ile Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 121

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 121

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Arg Tyr Asp

85 90 95

Gln Asn Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 122

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 122

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Val Tyr Ser Tyr Ile

85 90 95

Tyr Tyr Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 123

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 123

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Val Tyr Arg Tyr Ser

85 90 95

Leu Gly Val Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 124

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 124

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ser Ser Asp

85 90 95

Thr His Val Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 125

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 125

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Glu Tyr Thr

85 90 95

Glu Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 126

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 126

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Glu Tyr Ile

85 90 95

Glu Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 127

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 127

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ala Tyr Thr

85 90 95

Lys Asn Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 128

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 128

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Tyr Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Arg Tyr Asp

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 129

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 129

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Tyr Thr Tyr Asn

85 90 95

Ala Leu Leu Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 130

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 130

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Glu Tyr Ile

85 90 95

Glu Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 131

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 131

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Tyr Asp

85 90 95

Gln Gln Leu Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 132

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 132

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 133

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 133

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Tyr Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 134

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 134

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro

450

<210> 135

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 135

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 136

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 136

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Arg Ser Asn Tyr Lys Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Ala Lys Asn Phe Leu Asn Trp Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 137

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 137

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Thr Ser Lys Arg Leu Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Arg Tyr Glu

85 90 95

His Gln Val Ser Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 138

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 138

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 139

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 139

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 140

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 140

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 141

<211> 107

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 141

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 142

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 142

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 143

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 143

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 144

<211> 324

<212> PRT

<213> mouse (Mus musculus)

<400> 144

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 145

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 145

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Asp Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 146

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 146

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Glu Pro Asn

100 105 110

Glu Val Glu Asp Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asp Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 147

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 147

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 148

<211> 106

<212> PRT

<213> mouse (Mus musculus)

<400> 148

Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp

20 25 30

Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro

35 40 45

Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu

65 70 75 80

Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val

85 90 95

Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser

100 105

<210> 149

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 149

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 150

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 150

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 151

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 151

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Asp Leu Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ala Glu Glu Asp Gly Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 152

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 152

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Tyr Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 153

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 153

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 154

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 154

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 155

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 155

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 156

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 156

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 157

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 157

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 158

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 158

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

100 105

<210> 159

<211> 325

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 159

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu

325

<210> 160

<400>

000

<210> 161

<400>

000

<210> 162

<400>

000

<210> 163

<400>

000

<210> 164

<400>

000

<210> 165

<400>

000

<210> 166

<400>

000

<210> 167

<400>

000

<210> 168

<400>

000

<210> 169

<400>

000

<210> 170

<400>

000

<210> 171

<400>

000

<210> 172

<400>

000

<210> 173

<400>

000

<210> 174

<400>

000

<210> 175

<400>

000

<210> 176

<400>

000

<210> 177

<400>

000

<210> 178

<400>

000

<210> 179

<400>

000

<210> 180

<400>

000

<210> 181

<400>

000

<210> 182

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> artificially synthesized sequence

<400> 182

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 183

<211> 189

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 183

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln His His His His His His Gly Gly Gly Gly Ser Gly Leu

165 170 175

Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

180 185

Claims

1. An anticancer agent comprising, as an active ingredient, an anti-CD 137 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;

(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;

(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.

2. An anti-cancer agent comprising an anti-CD 137 antigen binding molecule comprising any combination of a VH and a VL selected from the following (a) to (m):

(g) VH comprising the amino acid sequence of SEQ ID NO: 49; and VL comprising the amino acid sequence of SEQ ID NO 57;

(j) VH comprising the amino acid sequence of SEQ ID NO: 51; and VL comprising the amino acid sequence of SEQ ID NO: 60;

(k) VH comprising the amino acid sequence of SEQ ID NO: 52; and VL comprising the amino acid sequence of SEQ ID NO 60;

3. The anti-cancer agent of claim 1 or 2, wherein the anti-CD 137 antigen-binding molecule is a human, humanized, or chimeric antibody, or an antigen-binding fragment of any thereof.

4. The anti-cancer agent of any one of claims 1 to 3, wherein the anti-CD 137 antigen-binding molecule comprises an altered Fc region, and the altered Fc region comprises any one combination of amino acid alterations according to EU numbering selected from the group consisting of:

L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K；

K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K；

L234Y/P238D/T250V/V264I/T307P/A330K/P343R/D413K；

L234Y/P238D/V264I/A330K/P343R/D413K；

L234Y/G237D/P238D/T250V/T307P/A330K/P343R/D413K；

L234Y/G237D/P238D/A330K/P343R/D413K；

L235W/G236N/H268D/Q295L/K326T/A330K/Q311R/P343R；

L234Y/P238D/T250V/V264I/T307P/A330K/Q311R/P343R；

L234Y/P238D/V264I/A330K/Q311R/P343R；

L234Y/G237D/P238D/T250V/T307P/A330K/Q311R/P343R；

L234Y/G237D/P238D/A330K/Q311R/P343R；

L235W/G236N/H268D/Q295L/K326T/A330K/P343R；

K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R；

L235W/G236N/H268D/Q295L/K326T/A330K/D413K；

K214R/G236N/H268D/A330K/P343R；

K214R/L235W/G236N/H268D/A330K/P343R；

K214R/G236N/H268D/A330K/D413K；

K214R/G236N/H268D/A330K/P343R/D413K；

K214R/L235W/G236N/H268D/A330K/P343R/D413K；

K214R/G236N/H268D/A330K/Q311R；

K214R/L235W/G236N/H268D/A330K/Q311R；

K214R/G236N/H268D/A330K/Q311R/P343R；

K214R/L235W/G236N/H268D/A330K/Q311R/P343R；

K214R/G236N/H268D/A330K/Q311R/D413K；

K214R/L235W/G236N/H268D/A330K/Q311R/D413K; and

K214R/L235W/G236N/H268D/Q295L/K326T/A330K/Q311R。

5. an anti-cancer agent comprising an anti-CD 137 antigen-binding molecule comprising any combination of VH, VL, CH, and CL selected from the group consisting of (i) to (xxxviii) below:

(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;

(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;

(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;

(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;

(xiii) VH comprising the amino acid sequence of SEQ ID NO 43; CH comprising the amino acid sequence of SEQ ID NO: 84; VL comprising the amino acid sequence of SEQ ID NO 54; and CL comprising the amino acid sequence of SEQ ID NO 63;

6. The anti-cancer agent of any one of claims 1 to 5, which is administered to a cancer patient having: (i) Selected from the group consisting of B cells, dendritic cells, natural killer cells, macrophages and CD8 ⁺ A solid cancer infiltrated by one or more of T cells, and/or (ii) regulatory T (Treg) cells or CD4 ⁺ T cell infiltrated solid cancer.

7. The anti-cancer agent of any one of claims 1 to 6, which is administered to a patient having a cancer refractory to treatment with an immune checkpoint inhibitor.

8. The anti-cancer agent of any one of claims 1 to 7, which is administered to a cancer patient having one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myelogenous leukemia, and pediatric cancer.

9. The anti-cancer agent of any one of claims 1 to 5, for use in combination with at least one other anti-cancer agent.

10. The anticancer agent of claim 9, wherein the other anticancer agent is at least one anticancer agent selected from: chemotherapeutic agents, T cell activation agonists, immune checkpoint inhibitors, T cell redirecting antigen binding molecules, anti-fibrotic agents, and angiogenesis inhibitors.

11. The anti-cancer agent of claim 10, wherein the other anti-cancer agent is an immune checkpoint inhibitor.

12. The anti-cancer agent of claim 10 or 11, wherein the other anti-cancer agent is at least one immune checkpoint inhibitor selected from an anti-PD 1 antibody, an anti-PDL 1 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, and an anti-LAG 3 antibody.

13. The anti-cancer agent of claim 10, wherein the other anti-cancer agent is a T-cell redirecting antigen-binding molecule.

14. The anticancer agent of claim 9, wherein the other anticancer agent is depleted and/or inactivated selected from regulatory T cells, CD4 ⁺ An agent for one or more cells of T cells, B cells, NK cells and macrophages.

15. The anti-cancer agent of any one of claims 9 to 14, which is administered to a cancer patient having one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle cancer, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, merkel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal gland cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, and pediatric cancer.