Antigen-binding molecule containing antigen-binding domain of which binding activity to antigen is changed depending on MTA, and library for obtaining the antigen-binding domain

Abstract

To provide an antigen-binding molecule capable of treating various diseases (for example, cancer) caused by a target tissue (for example, a tumor tissue) in a target tissue-specific manner.SOLUTION: It is found that methylthioadenosine (MTA) is a low-molecular-weight compound specific to a tumor tissue; an antigen-binding molecule containing an antigen-binding domain of which the binding activity to an antigen is changed depending on the concentration of MTA or an antigen-binding molecule containing the antigen-binding domain, is created, and further, a library containing a plurality of antigen-binding domains that are different from each other or an antigen-binding molecule containing the antigen-binding domains, is created; and it is found that the problem can be solved by using the library.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in a methylthioadenosine (MTA)-dependent manner, a method for producing and screening the antigen-binding domain or the antigen-binding molecule, a library for obtaining the antigen-binding domain or the antigen-binding molecule and a method for designing the library, and a pharmaceutical composition comprising the antigen-binding molecule. The present disclosure also relates to a method for designing a library for efficiently obtaining an antigen-binding domain whose antigen-binding activity changes in a low-molecular-weight compound-dependent manner. Furthermore, the present disclosure also relates to an antigen-binding molecule that specifically binds to MTA, and a method for measuring MTA concentration and a method for diagnosing a disease using the antigen-binding molecule. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Documents 1 and 2).

[0003] As cancer therapeutic drugs using antibody drugs, Rituxan against CD20 antigen, Cetuximab against EGFR antigen, Herceptin against HER2 antigen, etc. have been approved (Non-Patent Document 3). These antibody molecules bind to antigens expressed in cancer cells and exert cytotoxic activity against cancer cells by ADCC, etc. It is known that such cytotoxic activity by ADCC, etc. depends on the number of antigens expressed in the target cells of the therapeutic antibody (Non-Patent Document 4), so from the viewpoint of the effect of the therapeutic antibody, it is preferable that the expression amount of the target antigen is high. However, even if the expression amount of the antigen is high, if the antigen is expressed in normal tissue, it will exert cytotoxic activity such as ADCC against normal cells, and side effects will become a major problem. Therefore, it is preferable that the antigen targeted by the therapeutic antibody as a cancer therapeutic drug is specifically expressed in cancer cells.

[0004] Following the success of antibody drugs that exert cytotoxic activity through ADCC activity, improved second-generation antibody molecules that exert strong cytotoxic activity have been reported by enhancing ADCC activity by removing fucose from the N-glycosylated Fc region of natural human IgG1 (Non-Patent Document 5), enhancing binding to FcγRIIIa by amino acid substitution in the Fc region of natural human IgG1 (Non-Patent Document 6), etc. As antibody drugs that exert cytotoxic activity against cancer cells by a mechanism other than the above-mentioned NK cell-mediated ADCC activity, improved antibody molecules that exert stronger cytotoxic activity, such as antibody drug conjugates (ADCs) in which a drug with strong cytotoxic activity is conjugated to an antibody (Non-Patent Document 7), and low-molecular-weight antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to the cancer cells (Non-Patent Document 8), have also been reported.

[0005] While such antibody molecules that exert stronger cytotoxic activity can exert cytotoxic activity against cancer cells that do not express many antigens, they also exert cytotoxic activity against normal tissues that express few antigens. In fact, compared to cetuximab, which is a natural human IgG1 against the EGFR antigen, EGFR-BiTE, a bispecific antibody against CD3 and EGFR, can exert strong cytotoxic activity against cancer cells and exert antitumor effects by recruiting T cells to cancer cells. On the other hand, since EGFR is also expressed in normal tissues, it has been recognized that serious side effects occur when EGFR-BiTE is administered to cynomolgus monkeys (Non-Patent Document 9). In addition, bivatuzumab mertansine, an ADC in which mertansine is bound to an antibody against CD44v6, which is highly expressed in cancer cells, has been observed to cause severe skin toxicity and liver toxicity in clinical trials because CD44v6 is also expressed in normal tissues (Non-Patent Document 10).

[0006] In this way, when using an antibody that can exert a strong cytotoxic activity against cancer cells with low antigen expression, the target antigen needs to be expressed in an extremely cancer-specific manner, but the number of cancer antigens that are expressed in an extremely cancer-specific manner is thought to be limited, as HER2, the target antigen of Herceptin, and EGFR, the target antigen of Cetuximab, are also expressed in normal tissues. Therefore, although it is possible to strengthen the cytotoxic activity against cancer, side effects due to cytotoxicity against normal tissues may become a problem.

[0007] Recently, it has been shown that ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, extends the overall survival of metastatic melanoma (Non-Patent Document 11). However, because ipilimumab systemically inhibits CTLA4, while it enhances tumor immunity, it also shows serious autoimmune disease-like side effects due to systemic immune activation, which has become a problem (Non-Patent Document 12).

[0008] Various technologies applicable to second-generation antibody drugs have been developed, and technologies for improving effector function, antigen binding ability, pharmacokinetics, and stability, or reducing the risk of immunogenicity have been reported (Non-Patent Document 13), but few technologies have been reported that enable antibody drugs to act specifically on diseased tissues to resolve the above-mentioned side effects. For example, for diseased sites such as cancer tissues and inflammatory tissues, pH-dependent antibodies have been reported that utilize the fact that the pH in these diseased tissues is acidic (Patent Documents 1 and 2). However, the decrease in pH (i.e., increase in hydrogen ion concentration) in cancer tissues and inflammatory tissues compared to normal tissues is slight, making it difficult to produce antibodies that detect and act on slight increases in hydrogen ion concentration, which has an extremely small molecular weight. At the same time, there are cases where the pH is acidic in normal tissues such as osteoclast bone resorption lacunae regions and tissues other than the target lesion, and it was thought that there were still many challenges to be overcome in using pH conditions as an environmental factor specific to the diseased site. On the other hand, a method for producing an antibody that exhibits antigen binding activity only after being cleaved by a protease expressed in diseased sites such as cancer tissues and inflammatory tissues has been reported (Patent Document 3). However, since the cleavage of antibodies by proteases is irreversible, it was thought that the antibody cleaved at the lesion site could bind to the antigen in normal tissues by returning to the bloodstream. In addition, the cancer specificity of such proteases was also thought to be an issue. In order to overcome such issues, antigen-binding molecules whose binding activity to antigens changes depending on the concentration of disease-tissue-specific compounds have been reported (Patent Documents 4 and 5). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. WO2003 / 105757 [Patent Document 2] International Publication No. WO2012 / 033953 [Patent Document 3] International Publication No. WO2010 / 081173

Patent document 4

Patent document 5

Non-licensed literature

[0010] [Non-licensed document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-licensed document 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-licensed document 3] Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327

Non-licensed Document 4

Non-licensed Document 5

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

[0011] An object of the present disclosure is to newly discover small molecular weight compounds that are specifically present or produced in diseased tissues, to provide antigen-binding molecules whose binding to target antigens is controlled in a manner dependent on the small molecular weight compounds (small molecular weight compound-switch antigen-binding molecules), and to provide a method for efficiently obtaining such antigen-binding molecules in a short period of time. [Means for solving the problem]

[0012] The present inventors have conducted intensive research to achieve the above object, and have discovered methylthioadenosine (MTA) as a small molecule compound specific to cancer tissue, and have created an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. The present inventors have also found that the antigen-binding molecule or a pharmaceutical composition containing the antigen-binding molecule is useful for cancer treatment, and that the antigen-binding molecule is useful for cancer treatment including administration of the antigen-binding molecule, and that the antigen-binding molecule is useful in the manufacture of medicines for cancer treatment.

[0013] The present inventors have also created a method for screening and producing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. The present inventors have also succeeded in creating a library capable of efficiently screening an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. The present inventors have further succeeded in creating a library capable of screening an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner and / or a low molecular weight compound other than MTA, and have also created a method for screening and producing the antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner and / or a low molecular weight compound other than MTA. Furthermore, the present inventors also succeeded in obtaining an antigen-binding molecule that specifically binds to MTA itself.

[0014] The present disclosure is based on these findings and specifically includes the embodiments exemplified below. [A1] An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in a 5'-methylthioadenosine (MTA)-dependent manner. [A2] An antigen-binding molecule, in which the binding activity of the antigen-binding domain for the antigen in the presence of MTA is different from the binding activity for the antigen in the absence of MTA. [A3] The antigen-binding molecule according to [A1] or [A2], wherein the binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen is substantially unaffected by adenosine. [A4] The antigen-binding molecule according to [A1] to [A3], wherein the binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen is substantially unaffected by S-(5'-Adenosyl)-L-homocysteine ​​(SAH). [A5] The antigen-binding molecule according to [A1] to [A4], wherein the binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen is substantially unaffected by AMP, ADP, or ATP. [A6] The antigen-binding molecule according to [A1] or [A2], wherein the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen also changes depending on adenosine. [A7] The antigen-binding molecule according to [A1], [A2] or [A6], wherein the binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen also changes depending on S-(5'-Adenosyl)-L-homocysteine ​​(SAH). [A8] The antigen-binding molecule according to [A1], [A2], [A6] or [A7], wherein the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule for the antigen also changes depending on AMP, ADP and / or ATP. [A9] The antigen-binding molecule according to any one of [A1] to [A8], wherein the antigen-binding domain comprises an antibody variable region and / or a single domain antibody. [A10] The antigen-binding molecule according to any one of [A1] to [A9], wherein the antigen-binding molecule is an antibody. [A11] The antigen-binding molecule according to any one of [A1] to [A10], which contains an antibody Fc region. [A12] The antigen-binding molecule according to [A11], wherein the antibody Fc region is a native Fc region or a modified Fc region. [A13] The antigen-binding molecule of any one of [A1] to [A12], wherein the antigen-binding activity of the antigen-binding domain in the presence of MTA is stronger than the antigen-binding activity of the antigen-binding domain in the absence of MTA. [A14] The antigen-binding molecule of any one of [A1] to [A12], wherein the antigen-binding activity of the antigen-binding domain in the presence of MTA is weaker than the antigen-binding activity of the antigen-binding domain in the absence of MTA. [A15] The antigen-binding molecule of any one of [A1] to [A14], wherein the antigen-binding domain has amino acid residues that interact with MTA. [A16] The antigen-binding molecule according to [A15], wherein the amino acid residue that interacts with MTA interacts with MTA when bound to an antigen in the antigen-binding domain. [A17] The antigen-binding molecule according to [A15] or [A16], wherein the antigen-binding domain comprises an antibody variable region or a single domain antibody, and the amino acid residues that interact with the MTA are located in the CDR of the antibody variable region or the single domain antibody. [A18] The antigen-binding molecule of any one of [A15] to [A17], wherein the antigen-binding domain is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid positions selected from the group consisting of amino acid positions 34, 35a, 47, 52, 52e, and 101 in the heavy chain and positions 32, 34, 36, 46, 49, 50, 89, 90, 91, and 96 in the light chain, as specified by Kabat numbering, in the amino acid sequence of the antibody variable region. [A19] The antigen-binding molecule of any one of [A15] to [A18], wherein the antigen-binding domain is an antibody variable region comprising at least one amino acid selected from heavy chain W34, C35a, W47, F52, Y52e, and E101, and light chain R32, S34, Y36, L46, Y49, S50, A89, G90, L91, and P96 (Kabat numbering). [A20] The antigen-binding molecule of any one of [A1] to [A19], wherein the antigen-binding domain is an antibody variable region, and the antibody variable region contains at least one amino acid selected from the group consisting of the following amino acids (Kabat numbering): any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; A at heavy chain position 32; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 33 of the heavy chain; W located at heavy chain position 34; M located at heavy chain position 35; C located at heavy chain position 35a; C at heavy chain position 50; I located at heavy chain position 51; F located at heavy chain position 52; A located at heavy chain position 52a; any of A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at position 52b of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52c of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52d of the heavy chain; Y located at heavy chain position 52e; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52f of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52g of the heavy chain; S located at heavy chain position 53; G at heavy chain position 54; G at heavy chain position 55; S located at heavy chain position 56; T located at heavy chain position 57; Y at heavy chain position 58; Y at heavy chain position 59; A at heavy chain position 60; S located at heavy chain position 61; W at heavy chain position 62; A at heavy chain position 63; K located at heavy chain position 64; G at heavy chain position 65; G at heavy chain position 95; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 96 of the heavy chain; G at heavy chain position 97; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 99 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100 of the heavy chain; G located at position 100a of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100b of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100c of the heavy chain; E located at heavy chain position 101; L located at heavy chain position 102; Q at position 24 of the light chain; S at position 25 of the light chain; S at light chain position 26; E located at light chain position 27; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at light chain position 27a; V at light chain position 28; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the light chain; any of A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at position 31 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the light chain; L located at light chain position 33; S at light chain position 34; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 49 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 50 of the light chain; A at light chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the light chain; T at light chain position 53; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 54 of the light chain; P at light chain position 55; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 56 of the light chain; A at light chain position 89; G at position 90 of the light chain; L located at light chain position 91; Y at light chain position 92; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 93 of the light chain; G at light chain position 94; N at light chain position 95; I located at light chain position 95a; P at light chain position 96; A at position 97 of the light chain. [A21] The antigen-binding molecule of any one of [A15] to [A17], wherein the antigen-binding domain is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid positions selected from the group consisting of amino acid positions 34, 47, 50, 58, 95, 98, 99, and 100a in the heavy chain and positions 28, 91, 95b, 95c, and 96 in the light chain, as defined by Kabat numbering, in the amino acid sequence of the antibody variable region. [A22] The antigen-binding molecule of any one of [A15] to [A17], and [A21], wherein the antigen-binding domain is an antibody variable region containing at least one amino acid selected from heavy chain W34, W47, C50, Y58, E95, F98, G99, and G100a, and light chain Y28, T91, F95b, Y95c, and F96 (Kabat numbering). [A23] The antigen-binding molecule according to any one of [A1] to [A17] or [A21] to [A22], wherein the antigen-binding domain is an antibody variable region, and the antibody variable region contains at least one amino acid selected from the group consisting of the following amino acids (Kabat numbering): any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 33 of the heavy chain; W located at heavy chain position 34; M located at heavy chain position 35; C located at heavy chain position 35a; C at heavy chain position 50; I located at heavy chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the heavy chain; S located at heavy chain position 52a; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at heavy chain position 53; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 54 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 55 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 56 of the heavy chain; T located at heavy chain position 57; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 58 of the heavy chain; Y at heavy chain position 59; A at heavy chain position 60; S located at heavy chain position 61; W at heavy chain position 62; V located at heavy chain position 63; N at heavy chain position 64; G at heavy chain position 65; E located at heavy chain position 95; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 96 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 97 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 99 of the heavy chain; S located at heavy chain position 100; G located at position 100a of the heavy chain; A located at position 100b of the heavy chain; L located at position 100c of the heavy chain; N at heavy chain position 101; L located at heavy chain position 102; H at light chain position 24; S at position 25 of the light chain; S at light chain position 26; K at position 27 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at light chain position 27a; V located at light chain position 27b; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 28 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the light chain; L located at light chain position 33; A at light chain position 34; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 49 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 50 of the light chain; A at light chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 53 of the light chain; L located at light chain position 54; A at light chain position 55; S at light chain position 56; Q at light chain position 89; G at position 90 of the light chain; T at light chain position 91; Y at light chain position 92; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 93 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 94 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 95 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 95a of the light chain; F located at light chain position 95b; Y located at light chain position 95c; F at light chain position 96; A at position 97 of the light chain. [A24] The antigen-binding molecule of any one of [A15] to [A17], wherein the antigen-binding domain is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid positions selected from the group consisting of amino acid positions 33, 50, 52, 54, 56, 57, 58, 99, 100, and 100a in the heavy chain and positions 91, 95c, and 96 in the light chain, as defined by Kabat numbering, in the amino acid sequence of the antibody variable region. [A25] The antigen-binding molecule of any one of [A15] to [A17] and [A24], wherein the antigen-binding domain is an antibody variable region comprising at least one amino acid selected from heavy chain A33, I50, G52, D54, S56, T57, W58, G99, Y100, T100a, and light chain S91, Y95c, and N96 (Kabat numbering). [A26] The antigen-binding molecule according to any one of [A1] to [A17] or [A24] to [A25], wherein the antigen-binding domain is an antibody variable region, and the antibody variable region contains at least one amino acid selected from the group consisting of the following amino acids (Kabat numbering): any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y at position 26 of the heavy chain; any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 28 of the heavy chain; either A or L at heavy chain position 29; any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; any of D, E, F, H, N, P, R, or Y at heavy chain position 32; any of A, I, P, T, or V at position 33 of the heavy chain; any of A, E, F, H, I, K, L, M, N, Q, S, T, V, W or Y at position 34 of the heavy chain; G at position 35 of the heavy chain; either D, I or V at heavy chain position 50; I located at heavy chain position 51; G at heavy chain position 52; any of A, D, E, G, I, K, Q, or R at position 53 of the heavy chain; any of D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, or Y at position 54 of the heavy chain; any of A, D, E, F, G, or H at position 55 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y at position 56 of the heavy chain; any of A, D, E, G, H, I, K, L, N, P, Q, R, S, T, or V at position 57 of the heavy chain; W located at heavy chain position 58; any of A, D, E, F, G, H, I, K, L, Q, R, S, T, V, W, or Y at position 59 of the heavy chain; P located at heavy chain position 60; any of A, F, Q, R, S, T, V, W, or Y at position 61 of the heavy chain; W at heavy chain position 62; V located at heavy chain position 63; K located at heavy chain position 64; A, F, or G at heavy chain position 65; G at heavy chain position 95; any of A, E, F, G, H, K, L, Q, R, S, T, W, or Y at position 96 of the heavy chain; any of A, F, H, K, N, W, or Y at position 97 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, D, E, G, H, Q, or S at position 99 of the heavy chain; F or Y at heavy chain position 100; N, T or V at position 100a of the heavy chain N located at position 100b of the heavy chain; A located at position 100c of the heavy chain; F or W located at position 100d of the heavy chain; D located at heavy chain position 101; P located at heavy chain position 102; Q at position 24 of the light chain; S at position 25 of the light chain; S at light chain position 26; Q at position 27 of the light chain; S located at light chain position 27e; V located at light chain position 27f; any of A, E, F, H, I, K, L, N, R, S, T, V, W, or Y at position 28 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; N at light chain position 30; N at light chain position 31; any of A, E, F, G, H, S, or Y at position 32 of the light chain; L located at light chain position 33; S at light chain position 34; D at position 50 of the light chain; A at light chain position 51; S at light chain position 52; T at light chain position 53; L located at light chain position 54; A at light chain position 55; S at light chain position 56; H at light chain position 89; G at position 90 of the light chain; either A, S, or T at position 91 of the light chain; any of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at position 92 of the light chain; any of A, D, E, F, G, H, L, N, Q, R, S, T, V, or Y at position 93 of the light chain; any of A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 94 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, V, W, or Y at position 95a of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95b of the light chain; any of A, F, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95c of the light chain; D located at light chain position 96d; N at light chain position 96; A or G at light chain position 97; A, F, I, L or V at position 98 of the light chain. [A27] The antigen-binding molecule according to any one of [A1] to [A26], wherein the antigen is a molecule other than MTA, or a molecule other than MTA that has immunogenicity in the human body. [A28] The antigen-binding molecule according to any one of [A1] to [A27], wherein the antigen is any one of a peptide, a polypeptide, or a protein. [A29] The antigen-binding molecule of any one of [A1] to [A28], further comprising a second antigen-binding domain, wherein the second antigen-binding domain has binding activity to a second antigen different from the antigen bound by the antigen-binding domain. [A30] The antigen-binding molecule of [A29], wherein the binding activity of the second antigen-binding domain for the second antigen is substantially unaffected by MTA. [A31] The antigen-binding molecule according to [A29], wherein the binding activity of the second antigen-binding domain to the second antigen changes in an MTA-dependent manner. [A32] The antigen-binding molecule of [A31], wherein the binding activity of the second antigen-binding domain for the second antigen in the presence of MTA is different from the binding activity of the second antigen-binding domain for the second antigen in the absence of MTA. [A33] The antigen-binding molecule according to any one of [A1] to [A32], wherein the antigen is a membrane molecule or a soluble molecule. [A34] The antigen-binding molecule according to [A33], wherein the antigen is a membrane molecule and is expressed in disease tissue. [A35] The antigen-binding molecule according to [A33], wherein the antigen is a soluble molecule and is expressed in cancer tissue. [A36] The antigen-binding molecule according to [A35], wherein the antigen expressed in the cancer tissue is an antigen expressed in cancer cells, or an antigen expressed in cancer stromal cells or immune tissue in the cancer tissue. [A37] The antigen-binding molecule according to [A35] or [A36], wherein the cancer tissue is a cancer tissue in which MTA accumulates. [A38] The antigen-binding molecule according to any one of [A35] to [A37], wherein the cancer tissue is a cancer tissue in which a gene encoding MTA phospholylase (MTAP) is deleted or has reduced expression, or which has a mutation or splicing variant that reduces the enzyme activity. [A39] The antigen-binding molecule according to any one of [A35] to [A37], wherein the cancer tissue is a cancer tissue in which MTAP activity is deficient or reduced. [A40] The antigen-binding molecule according to any one of [A1] to [A39], wherein the antigen is a membrane-type molecule, and the antigen-binding molecule exhibits cytotoxic activity against cells expressing the antigen. [A41] The antigen-binding molecule according to [A40], which exhibits at least one cytotoxic activity selected from ADCC activity, ADCP activity and CDC activity. [A42] The antigen-binding molecule according to [A1] to [A39], which has agonistic activity against the antigen. [A43] The antigen-binding molecule according to any one of [A29] to [A32], wherein one of the antigen and the second antigen is an antigen expressed in a target cell, and the other is an antigen expressed in an effector cell. [A44] The antigen-binding molecule according to [A43], wherein the target cell is a cancer cell. [A45] The antigen-binding molecule according to [A44], wherein the cancer cells are cancer cells in which the gene encoding MTAP is deleted or underexpressed, or which have a mutation or splicing variant that reduces the enzyme activity, or cancer cells present in the vicinity of cancer cells in which the gene encoding MTAP is deleted or underexpressed, or which have a mutation or splicing variant that reduces the enzyme activity. [A46] The antigen-binding molecule according to [A43], wherein the target cell is a cell other than a cancer cell that is present around a cancer cell in which the gene encoding MTAP is deleted or has reduced expression, or which has a mutation or splicing variant that reduces the enzyme activity. [A47] The antigen-binding molecule according to [A43], wherein the non-cancer cells present around the cancer cells are cancer-associated fibroblasts (CAFs) or tumor-associated macrophages (TAMs). [A48] The antigen-binding molecule according to any one of [A43] to

[47] , wherein the effector cell is a T cell. [A49] The antigen-binding molecule according to [A48], wherein the antigen expressed in the effector cell is a T cell receptor (TCR) complex. [A50] The antigen-binding molecule of either [A48] or [A49], wherein the antigen expressed in the effector cells is CD3. [A51] The antigen-binding molecule according to any one of [A48] to [A50], which activates effector cells and thereby induces cytotoxic activity against target cells. [A52] The antigen-binding molecule of any one of [A48] to [A51], which has TDCC activity. [A53] The antigen-binding molecule according to any one of [A1] to [A39], wherein the antigen is a soluble molecule, and the antigen-binding molecule exhibits neutralizing activity against the antigen. [A54] The antigen-binding molecule of any one of [A1] to [A53], wherein the KD value of the antigen-binding domain for the antigen in the absence of MTA is different from the KD value of the antigen-binding domain for the antigen in the presence of MTA. [A55] A pharmaceutical composition comprising the antigen-binding molecule of any one of [A1] to [A54]. [A56] A pharmaceutical composition for treating cancer, comprising as an active ingredient the antigen-binding molecule according to any one of [A1] to [A55]. [A57] The pharmaceutical composition according to [A56], wherein the cancer is a cancer in which MTA accumulates in tissues. [A58] The pharmaceutical composition according to any one of [A56] to [A57], wherein the cancer is a cancer in which the gene encoding MTAP is deleted or has reduced expression, or has a mutation or splicing variant that reduces enzymatic activity. [A59] The pharmaceutical composition according to any one of [A56] to [A58], wherein the cancer is a cancer in which MTAP activity is deficient or reduced. [A60] A method for producing an antigen-binding molecule according to any one of [A1] to [A54]. [A61] A polynucleotide encoding the antigen-binding molecule of any one of [A1] to [A52]. [A62] A vector comprising the polynucleotide according to [A61] [A63] A cell harboring the vector described in [A62]. [A64] An antigen-binding molecule obtained by culturing the cell according to [A63] and recovering the antigen-binding molecule from the culture supernatant. [G1] The antigen-binding molecule according to any one of [A1] to [A54], which has a high plasma retention and / or a low plasma antigen accumulation ability compared to a control antigen-binding molecule that does not bind to MTA in a concentration-dependent manner. [G2] A pharmaceutical formulation comprising the antigen-binding molecule according to [G1] and a pharma- ceutically acceptable carrier. [G3] A method for producing an antigen-binding molecule having higher plasma retention and / or lower plasma antigen accumulation capacity compared to a control antigen-binding molecule, the method comprising: (a) producing an antigen-binding molecule whose antigen-binding activity increases as the MTA concentration increases; and (b) measuring the plasma retention and / or plasma antigen accumulation capacity of the antigen-binding molecule produced in (a).

[0015] The present disclosure also encompasses the embodiments described, by way of example, below. [B1] A library consisting of antigen-binding molecules comprising multiple antigen-binding domains whose sequences differ from one another and / or nucleic acids encoding antigen-binding molecules comprising multiple antigen-binding domains whose sequences differ from one another, wherein the library is mainly composed of antigen-binding molecules comprising antigen-binding domains having amino acid residues that interact with MTA and / or nucleic acids encoding the antigen-binding molecules. [B2] The library according to [B1], wherein the antigen-binding domain is an antibody variable region. [B3] The library described in [B2], comprising nucleic acids encoding a plurality of antibody variable region variants which have different sequences from one another and which have amino acids different from those located at one or more amino acid sites in an unmodified antibody variable region which has binding activity to MTA, and / or a plurality of antibody variable region variants which have different sequences from one another and which have amino acids different from those located at one or more amino acid sites in an unmodified antibody variable region which has binding activity to MTA. [B4] The library according to [B3], wherein the amino acid site in the modified antibody variable region having an amino acid different from that in the unmodified antibody variable region is one or more amino acid sites selected from the group consisting of the following amino acid sites: 1) an amino acid site corresponding to an amino acid site in the unmodified antibody variable region that is not involved in binding to MTA; 2) an amino acid site that does not significantly reduce the binding of the antibody variable region variant to MTA, as compared to the antibody variable region variant; and 3) An amino acid site that is likely to contribute to MTA-dependent binding of the antibody variable region variant to an antigen. [B5] The library according to [B3], wherein the amino acid site in the modified antibody variable region having an amino acid different from that in the unmodified antibody variable region is one or more amino acid sites selected from the group consisting of the following amino acid sites: 1) an amino acid site corresponding to an amino acid site in the unmodified antibody variable region that is not involved in binding to MTA; 2) an amino acid site that does not significantly reduce the binding of the antibody variable region variant to MTA, as compared to the antibody variable region variant; 3) an amino acid site corresponding to an amino acid site exposed on the surface of the unmodified antibody variable region; and 4) An amino acid site corresponding to an amino acid site located in a region of the unmodified antibody variable region that has a large rate of structural change upon MTA binding / unbinding. [B6] The library according to any of [B3] to [B5], wherein the unmodified antibody variable region does not substantially bind to adenosine and / or S-(5'-Adenosyl)-L-homocysteine ​​(SAH). [B7] The library according to any one of [B3] to [B6], wherein the unmodified antibody variable region is any one of the following: a) an antibody variable region comprising a heavy chain variable region set forth in SEQ ID NO:46 and a light chain variable region set forth in SEQ ID NO:47; b) an antibody variable region comprising a heavy chain variable region set forth in SEQ ID NO:50 and a light chain variable region set forth in SEQ ID NO:51; c) an antibody variable region comprising a heavy chain variable region set forth in SEQ ID NO:48 and a light chain variable region set forth in SEQ ID NO:49; d) An antibody variable region comprising a heavy chain variable region set forth in SEQ ID NO:52 and a light chain variable region set forth in SEQ ID NO:53. [B8] The multiple antibody variable regions include any one of the following: H-CDR1 comprising XAXWMC (SEQ ID NO:65); H-CDR2 comprising CIFAXXXYXXSGGSTYYASWAKG (SEQ ID NO:66); H-CDR3 comprising GXGXXXGXXDEL (SEQ ID NO:67); L-CDR1 comprising QSSEXVXXXXLS (SEQ ID NO:68); L-CDR2 comprising XAXTXPX (SEQ ID NO:69); and L-CDR3 comprising AGLYXGNIPA (SEQ ID NO:70); wherein X represents any amino acid, and X present at different positions does not have to be the same amino acid. [B9] The multiple antibody variable regions include any one of the following: H-CDR1 comprising XAXWMC (SEQ ID NO:65); CIFAX 1 H-CDR2 comprising XXYXXSGGSTYYASWAKG (SEQ ID NO:71); H-CDR3 comprising GXGXXXGXXDEL (SEQ ID NO:67); QSSEXVXXX 1 L-CDR1 containing XLS (SEQ ID NO:72); L-CDR2 comprising XAXTXPX (SEQ ID NO:69); and L-CDR3 comprising AGLYXGNIPA (SEQ ID NO:70); an antibody variable region comprising X is any amino acid, X 1 are amino acids selected from A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T and V, and X or X 1 The library according to [B2], wherein the amino acids do not have to be the same kind. [B10] The multiple antibody variable regions include any one of the following: H-CDR1 comprising XXAXWMC (SEQ ID NO:73); CIFAX 1H-CDR2 comprising XXYXXSGGSTYYASWAKG (SEQ ID NO:71); H-CDR3 comprising GXGXXXGXXDEL (SEQ ID NO:67); QSSEXVXXX 1 L-CDR1 containing XLS (SEQ ID NO:72); L-CDR2 comprising XAXTXPX (SEQ ID NO:69); and L-CDR3 comprising AGLYXGNIPA (SEQ ID NO:70); an antibody variable region comprising X is any amino acid, X 1 are amino acids selected from A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T and V, and X or X 1 The library according to [B2], wherein the amino acids do not have to be the same kind. [B11] The multiple antibody variable regions include any one of the following: H-CDR1 comprising XXXWMC (SEQ ID NO:74); H-CDR2 comprising CIXSXXXXTXYASWVNG (SEQ ID NO:75); H-CDR3 comprising EXXXXSGALNL (SEQ ID NO:76); L-CDR1 comprising HSSKXVXXXXXLA (SEQ ID NO:77); L-CDR2 comprising XAXXLAS (SEQ ID NO:78); and L-CDR3 comprising QGTYXXXXFYFA (SEQ ID NO:79); wherein X represents any amino acid, and X present at different positions does not have to be the same amino acid. [B12] The library according to any one of [B3] to [B5], wherein an unmodified antibody variable region also has binding activity to adenosine. [B13] The library according to [B12], wherein the unmodified antibody variable region also has binding activity to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP and / or ATP. [B14] The library described in any one of [B3] to [B5] and [B12], wherein the unmodified antibody variable region is an antibody variable region having a heavy chain variable region shown in SEQ ID NO: 31 and a light chain variable region shown in SEQ ID NO: 32. [B15] The multiple antibody variable regions include any one of the following: X 2 X 3 X 4 X 5 H-CDR1 comprising G (SEQ ID NO: 80); X 6 IGX 7 X 8 X 9 X 10 X 11 WX 12 PX 13 WVKX 14 H-CDR2 comprising (SEQ ID NO:81); GX 15 X 16 X 17 X 18 X 19 X 20 NAX 21 H-CDR3 comprising DP (SEQ ID NO: 82); QSSQSVX 22 X 23 NNX 24 L-CDR1 comprising LS (SEQ ID NO: 83); L-CDR2 comprising DASTLAS (SEQ ID NO:84); and HGX 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 DNX 33 L-CDR3 comprising (SEQ ID NO: 85); an antibody variable region comprising X 2 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X 3 is an amino acid selected from D, E, F, H, K, N, P, R and Y; X 4is an amino acid selected from A, I, P, T and V; X 5 is an amino acid selected from A, E, F, H, I, K, L, M, N, Q, S, T, V, W and Y; X 6 is an amino acid selected from D, I and V; X 7 is an amino acid selected from A, D, E, G, I, K, Q and R; X 8 is an amino acid selected from D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W and Y; X 9 is an amino acid selected from A, D, E, F, G, H and S; X 10 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X 11 is an amino acid selected from A, D, E, G, H, I, K, L, N, P, Q, R, S, T and V; X 12 is an amino acid selected from A, D, E, F, G, H, I, K, L, Q, R, S, T, V, W and Y; X 13 is an amino acid selected from A, F, Q, R, S, T, V, W and Y; X 14 is an amino acid selected from A, F and G, X 15 is an amino acid selected from A, E, F, G, H, K, L, Q, R, S, T, W and Y; X 16 is an amino acid selected from F, H, K, N, W and Y; X 17 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W and Y; X 18 is an amino acid selected from A, D, E, G, H, Q and S; X 19 is an amino acid selected from F and Y; X 20 is an amino acid selected from N, T and V; X21 is an amino acid selected from F and W, X 22 is an amino acid selected from A, E, F, H, I, K, L, N, R, S, T, V, W and Y; X 23 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W and Y; X 24 is an amino acid selected from A, E, F, G, H, S and Y; X 25 is an amino acid selected from A, S and T, X 26 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y; X 27 is an amino acid selected from A, D, E, F, G, H, L, N, Q, R, S, T, V and Y; X 28 is an amino acid selected from A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W and Y; X 29 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, and Y; X 30 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, V, W and Y; X 31 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W and Y; X 32 is an amino acid selected from A, F, H, I, K, L, N, P, Q, R, S, T, V, W and Y; X 33 The library according to [B2], wherein is an amino acid selected from A and G. [B16] A library, in which nucleic acids encoding antigen-binding molecules comprising an antigen-binding domain that binds to MTA are enriched from the library according to [B15]. [B17] The library according to [B16], wherein the binding of the antigen-binding domain to MTA is binding to MTA in the absence of an antigen. [B18] The concentration comprises the following steps (1) and (2): (1) contacting an antigen-binding domain displayed from the library described in [B15] with an MTA; and (2) selecting the antigen-binding domain bound to the MTA in step (1); The library according to [B16] or [B17], [B19] A library in which nucleic acids encoding antigen-binding domains that bind to adenosine are enriched from the library according to [B15]. [B20] The library according to [B19], wherein the binding of the antigen-binding domain to adenosine is binding to adenosine in the absence of an antigen. [B21] The concentration comprises the following steps (1) and (2): (1) contacting an antigen-binding domain displayed from the library according to [B15] with adenosine; and (2) selecting an antigen-binding domain bound to adenosine in the step (1); The library according to [B19] or [B20], comprising:

[0016] The present disclosure also encompasses the embodiments described, by way of example, below. [C1] The following steps (a) and (b): (a) identifying an amino acid site that satisfies at least one of the following (i) to (vi) in an antigen-binding domain having binding activity to MTA: (i) an amino acid site exposed on the surface of the antigen-binding domain; (ii) an amino acid site located in a region where the rate of conformational change is large when the antigen-binding domain is compared between its structure when bound to MTA and its structure when not bound to MTA; (iii) amino acid sites not involved in binding to MTA; (iv) an amino acid site that does not significantly reduce binding to MTA; (v) an amino acid site that has diversity in amino acid frequency in the animal species to which the antigen-binding domain belongs; or (vi) Amino acid sites that are not important for the formation of the canonical structure; (b) designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain that have different sequences and have amino acid modifications at one or more amino acid sites identified in step (a); A method for producing a library comprising: [C2] The amino acid modification in step (b) satisfies at least one of the following (1) to (3): (1) When the structure of the antigen-binding domain variant having the amino acid modification is compared between when bound to MTA and when not bound to MTA, the rate of structural change at the amino acid site where the amino acid modification is located is large after the modification; (2) when the structure of the antigen-binding domain variant having the amino acid alteration is compared between when bound to MTA and when not bound to MTA, the presence of the altered amino acid is not inhibiting the structural change of the antigen-binding domain variant; (3) The binding activity of the antigen-binding domain modified with the amino acid modification to MTA is not significantly reduced compared to that of the unmodified antigen-binding domain; The manufacturing method described in [C1]. [C3] The method of any one of [C1] to [C2], wherein the antigen-binding domain having binding activity to MTA does not substantially bind to adenosine. [C4] The method according to [C3], wherein the antigen-binding domain having binding activity to MTA does not bind to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, or / and ATP. [C5] A library produced by the production method according to any one of [C1] to [C4]. [C6] The following steps (a) and (b): (a) identifying an amino acid site that satisfies at least one of the following (i) to (ii) in an antigen-binding domain having binding activity to a low molecular weight compound: (i) an amino acid site exposed on the surface of the antigen-binding domain; (ii) an amino acid site located in a region where a rate of conformational change is large when the antigen-binding domain is compared between a structure bound to the small molecular weight compound and a structure not bound to the small molecular weight compound; (b) designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain that have different sequences and have amino acid modifications at one or more amino acid sites identified in step (a); A method for producing a library comprising: [C7] The amino acid modification in step (b) satisfies at least one of the following (1) to (3): (1) when the structure of the antigen-binding domain variant having the amino acid alteration is compared between when it is bound to the low molecular weight compound and when it is not bound to the low molecular weight compound, the rate of structural change at the amino acid site where the altered amino acid is located is large after the alteration; (2) when the structure of the antigen-binding domain variant having the amino acid alteration bound to the low molecular weight compound is compared with the structure of the antigen-binding domain not bound to the low molecular weight compound, the presence of the altered amino acid is not inhibited from changing the structure of the antigen-binding domain variant; (3) the binding activity of the modified antigen-binding domain having the amino acid modification(s) towards the small molecule compound is not significantly reduced, as compared to that of the unmodified antigen-binding domain; The manufacturing method described in [C5]. [C8] The following steps (a) and (b): (a) identifying an amino acid site in an antigen-binding domain that interacts with a low molecular weight compound, the amino acid site satisfying at least one of the following (i) to (iv): (i) an amino acid site exposed on the surface of the antigen-binding domain; (ii) an amino acid site located in a region where a rate of conformational change is large when the antigen-binding domain is compared between a structure bound to the small molecular weight compound and a structure not bound to the small molecular weight compound; (iii) an amino acid site that is not involved in binding to the low molecular weight compound; (iv) an amino acid site that does not significantly reduce the binding to the low molecular weight compound; (v) an amino acid site that has diversity in amino acid frequency in the animal species to which the antigen-binding domain belongs; or (vi) Amino acid sites that are not important for the formation of the canonical structure; (b) designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain that have different sequences and have amino acid modifications at one or more amino acid sites identified in step (a) that satisfy at least one of the following (1) or (2): (1) when the structure of the antigen-binding domain variant having the amino acid alteration is compared between when it is bound to the low molecular weight compound and when it is not bound to the low molecular weight compound, the rate of structural change at the amino acid site where the altered amino acid is located is large after the alteration; (2) when the structure of the antigen-binding domain variant having the amino acid alteration bound to the low molecular weight compound is compared with the structure of the antigen-binding domain not bound to the low molecular weight compound, the presence of the altered amino acid is not inhibited from changing the structure of the antigen-binding domain variant; A method for producing a library comprising: [C9] The method for producing a compound according to any one of [C6] to [C8], wherein the low molecular weight compound is at least one selected from adenosine, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and S-(5'-Adenosyl)-L-homocysteine ​​(SAH). [C10] A library produced by the production method according to any one of [C6] to [C8].

[0017] The present disclosure also encompasses the embodiments described, by way of example, below. [D1] A method for screening for antigen-binding molecules that contain an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. [D2] The method according to [D1], comprising comparing the antigen-binding activity of the antigen-binding domain in the presence of MTA at a first concentration with the antigen-binding activity in the presence of MTA at a concentration (second concentration) different from the first concentration. [D3] The method according to [D1] or [D2], comprising a step of selecting an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity in the presence of a first concentration of MTA differs from that in the presence of a second concentration of MTA. [D4] The method according to any one of [D2] to [D3], comprising a step of selecting an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is higher than its antigen-binding activity in the presence of MTA at the second concentration. [D5] The method according to any one of [D2] to [D3], comprising a step of selecting an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is lower than its antigen-binding activity in the presence of MTA at the second concentration. [D6] The following steps (a) to (c): (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) placing the antigen-binding molecule comprising the antigen-binding domain bound in step (a) in the presence of a second concentration of MTA; and (c) isolating the antigen-binding molecule comprising the antigen-binding domain dissociated in step (b); The method according to [D1], comprising: [D7] The following steps (a) to (d); (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) confirming that the antigen-binding molecule comprising the antigen-binding domain in step (a) has bound to the antigen; (c) placing the antigen-binding molecule comprising the antigen-binding domain bound to the antigen in the presence of a second concentration of MTA; and (d) isolating an antigen-binding molecule comprising an antigen-binding domain having an antigen-binding activity weaker than the standard determined in step (b) in step (c); The method according to [D1], comprising: [D8] The method according to [D6] or [D7], wherein the antigen-binding domain is confirmed to be capable of binding to MTA before carrying out the step (a). [D9] The following steps (a) to (d): (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) confirming that the antigen-binding molecule comprising the antigen-binding domain in step (a) does not bind to an antigen; (c) allowing an antigen-binding molecule comprising an antigen-binding domain that does not bind to the antigen to bind to the antigen in the presence of a second concentration of MTA; and (d) isolating an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (c); The method according to [D1], comprising: [D10] The following steps (a) to (c): (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) placing the antigen-binding molecule comprising the antigen-binding domain bound in step (a) in the presence of a second concentration of MTA; and (c) isolating the antigen-binding molecule comprising the antigen-binding domain dissociated in step (b); The method according to [D1], comprising: [D11] The following steps (a) to (d); (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) selecting an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (a); (c) placing the antigen-binding molecule containing the antigen-binding domain selected in step (b) in the presence of a second concentration of MTA; and (d) isolating in the step (c) an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity is weaker than the criterion selected in the step (b); The method according to [D1], comprising: [D12] Before the step (a), the following steps (1) and (2) are carried out: (1) contacting a library displaying antigen-binding molecules containing antigen-binding domains with an MTA; and (2) selecting an antigen-binding molecule comprising the antigen-binding domain bound to the MTA in step (1); wherein the library contacted with the antigen in the presence of a first concentration of MTA in the step (a) is a library displaying antigen-binding molecules comprising the antigen-binding domain selected in the steps (1) and (2). [D13] The following steps (a) to (d): (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) selecting an antigen-binding molecule comprising an antigen-binding domain that does not bind to the antigen in step (a); (c) allowing the antigen-binding molecule containing the antigen-binding domain selected in step (b) to bind to an antigen in the presence of a second concentration of MTA; and (d) isolating an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (c); The method according to [D1], comprising: [D14] The method according to any one of [D2] to [D8], wherein the first concentration is higher than the second concentration. [D15] The method according to [D1], which comprises comparing the antigen-binding activity of the antigen-binding domain in the presence of MTA with the antigen-binding activity in the absence of MTA. [D16] The method according to [D1] or [D15], comprising a step of selecting an antigen-binding domain having different antigen-binding activity in the presence and absence of MTA. [D17] The method according to [D1] or [D15], comprising a step of selecting an antigen-binding domain whose antigen-binding activity in the presence of MTA is higher than its antigen-binding activity in the absence of MTA. [D18] The following steps (a) to (c): (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of an MTA; (b) placing the antigen-binding molecule comprising the antigen-binding domain bound in step (a) in the absence of MTA; and (c) isolating the antigen-binding molecule comprising the antigen-binding domain dissociated in step (b); The method according to [D1], comprising: [D19] The following steps (a) to (d); (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of an MTA; (b) confirming that the antigen-binding molecule comprising the antigen-binding domain in step (a) has bound to the antigen; (c) placing the antigen-binding molecule comprising the antigen-binding domain bound to the antigen in the absence of an MTA; and (d) isolating an antigen-binding molecule comprising an antigen-binding domain having an antigen-binding activity weaker than the standard determined in step (b) in step (c); The method according to [D1], comprising: [D20] The method according to [D18] or [D19], wherein the antigen-binding molecule comprising the antigen-binding domain is confirmed to be capable of binding to an MTA before carrying out the step (a). [D21] The following steps (a) to (d): (a) contacting an antigen-binding molecule comprising an antigen-binding domain with an antigen in the presence of an MTA; (b) confirming that the antigen-binding molecule comprising the antigen-binding domain in step (a) does not bind to an antigen; (c) binding an antigen-binding molecule comprising an antigen-binding domain that does not bind to the antigen to the antigen in the absence of an MTA; and (d) isolating an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (c); The method according to [D1], comprising: [D22] The following steps (a) to (c): (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of an MTA; (b) placing the antigen-binding molecule comprising the antigen-binding domain bound in step (a) in the absence of MTA; and (c) isolating the antigen-binding molecule comprising the antigen-binding domain dissociated in step (b); The method according to [D1], comprising: [D23] The following steps (a) to (d); (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of MTA; (b) selecting an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (a); (c) placing the antigen-binding molecule containing the antigen-binding domain selected in step (b) in the absence of MTA; and (d) isolating in the step (c) an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity is weaker than the criterion selected in the step (b); The method according to [D1], comprising: [D24] Before the step (a), the following steps (1) and (2) are carried out: (1) contacting a library displaying antigen-binding molecules containing antigen-binding domains with an MTA; and (2) selecting an antigen-binding molecule comprising the antigen-binding domain bound to the MTA in step (1); The method according to [D22] or [D23], wherein the library contacted with the antigen in the presence of MTA in the step (a) is a library displaying antigen-binding molecules comprising the antigen-binding domain selected in the steps (1) and (2). [D25] The following steps (a) to (d): (a) contacting a library displaying antigen-binding molecules comprising antigen-binding domains with an antigen in the presence of an MTA; (b) selecting an antigen-binding molecule comprising an antigen-binding domain that does not bind to the antigen in step (a); (c) allowing the antigen-binding molecule containing the antigen-binding domain selected in step (b) to bind to an antigen in the absence of an MTA; and (d) isolating an antigen-binding molecule comprising the antigen-binding domain bound to the antigen in step (c); The method according to [D1], comprising: [D26] The method according to any one of [D10] to [D14] or [D22] to [D25], wherein the library displaying an antigen-binding molecule comprising the antigen-binding domain is a naive human antibody display library, a synthetic human antibody display library, a library according to any one of [B1] to [B14], or a library according to [C5]. [D27] The method according to any one of [D1] to [D26], wherein the antigen-binding domain is an antibody variable region or a single domain antibody.

[0018] The present disclosure also encompasses the embodiments described, by way of example, below. [F1] A method for producing an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. [F2] The method described in [F1], which comprises comparing the antigen-binding activity of the antigen-binding domain in the presence of MTA at a first concentration with the antigen-binding activity in the presence of MTA at a concentration (second concentration) different from the first concentration. [F3] The method according to [F1] or [F2], comprising a step of selecting an antigen-binding domain whose antigen-binding activity in the presence of a first concentration of MTA differs from that in the presence of a second concentration of MTA. [F4] The method according to any one of [F2] to [F3], comprising a step of selecting an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is higher than its antigen-binding activity in the presence of MTA at the second concentration. [F5] The method according to any one of [F2] to [F3], comprising a step of selecting an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is lower than its antigen-binding activity in the presence of MTA at the second concentration. [F6] The method according to any one of [F3] to [F5], further comprising the steps of culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the selected antigen-binding domain is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium. [F7] The following steps (a) to (d): (a) contacting the antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) placing the antigen-binding domain bound in step (a) in the presence of a second concentration of MTA; (c) isolating the antigen-binding domain dissociated in step (b); and (d) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (c) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F8] The following steps (a) to (e); (a) contacting the antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) confirming that the antigen-binding domain has bound to the antigen in step (a); (c) placing the antigen-binding domain bound to the antigen in the presence of a second concentration of MTA; (d) isolating the antigen-binding domains in step (c) whose antigen-binding activity is weaker than the standard determined to bind to the antigen in step (b); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F9] The method according to [F7] or [F8], wherein the antigen-binding domain is confirmed to be capable of binding to MTA before carrying out the step (a). [F10] The following steps (a) to (e): (a) contacting the antigen-binding domain with an antigen in the presence of a first concentration of MTA; (b) confirming that the antigen-binding domain in step (a) does not bind to the antigen; (c) allowing the antigen-binding domain that does not bind to the antigen to bind to the antigen in the presence of a second concentration of MTA; (d) isolating the antigen-binding domain that bound to the antigen in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F11] The following steps (a) to (d): (a) contacting a library displaying antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) placing the antigen-binding domain bound in step (a) in the presence of a second concentration of MTA; (c) isolating the antigen-binding domain dissociated in step (b); and (d) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (c) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F12] The following steps (a) to (e); (a) contacting a library displaying antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) selecting an antigen-binding domain that binds to the antigen in step (a); (c) placing the antigen-binding domain selected in step (b) in the presence of a second concentration of MTA; (d) isolating the antigen-binding domain in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F13] Prior to the step (a), the following steps (1) and (2) are performed: (1) contacting a library displaying antigen-binding domains with an MTA; and (2) selecting the antigen-binding domain bound to the MTA in step (1); The method according to [F11] or [F12], wherein the library contacted with the antigen in the presence of MTA at a first concentration in the step (a) is a library displaying antigen-binding domains selected in the steps (1) and (2). [F14] The following steps (a) to (e): (a) contacting a library displaying antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) selecting an antigen-binding domain that does not bind to the antigen in step (a); (c) allowing the antigen-binding domain selected in step (b) to bind to an antigen in the presence of a second concentration of MTA; (d) isolating the antigen-binding domain that bound to the antigen in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F15] The method according to any one of [F2] to [F14], wherein the first concentration is higher than the second concentration. [F16] The method according to [F1], which comprises comparing the antigen-binding activity of the antigen-binding domain in the presence of MTA with the antigen-binding activity in the absence of MTA. [F17] The method according to [F1] or [F16], comprising a step of selecting an antigen-binding domain having different antigen-binding activity in the presence and absence of MTA. [F18] The method according to [F1] or [F16], comprising a step of selecting an antigen-binding domain that has higher antigen-binding activity in the presence of MTA than in the absence of MTA. [F19] The method according to any one of [F16] to [F18], further comprising the steps of culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the selected antigen-binding domain is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium. [F20] The following steps (a) to (d): (a) contacting the antigen-binding domain with an antigen in the presence of an MTA; (b) placing the antigen-binding domain bound in step (a) in the absence of MTA; (c) isolating the antigen-binding domain dissociated in step (b); and (d) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (c) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F21] The following steps (a) to (e); (a) contacting the antigen-binding domain with an antigen in the presence of an MTA; (b) confirming that the antigen-binding domain has bound to the antigen in step (a); (c) placing the antigen-binding domain bound to the antigen in the absence of an MTA; (d) isolating the antigen-binding domains in step (c) whose antigen-binding activity is weaker than the standard determined to bind to the antigen in step (b); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F22] The method according to [F20] or [F21], wherein the antigen-binding domain is confirmed to be capable of binding to MTA before carrying out the step (a). [F23] The following steps (a) to (e): (a) contacting an antigen-binding domain with an antigen in the presence of an MTA; (b) confirming that the antigen-binding domain in step (a) does not bind to the antigen; (c) binding the antigen-binding domain that does not bind to the antigen to the antigen in the absence of an MTA; (d) isolating the antigen-binding domain that bound to the antigen in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F24] The following steps (a) to (d): (a) contacting a library displaying antigen-binding domains with an antigen in the presence of an MTA; (b) placing the antigen-binding domain bound in step (a) in the absence of MTA; (c) isolating the antigen-binding domain dissociated in step (b); and (d) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (c) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F25] The following steps (a) to (e); (a) contacting a library displaying antigen-binding domains with an antigen in the presence of an MTA; (b) selecting an antigen-binding domain that binds to the antigen in step (a); (c) placing the antigen-binding domain selected in step (b) in the absence of MTA; (d) isolating the antigen-binding domain in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F26] Prior to the step (a), the following steps (1) and (2) are performed: (1) contacting a library displaying antigen-binding domains with an MTA; and (2) selecting the antigen-binding domain bound to the MTA in step (1); The method according to [F24] or [F25], wherein the library contacted with the antigen in the presence of MTA in the step (a) is a library displaying antigen-binding domains selected in the steps (1) and (2). [F27] The following steps (a) to (e): (a) contacting a library displaying antigen-binding domains with an antigen in the presence of an MTA; (b) selecting an antigen-binding domain that does not bind to the antigen in step (a); (c) binding the antigen-binding domain selected in step (b) to an antigen in the absence of an MTA; (d) isolating the antigen-binding domain that bound to the antigen in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding an antigen-binding molecule comprising the antigen-binding domain isolated in step (d) is operably linked, and recovering the antigen-binding molecule comprising the antigen-binding domain from the cell culture medium; The method according to [F1], comprising: [F28] The method according to any one of [F11] to [F15] or [F24] to [F27], wherein the library displaying the antigen-binding domain is a naive human antibody display library, or a synthetic human antibody display library, or a library described in any one of [B1] to [B14], or a library described in [C5]. [F29] The method according to any one of [F1] to [F28], wherein the antigen-binding domain is an antibody variable region or a single domain antibody.

[0019] The present disclosure also encompasses the embodiments described, by way of example, below. [E1] An antigen-binding molecule that specifically binds to MTA. [E2] The antigen-binding molecule of [E1], which binds to MTA and does not substantially bind to adenosine. [E3] The antigen-binding molecule of [E1] or [E2], which binds to MTA, and which does not substantially bind to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, or / and ATP. [E4] A method for measuring an MTA concentration, using the antigen-binding molecule according to any one of [E1] to [E3]. [E5] The measurement method according to [E4], wherein the MTA concentration is the MTA concentration in tissue. [E6] The method according to any one of [E4] and [E5], in which the MTA concentration is measured using an antigen-antibody reaction. [E7] A measurement method according to any one of [E4] to [E5], which uses an immunohistochemical staining method. [E8] A measurement method according to any one of [E4] to [E5], which uses an imaging method. [E9] A measurement method described in any one of [E4] to [E5], which uses an in vivo imaging method. [E10] A method for diagnosing a disease, using the antigen-binding molecule according to any one of [E1] to [E3]. [E11] The method according to [E10], wherein the diagnosis is for determining the presence or absence of a disease or predicting the efficacy of treatment for a disease. [E12] The method according to any one of [E10] to [E11], wherein the disease is cancer. [E13] The method according to [E12], wherein the cancer is a cancer in which MTA accumulates in cancer tissue. [E14] The method according to any one of [E12] or [E13], wherein the cancer is a cancer tissue in which the gene encoding MTAP is deleted or underexpressed, or which has a mutation or splicing variant that reduces enzymatic activity. [E15] A diagnostic kit for a disease, comprising the antigen-binding molecule according to any one of [E1] to [E3]. [E16] The kit according to [E15], wherein the diagnosis is for determining the presence or absence of a disease or predicting the effectiveness of treatment for a disease. [E17] The kit according to any one of [E15] to [E16], wherein the disease is cancer. [E18] The kit according to [E17], wherein the cancer is a cancer in which MTA accumulates in cancer tissue. Effect of the Invention

[0020] The antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner according to the present disclosure, and a pharmaceutical composition comprising the same, do not act systemically in normal tissues or blood, but act reversibly on cancer, thereby exerting medicinal efficacy while avoiding side effects and enabling the treatment of cancer. Furthermore, by using the library of the present disclosure comprising antigen-binding molecules comprising multiple antigen-binding domains whose sequences differ from one another and whose antigen-binding activity changes in an MTA-dependent manner, it is possible to efficiently obtain, in a short period of time, various antigen-binding molecules whose antigen-binding activity changes in an MTA-dependent manner, which are useful for treating cancer tissue-specific diseases, as described above. [Brief description of the drawings]

[0021] [Figure 1] 1 shows the intracellular MTA concentration of each cell line. The vertical axis indicates the intracellular MTA concentration, and the horizontal axis indicates the cell line name and MTAP deficiency status. MTAP- means an MTAP-deficient cell line, and MTAP+ means an MTAP-non-deficient cell line. [Diagram 2] 1 shows the MTA concentration in the culture medium when each cell line was cultured. The vertical axis indicates the MTA concentration in the culture medium, and the horizontal axis indicates the culture time, the cell line name, and the MTAP deficiency state. MTAP- means an MTAP-deficient cell line, and MTAP+ means an MTAP-non-deficient cell line. [Diagram 3] This is a diagram showing the MTA concentration in the culture medium when each cell line was cultured in a medium to which MTA had been added beforehand. The vertical axis is the MTA concentration in the culture medium, and the horizontal axis is the culture time. Each spot represents each measured data. The graph on the left shows the MTA concentration in the culture medium when HT-1376, a non-MTAP-deficient cell line, was cultured, and the graph on the right shows the MTA concentration in the culture medium when SK-MES-1, a non-MTAP-deficient cell line, was cultured. [Figure 4] 1 shows the MTA concentration in tumors of tumor-bearing mice. The vertical axis shows the MTA concentration in the tumor, and the horizontal axis shows the name of the cell line transplanted into the mouse. Each spot shows each measured data. [Diagram 5]This is a diagram showing the relationship between the amount of MTAP DNA in human clinical samples and the MTA concentration in tissues. The graph on the left shows the results of measuring clinical samples of bladder cancer, and the graph on the right shows the results of measuring clinical samples of esophageal cancer. Each spot in the graph represents a clinical sample, and the light-colored spots represent samples in which the MTA concentration in tissues was below the detection limit. The vertical axis is the MTA concentration in tissues, and the horizontal axis is ΔCt, which is the Ct value of the MTAP gene minus the Ct value of the ΨX4 gene. [Figure 6] This is a diagram showing the extracellular MTA concentration in the tissues of tumor-bearing mice. The graph on the left shows the extracellular MTA concentration in the tumor, and the graph on the right shows the extracellular MTA concentration in the liver, which is a normal tissue. The vertical axis shows the extracellular MTA concentration in the tissue, and the horizontal axis shows the name of the cell line transplanted into the mouse. Each spot shows each measured data, and the hollow spots show data below the lower limit of MTA quantification. [Figure 7] 1 shows the amount of C03H-BH076N17 / C03L-KT0 bound to hIL-6R under different concentrations of MTA or adenosine, with the ordinate showing the amount of hIL-6R bound per amount of solid-phase antibody and the abscissa showing the concentration of MTA or adenosine. [Figure 8] This is a diagram showing the binding mode of SMB0002hFab and adenosine. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and adenosine in a ball-and-stick model. Amino acid residues that form interactions with adenosine are shown in stick models. The dashed lines and their numerical values ​​indicate the distance between the hydrogen bonds, CH-π interactions, or π-π interactions between each amino acid residue and adenosine. The unit is Å. [Figure 9] This is a diagram of the variable regions extracted from two molecules, SMB0002hFab_1 and SMB0002hFab_2, contained in the asymmetric unit of the crystal structure of SMB0002hFab, superimposed on each other. In the figure, SMB0002hFab_1 is shown in gray and SMB0002hFab_2 is shown in black. [Figure 10]This figure shows the adenosine and variable regions extracted from SMB0002hFab_1, one of the two molecules in the asymmetric unit of the crystal structure of SMB0002hFab, and the complex of SMB0002hFab and adenosine (SMB0002hFab-adenosine complex), which were then superimposed. In the figure, SMB0002hFab_1 is shown in gray, and the SMB0002hFab-adenosine complex is shown in black. [Figure 11] This figure shows the adenosine and variable regions extracted from SMB0002hFab_2, one of the two molecules in the asymmetric unit of the crystal structure of SMB0002hFab, and the complex of SMB0002hFab and adenosine (SMB0002hFab-adenosine complex), which were superimposed. In the figure, SMB0002hFab_2 is shown in gray, and the SMB0002hFab-adenosine complex is shown in black. [Figure 12] 1 shows the amount of binding to MTA of clones obtained after panning of a heavy chain variable region phage display library against MTA, with the vertical axis showing absorbance by phage ELISA when MTA was not immobilized and the horizontal axis showing absorbance when MTA was immobilized. [Figure 13] 1 shows the amount of binding to MTA of clones obtained after panning of a light chain variable region phage display library against MTA, with the vertical axis showing absorbance by phage ELISA when MTA was not immobilized and the horizontal axis showing absorbance when MTA was immobilized. [Figure 14] This is a graph showing the binding amounts of antibodies that bind to antigens in an MTA-dependent manner to hIL-6R, hIL-6, and hIgA in the presence of MTA or adenosine using SPR, where the vertical axis indicates the binding amount (-100 to 200 RU), and the horizontal axis indicates the reaction time (-100 to 1200 seconds, 0 second is the start time of the antigen reaction). [Figure 15] This is a diagram of the variable region and MTA extracted from the crystal structure of the complex of MTA0303Fab and MTA, in which the heavy chain of the antibody is shown in black, the light chain in grey, and the MTA in a ball-and-stick model. [Figure 16]This is a diagram showing the binding mode between MTA0303Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in grey, and MTA in a ball-and-stick model. Amino acid residues that form interactions with MTA are shown in stick models. The dashed lines and their numerical values ​​indicate the distances between the hydrogen bonds, CH-π interactions, π-π interactions, and sulfur-π interactions between each amino acid residue and MTA. The unit is Å. [Figure 17] This is a diagram showing the binding mode between MTA0303Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in grey, and the MTA in a ball-and-stick model. Amino acid residues that form interactions with the MTA are shown in stick models. The dashed lines and their associated numbers indicate the hydrogen bond distances between the amino acid residues and the MTA. The unit is Å. [Figure 18] This is a diagram of the variable region and MTA extracted from the crystal structure of the complex of MTA0330Fab and MTA, in which the antibody heavy chain is shown in black, the light chain in grey, and the MTA in a ball-and-stick model. [Figure 19] This is a diagram showing the binding mode between MTA0330Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in grey, and MTA in a ball-and-stick model. Amino acid residues that form interactions with MTA are shown in stick models. The dashed lines and their numerical values ​​indicate the distance between hydrogen bonds, CH-π interactions, or π-π interactions between each amino acid residue and MTA. The unit is Å. [Figure 20] This is a diagram showing the crystal structure of the complex of MTA0303Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in light gray, and MTA in a stick model. The Cα atoms of isoleucine, leucine, valine, and alanine contained in the heavy chain of the antibody are shown as dark gray spheres. The Cα atoms of isoleucine, leucine, valine, and alanine contained in the light chain are shown as white spheres. [Figure 21] This is a diagram showing the crystal structure of the complex of MTA0330Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in light gray, and MTA in a stick model. The Cα atoms of isoleucine, leucine, valine, and alanine contained in the heavy chain of the antibody are shown as dark gray spheres. The Cα atoms of isoleucine, leucine, valine, and alanine contained in the light chain are shown as white spheres. [Figure 22] Figure 1 shows an overlay of 1H-15N TROSY spectra of MTA0303Fab in the MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum shown in grey. [Figure 23] Figure 1 shows an overlay of 1H-13C SOFAST-HMQC spectra of MTA0303Fab in its MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum shown in grey. [Figure 24] Figure 1 shows an overlay of 1H-15N TROSY spectra of MTA0330Fab in the MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum shown in grey. [Diagram 25] Figure 1 shows an overlay of 1H-13C SOFAST-HMQC spectra of MTA0330Fab in its MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum shown in grey. [Figure 26] FIG. 1 shows that a small molecule switch antibody does not bind to an antigen in a normal environment in which small molecules are not present, but binds to the antigen in a target tissue in which small molecules are present at high concentrations. [Figure 27] This is a diagram showing how a small molecule fulfills its switching function by being sandwiched between a complex of an antibody and an antigen. In the absence of a small molecule, the interaction between the antibody and antigen is insufficient and the antibody cannot bind to the antigen, but in the presence of a small molecule, the antibody becomes able to bind to the antigen by being sandwiched between the antibody and antigen. [Figure 28] A diagram showing the T cell activation ability of a bispecific antibody having an antigen-binding domain that binds to IL-6R in an MTA-dependent manner and an antigen-binding domain that binds to CD3 in the presence or absence of MTA or ADO, tested using NFAT-RE-luc2-Jurkat cells. The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the relative light units (RLU). [Figure 29]This figure shows the amount of hIL-6R binding of anti-IL-6R antibodies that bind to IL-6R in an MTA-dependent manner under different MTA concentrations, measured using Biacore T200. The vertical axis shows the amount of hIL-6R binding per amount of solid-phase antibody, and the horizontal axis shows the MTA concentration. [Diagram 30] This is a sensorgram showing the time-dependent change in the amount of binding of an anti-IL-6R antibody that binds to IL-6R in an MTA-dependent manner to the antigen (hIL-6R), measured using the Octet RED384 system. The upper and lower panels show the results of measurements using 100 μM and 10 μM MTA, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The following definitions and detailed description are provided to facilitate understanding of the disclosure described herein. amino acid As used herein, amino acids are represented by one-letter or three-letter codes, or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0023] Amino acid modification For modifying amino acids in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. In addition, as a method for modifying amino acids by substituting amino acids other than natural amino acids, several known methods can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also preferably used.

[0024] As used herein, the meaning of the term "and / or" used to describe the site of amino acid modification includes any combination of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following amino acid modification variations: (a) 33rd place, (b) 55th place, (c) 96th place, (d) 33rd and 55th place, (e) 33rd and 96th place, (f) 55th and 96th place, (g) 33rd, 55th and 96th place.

[0025] In the present specification, as an expression for an amino acid modification, an expression in which the one-letter code or three-letter code of the amino acid before and after the modification is written before and after a number representing a specific position can be appropriately used. For example, the modification N100bL or Asn100bLeu used when adding a substitution of an amino acid contained in an antibody variable region represents a substitution of Asn at position 100b represented by Kabat numbering with Leu. That is, the number represents the position of the amino acid represented by Kabat numbering, the one-letter code or three-letter code of the amino acid written before it represents the amino acid before the substitution, and the one-letter code or three-letter code of the amino acid written after it represents the amino acid after the substitution. Similarly, the modification P238D or Pro238Asp used when adding an amino acid substitution to the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 represented by EU numbering with Asp. That is, the number indicates the position of the amino acid according to EU numbering, the one-letter or three-letter code of the amino acid written before it indicates the amino acid before substitution, and the one-letter or three-letter code of the amino acid written after it indicates the amino acid after substitution.

[0026] antigen As used herein, the structure of an "antigen" is not limited to a specific structure, so long as it contains an epitope to which an antigen-binding domain binds. In one embodiment, the antigen is a peptide of 4 or more amino acids, or a polypeptide, or a protein. Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, 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, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Complement control factor (Decay acceleratingfactor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, ephrin B2 / EphB4 , 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, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, G DF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF -9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, 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 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 , IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like proliferation factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-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 (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NCADHERIN, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, 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, PDK-1, P ECAM, 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, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-likereceptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP High-pressure washer CA125, high-pressure washers and wheelbarrows TW EAK, TXB2, Ung, uPAR, uPAR-1, VC AM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGF R, VEGFR-3(flt-4), VEGI, VIM, VLA, VLA-1, VLA-4, and VNR Liquid classes WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WN T7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9A、WNT9B、WNT10A、WNT10B、WNT11、WNT16、X CL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1. DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, low-density LDL, PCSK9, prekallikrein RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B tau VAP1 IL-31 IL-31R Nav1.1 Nav 1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1 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, fibrinogen, fibrin, prothrombin, thrombin, antioxidant factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, and factor IX、factor IXa、factor X、factor Xa、factor XI、factor XIa、factor XII、factorExamples of antigens include receptors for hormones and growth factors, as well as antigens expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues. is preferred.

[0027] The above examples of antigens include receptors, but even when these receptors exist in a soluble form in biological fluids, they can be used as antigens to which antigen-binding molecules comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner according to the present disclosure bind. A non-limiting example of such a soluble receptor is a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

[0028] Examples of the antigen include membrane molecules expressed on the cell membrane and soluble molecules secreted from cells to the outside. When an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner according to the present disclosure binds to a soluble molecule secreted from a cell, the antigen-binding molecule preferably has neutralizing activity as described below.

[0029] There is no limitation on the solution in which the soluble molecule exists, and the soluble molecule may exist in biological fluids, i.e., all fluids that fill the vessels or tissues / cells in a living body. In a non-limiting embodiment, the soluble molecule to which the antigen-binding molecule of the present disclosure binds may exist in extracellular fluid. In vertebrates, the term "extracellular fluid" refers to a collective term for plasma, interstitial fluid, lymphatic fluid, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, aspirate, or components in bones and cartilage such as synovial fluid, alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor), and other intercellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activities of cells, and fluids in the digestive tract and other body cavities).

[0030] When an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner according to the present disclosure binds to a membrane-type molecule expressed on a cell membrane, preferred examples of the antigen-binding molecule include antigen-binding molecules that have cytotoxic activity or that bind or have the ability to bind a cytotoxic substance, as described below. Also preferred as a non-limiting embodiment are antigen-binding molecules that have neutralizing activity instead of or in addition to the property of having cytotoxic activity or binding or having the ability to bind a cytotoxic substance.

[0031] Antigen-binding domain As used herein, the "antigen-binding domain" may be any domain with any structure as long as it binds to a target antigen. Examples of such domains include the variable regions of the heavy and light chains of antibodies, a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication No. WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed in cell membranes (International Publication No. WO2002 / 032925), Affibody using an IgG binding domain consisting of a bundle of three helices consisting of 58 amino acids of Protein A as a scaffold (International Publication No. WO1995 / 001937), DARPins (Designed Ankyrin Repeats: AR), which are regions exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a turn containing 33 amino acid residues and two antiparallel helices and a loop subunit are repeatedly stacked, and the like. Suitable examples of such proteins include those described in WO 2002 / 020565, Anticalin, which is a four loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich-repeat (LRR) modules are repeatedly stacked in the variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and serves as the adaptive immune system of jawless fish such as lampreys and hagfish (WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present disclosure include an antigen-binding domain comprising the variable regions of the heavy and light chains of an antibody. Preferred examples of such an antigen-binding domain include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab", or "F(ab')2".

[0032] antigen binding molecule In the present disclosure, an antigen-binding molecule containing an antigen-binding domain is used in the broadest sense, and specifically, various molecular types are included as long as they contain an antigen-binding domain. The antigen-binding molecule may be a molecule consisting of only an antigen-binding domain, or may be a molecule containing an antigen-binding domain and other domains. For example, when the antigen-binding molecule is a molecule in which an antigen-binding domain and an Fc region are bound, examples include a complete antibody and an antibody fragment. The antibody may include a single monoclonal antibody (including agonist and antagonist antibodies), a human antibody, a humanized antibody, a chimeric antibody, and the like. The antigen-binding molecule of the present disclosure may also include a scaffold molecule in which a three-dimensional structure such as an existing stable α / β barrel protein structure is used as a scaffold (foundation), and only a partial structure of the structure is compiled into a library for constructing an antigen-binding domain.

[0033] antibody In the present specification, an antibody refers to an immunoglobulin that is naturally occurring or partially or completely synthetically produced. Antibodies can be isolated from natural sources such as plasma or serum in which they are naturally present, or from culture supernatants of hybridoma cells that produce antibodies, or can be partially or completely synthesized using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine classes (isotypes) of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, the antibody of the present disclosure may include IgG1, IgG2, IgG3, and IgG4. As for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present disclosure. In particular, for the sequence of human IgG1, the amino acid sequence at positions 356-358 as defined by EU numbering may be DEL or EEM. In addition, for the human Igκ (Kappa) constant region and the human Igλ (Lambda) constant region, multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present disclosure.

[0034] EU numbering and Kabat numbering According to the method used in the present disclosure, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991). In the present specification, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids of the variable region are represented according to the Kabat numbering, and the amino acids of the constant region are represented according to the EU numbering based on the Kabat amino acid positions.

[0035] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0036] Hypervariable region The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen contact residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0037] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0038] Fc area The Fc region comprises an amino acid sequence derived from the constant region of an antibody heavy chain. The Fc region is a portion of the heavy chain constant region of an antibody, including the hinge, CH2 and CH3 domains, from the N-terminus of the hinge region of the papain cleavage site at approximately amino acid position 216 according to EU numbering. The Fc region may be obtained from human IgG1, but is not limited to a specific subclass of IgG. A suitable example of the Fc region is an Fc region having binding activity to FcRn in an acidic pH range, as described below. Another suitable example of the Fc region is an Fc region having binding activity to Fcγ receptor, as described below. A non-limiting embodiment of such an Fc region is an Fc region represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).

[0039] Low molecular weight antibody The antibody used in the present disclosure is not limited to a full-length antibody molecule, and may be a minibody or a modified version thereof. Minibodies include antibody fragments in which a part of a full-length antibody (e.g., a whole antibody such as whole IgG) is deleted, and are not particularly limited as long as they have binding activity to an antigen. Minibodies of the present disclosure are not particularly limited as long as they are a part of a full-length antibody, but preferably contain a heavy chain variable region (VH) or / and a light chain variable region (VL). The amino acid sequence of VH or VL may be substituted, deleted, added, and / or inserted. Furthermore, as long as they have binding activity to an antigen, a part of VH or / and VL may be deleted. The variable region may be chimerized or humanized. Specific examples of antibody fragments include Fab, Fab', F(ab')2, Fv, and the like. Specific examples of minibodies include Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), diabody, sc(Fv)2 (single chain (Fv)2), etc. Multimers of these antibodies (e.g., dimers, trimers, tetramers, polymers) are also included in the minibodies of the present disclosure.

[0040] Antibody fragments can be produced by treating antibodies with enzymes such as papain or pepsin, or genes encoding these antibody fragments can be constructed and introduced into an expression vector, which can then be expressed in a suitable host cell (see, for example, Co et al. (J. Immunol. (1994) 152, 2968-2976), Better and Horwitz (Methods in Enzymology (1989) 178, 476-496), Plueckthun and Skerra et al. (Methods in Enzymology (1989) 178, 476-496), Lamoyi (Methods in Enzymology (1989) 121, 652-663), Rousseaux et al. (Methods in Enzymology (1989) 121, 663-669) and Bird et al. (TIBTECH (1991) 9, 132-137).

[0041] Diabodies refer to bivalent minibodies constructed by gene fusion (Holliger et al. (Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993), European Patent Publication EP404097, and PCT Publication WO1993 / 011161, etc.). Diabodies are dimers composed of two polypeptide chains, and usually, each polypeptide chain is linked by a linker of, for example, about 5 residues, which is so short that VL and VH cannot bind to each other in the same chain. VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment because the linker between them is short, so that they form a dimer, and thus diabodies have two antigen-binding sites.

[0042] An scFv can be obtained by linking the heavy chain variable region and the light chain variable region of an antibody. In this scFv, the heavy chain variable region and the light chain variable region are linked via a linker, preferably a peptide linker (Huston et al. (Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883)). The heavy chain variable region and the light chain variable region in an scFv may be derived from any of the antibodies described herein. There are no particular limitations on the peptide linker linking the variable regions, and for example, any single-chain peptide consisting of about 3 to 25 residues, or a peptide linker described below, can be used. The variable regions can be linked by the PCR method described above. The entire or a portion of the DNA sequence encoding the heavy chain or the heavy chain variable region of the antibody and the DNA sequence encoding the light chain or the light chain variable region are used as a template, and sequences corresponding to the sequences at both ends of the DNA sequence are then subjected to PCR. The DNA encoding the scFv can be amplified by PCR using a pair of primers having the same sequence. Next, DNA encoding a peptide linker portion and a pair of primers having sequences designed so that both ends of the primers are linked to the H chain and L chain, respectively, are combined to perform a PCR reaction, thereby obtaining DNA having a desired sequence. Once the DNA encoding the scFv is prepared, an expression vector containing the same and a recombinant cell transformed with the expression vector can be obtained according to a conventional method, and the resulting recombinant cell can be cultured to express the DNA encoding the scFv, thereby obtaining the scFv.

[0043] sc(Fv)2 is a minibody in which two VHs and two VLs are linked together via a linker or the like to form a single chain (Hudson et al. (J. Immunol. Methods (1999) 231, 177-189)). sc(Fv)2 can be prepared, for example, by linking scFvs with a linker.

[0044] Furthermore, an antibody characterized in that the two VHs and two VLs are arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH] linker [VL] linker [VH] linker [VL]). The order of the two VHs and two VLs is not particularly limited to the above arrangement, and they may be arranged in any order. For example, the following arrangements may be mentioned. -[VL] linker [VH] linker [VH] linker [VL] -[VH] linker [VL] linker [VL] linker [VH] -[VH] linker [VH] linker [VL] linker [VL] -[VL] linker [VL] linker [VH] linker [VH] -[VL] linker [VH] linker [VL] linker [VH]

[0045] The linker for linking the antibody variable region may be the same as the linkers described in the section on antigen-binding molecules. For example, the following sc(Fv)2 is particularly preferred in the present disclosure: -[VH] peptide linker (15 amino acids) [VL] peptide linker (15 amino acids) [VH] peptide linker (15 amino acids) [VL] When linking four antibody variable regions, three linkers are usually required, and the same linkers may be used for all of them, or different linkers may be used.

[0046] To obtain such minibodies, antibodies may be treated with an enzyme, such as papain or pepsin, to generate antibody fragments, or DNA encoding these antibody fragments or minibodies may be constructed and introduced into an expression vector, which may then be expressed in an appropriate host cell (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 652-663). 663-669; see Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0047] Furthermore, a non-limiting embodiment of the antibody in the present disclosure includes a chimeric antigen receptor in which a fusion of an antibody or a fragment thereof that recognizes an antigen instead of a T cell receptor and a T cell signal domain is incorporated into a T cell, and a T cell into which the chimeric antigen receptor has been incorporated, but is not limited to this.

[0048] Single Domain Antibodies A suitable example of an antigen-binding domain of the present invention is a single domain antibody (sdAb).

[0049] As used herein, the term "single domain antibody" is not limited by its structure as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing VH and VL, whereas single domain antibodies are known to be able to exhibit antigen-binding activity by their own domain structure alone, without pairing with other domains. Single domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer.

[0050] Examples of single domain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack light chains, such as camelid VHH and shark VNAR, or antibody fragments that contain all or a part of the VH domain or all or a part of the VL domain of an antibody. Examples of single domain antibodies that are antibody fragments that contain all or a part of the VH / VL domains of an antibody include, but are not limited to, single domain antibodies artificially created starting from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0051] Single domain antibodies can be obtained from animals capable of producing single domain antibodies or by immunizing animals capable of producing single domain antibodies. Examples of animals capable of producing single domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and US Patent Publication US2011 / 0123527 A1. Humanized single domain antibodies can also be obtained by making the framework sequence of a single domain antibody obtained from an animal a human germline sequence or a sequence similar thereto. Humanized single domain antibodies (e.g., humanized VHH) are also an embodiment of the single domain antibody of the present invention. "Humanized single domain antibody" refers to a chimeric single domain antibody comprising amino acid residues from non-human CDR and human FR. In one embodiment, all or substantially all CDRs of a humanized single domain antibody correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. In a humanized antibody, even if some of the residues in the FR do not correspond to those of a human antibody, it is considered as an example in which substantially all of the FRs correspond to those of a human antibody. For example, when humanizing a VHH, which is one embodiment of a single domain antibody, some of the residues in the FR must be residues that do not correspond to those of a human antibody (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284.).

[0052] Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11 (1691-1701) are among the most famous.

[0053] Method for producing antibodies having desired binding activity to antigens Methods for producing antibodies that are not dependent on low molecular weight compounds including MTA and have the desired binding activity against antigens that are different molecules from low molecular weight compounds are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0054] The anti-IL-6R antibody can be obtained as a polyclonal or monoclonal antibody by known means. As the anti-IL-6R antibody, a monoclonal antibody derived from a mammal can be preferably produced. The monoclonal antibody derived from a mammal includes those produced by a hybridoma and those produced by a host cell transformed with an expression vector containing an antibody gene by a genetic engineering technique. The monoclonal antibody of the present invention includes a "humanized antibody" and a "chimeric antibody".

[0055] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Hybridomas producing anti-IL-6R antibodies can then be selected by screening for monoclonal antibody-producing cells using a conventional screening method.

[0056] Specifically, monoclonal antibodies are produced, for example, as follows. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for obtaining antibodies, can be obtained by expressing the IL-6R gene, the nucleotide sequence of which is disclosed in SEQ ID NO: 2. That is, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968), is expressed instead of the IL-6R protein represented by SEQ ID NO: 1. Purified natural IL-6R protein can also be used as a sensitizing antigen.

[0057] The purified IL-6R protein can be used as a sensitizing antigen for immunization of a mammal. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. It can also be obtained by decomposing the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be selected from the amino acid sequence corresponding to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids constituting the peptide to be used as a sensitizing antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, a peptide of 8 to 50, preferably 10 to 30 residues can be used as a sensitizing antigen.

[0058] In addition, a fusion protein in which a desired partial polypeptide or peptide of the IL-6R protein is fused with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). A method for obtaining IL-6R used as a sensitizing antigen and a method for immunization using the same are also specifically described in International Publications WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0059] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. In general, rodents such as mice, rats, hamsters, rabbits, monkeys, etc. are preferably used.

[0060] The above-mentioned animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, the immunization is carried out by administering the sensitizing antigen to the mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, etc., is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. In addition, a suitable carrier may be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0061] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the body of the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages: - The structure of membrane proteins such as IL-6R can be maintained to provide immune stimulation. -There is no need to purify the immunogen.

[0062] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be suitably used. As a method for administering a vector to a living body, a commonly used method can be used. For example, DNA immunization is performed by introducing gold particles to which an expression vector is adsorbed into the cells of an individual animal to be immunized using a gene gun. Furthermore, an antibody that recognizes IL-6R can also be produced using the method described in International Publication WO2003 / 104453.

[0063] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As preferred immune cells, splenocytes in particular can be used.

[0064] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells are preferably provided with an appropriate selection marker for screening. The selection marker refers to a trait that allows (or does not allow) a cell to survive under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, they can continue to synthesize DNA by utilizing the salvage circuit of normal cells, and therefore grow even in HAT selection medium.

[0065] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0066] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

[0067] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0068] More specifically, the cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

[0069] The ratio of immune cells to myeloma cells may be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, or other usual culture medium used for this type of cell culture may be used, and serum supplements such as fetal calf serum (FCS) may be suitably added.

[0070] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., average molecular weight of about 1000 to 6000) pre-warmed to about 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, the appropriate culture medium listed above is successively added, and the procedure of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like that are undesirable for hybridoma growth.

[0071] The hybridomas thus obtained can be selected by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture can be continued using the above HAT medium for a sufficient time (usually several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die. Then, screening and single cloning of hybridomas producing the desired antibody are performed by a conventional limiting dilution method.

[0072] The hybridomas thus obtained can be selected by using a selection medium according to the selection marker possessed by the myeloma used in the cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing in HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used in the cell fusion, cells that have successfully fused with normal cells can selectively grow in the HAT culture medium. The culture is continued using the above-mentioned HAT culture medium for a sufficient time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Next, screening and single cloning of hybridomas that produce the desired antibody can be performed by the usual limiting dilution method.

[0073] Screening and monocloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such a monoclonal antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows the binding of an antibody to a cell surface to be measured by analyzing cells contacted with a fluorescent antibody with laser light and measuring the fluorescence emitted by individual cells.

[0074] To screen for hybridomas producing the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. The preferred cells for screening are mammalian cells in which IL-6R is forcibly expressed. By using non-transformed mammalian cells used as host cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas producing antibodies that do not bind to host cells but bind to IL-6R-forcibly expressing cells, hybridomas producing IL-6R monoclonal antibodies can be obtained.

[0075] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of a hybridoma is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. When the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody having the binding ability to the antigen and are selected by these screenings can be cloned by limiting dilution or the like.

[0076] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium, and can be stored for a long period of time in liquid nitrogen.

[0077] The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining highly pure antibodies.

[0078] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody gene is incorporated into an appropriate vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0079] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from a hybridoma cell that produces the anti-IL-6R antibody. To do this, generally, total RNA is first extracted from the hybridoma. For example, the following method can be used to extract mRNA from cells. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0080] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from a hybridoma. cDNA encoding an antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo Co., Ltd.) or the like. In addition, for the synthesis and amplification of cDNA, a SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be appropriately used. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites (described below) can be introduced at both ends of the cDNA.

[0081] The desired cDNA fragment is purified from the obtained PCR product and then ligated to a vector DNA. The recombinant vector is thus prepared, introduced into E. coli or the like, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector has the nucleotide sequence of the desired cDNA is confirmed by a known method, such as the dideoxynucleotide chain termination method.

[0082] To obtain genes encoding variable regions, it is easy to use the 5'-RACE method using primers for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template to obtain a 5'-RACE cDNA library. A commercially available kit such as the SMART RACE cDNA Amplification Kit can be used to synthesize the 5'-RACE cDNA library.

[0083] The resulting 5'-RACE cDNA library is used as a template to amplify antibody genes by PCR. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0084] Specifically, for example, when the objective is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains and κ and λ chains as light chains can be used. To amplify the variable region genes of IgG, a primer that anneals to a portion corresponding to the constant region close to the variable region is generally used as the 3' primer. On the other hand, a primer included in the 5' RACE cDNA library construction kit is used as the 5' primer.

[0085] Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The desired antibody can be screened using the binding activity of the reconstituted immunoglobulin to IL-6R as an index. For example, when the aim is to obtain an antibody against IL-6R, it is more preferable that the binding of the antibody to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, as follows; (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) Selecting an antibody that binds to IL-6R-expressing cells.

[0086] Methods for detecting the binding between an antibody and IL-6R-expressing cells are known. Specifically, the binding between an antibody and IL-6R-expressing cells can be detected by techniques such as FACS described above. Fixed specimens of IL-6R-expressing cells can be appropriately used to evaluate the binding activity of an antibody.

[0087] As a method for screening antibodies using binding activity as an index, a panning method using a phage vector is also preferably used. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell group, a screening method using a phage vector is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form a single chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contacting this phage with a desired antigen, the phage bound to the antigen can be recovered to recover DNA encoding an scFv having the desired binding activity. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.

[0088] After obtaining a cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene. Furthermore, in order to insert one copy of the digested fragment into a vector in the correct direction, it is preferable to insert a restriction enzyme that gives a sticky end. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. At this time, if a gene encoding an antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody whose constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibody of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region, and the two genes digested with the same combination of restriction enzymes are fused in frame to construct a chimeric antibody expression vector.

[0089] To produce an anti-IL-6R monoclonal antibody, an antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for expressing an antibody include, for example, enhancers and promoters. In addition, a suitable signal sequence can be added to the amino terminus so that the expressed antibody is secreted outside the cell. In the examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted outside the cell as a mature polypeptide. Next, a suitable host cell is transformed with this expression vector to obtain a recombinant cell expressing DNA encoding an anti-IL-6R antibody.

[0090] For the expression of antibody genes, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. An antibody molecule comprising an H chain and an L chain can be expressed by simultaneously transforming (co-transfecting) the same host cell with vectors incorporating the H chain and the L chain. Alternatively, a host cell can be transformed by incorporating DNAs encoding the H chain and the L chain into a single expression vector (see International Publication WO 1994 / 011523).

[0091] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. Any of these expression systems can be applied to isolate the antigen-binding domain of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells include the following cells: (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / 0, NS0, etc.), BHK (baby hamster kidney cell line), Hela, Vero, HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA), PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)) (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0092] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. For transformation of plant cells, callus cultured cells can be appropriately used.

[0093] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, and Pichia genus such as Pichia pastoris. - Filamentous fungi: Aspergillus genus, such as Aspergillus niger.

[0094] In addition, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be appropriately used. An expression vector containing the target antibody gene is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0095] In addition to the above-mentioned host cells, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by in-frame insertion into a gene encoding a protein that is produced specifically in milk. For example, goat β-casein can be used as a protein secreted into milk. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The desired antibody can be obtained as a fusion protein with a milk protein from the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. Also, a hormone can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0096] When the antigen-binding molecule described herein is administered to humans, an antigen-binding domain derived from a recombinant antibody that has been artificially modified for the purpose of reducing heterologous antigenicity against humans, etc., may be appropriately used as the antigen-binding domain in the antigen-binding molecule. Examples of recombinant antibodies include humanized antibodies. These modified antibodies are appropriately produced using known methods.

[0097] As a method for producing an antibody having a desired binding activity to a specific low molecular weight compound, an antibody having a desired binding activity to a low molecular weight compound can be obtained by a method similar to a method for producing an antibody that binds to a normal protein antigen. As an embodiment of a method for producing a sensitizing antigen used to obtain an antibody against a low molecular weight compound, a method of linking Mariculture Keyhole Limpet Hemocyanin (KLH) to a low molecular weight compound is exemplified. As an example of a non-limiting sensitizing antigen produced to obtain an antibody against MTA, 6'-MTA- Keyhole Limpet Hemocyanin (6'-MTA-KLH) is exemplified. Mariculture Keyhole Limpet Hemocyanin (KLH) is a highly antigenic protein that can be recognized by T cell receptors expressed on helper T cells, and is known to activate antibody production, so it is expected that linking it to MTA will enhance the production of antibodies against MTA. The low molecular weight compound to be linked to KLH is not limited to MTA, and it is possible to prepare sensitizing antigens by a similar method for various low molecular weight compounds that can be synthesized, including, but not limited to, AMP, ADP, ATP, adenosine, SAH, etc. International Publication WO2013 / 180200 also discloses the design of low molecular weight compound immunogens. Furthermore, the antigen to be linked to the low molecular weight compound is not limited to KLH, and for example, one linked to biotin or the like may be used as the sensitizing antigen. Furthermore, any antigen other than KLH or biotin that can be linked to a low molecular weight compound may be used. The present disclosure also encompasses the embodiments described, by way of example, below. [1] Biotinylated MTA. [2] Biotin-2'-MTA. [3] 6'-MTA-biotin. [4] Use of the biotinylated MTA according to any one of [1] to [3] for screening for an antigen-binding molecule that binds to the MTA. [5] Use of a biotinylated MTA according to any one of [1] to [3] as an immunogen for obtaining an antigen-binding molecule that binds to the MTA. [6] A method for screening for an antigen-binding molecule that binds to MTA using the biotinylated MTA described in [1] to [3].

[0098] Multispecific or multiparatopic antigen-binding molecules An antigen-binding molecule that contains at least two antigen-binding domains, at least one of which binds to a first epitope in an antigen molecule and at least one other antigen-binding domain that binds to a second epitope in an antigen molecule, is called a multispecific antigen-binding molecule from the viewpoint of the specificity of the reaction. When an antigen-binding molecule binds to two different epitopes through two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a bispecific antigen-binding molecule. When an antigen-binding molecule binds to three different epitopes through three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a trispecific antigen-binding molecule.

[0099] The paratope in the antigen-binding domain that binds to a first epitope in an antigen molecule and the paratope in the antigen-binding domain that binds to a second epitope that has a structure different from that of the first epitope are different in structure. Therefore, an antigen-binding molecule that contains at least two antigen-binding domains, in which at least one antigen-binding domain binds to a first epitope in an antigen molecule and at least one other antigen-binding domain binds to a second epitope in an antigen molecule, is called a multiparatopic antigen-binding molecule from the viewpoint of the specificity of its structure. When an antigen-binding molecule binds to two different epitopes through two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a double paratopic antigen-binding molecule. When an antigen-binding molecule binds to three different epitopes through three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a triple paratopic antigen-binding molecule.

[0100] Bispecific antibodies and methods for producing same Multivalent, multispecific or multiparatopic antigen-binding molecules comprising one or more antigen-binding domains and methods for preparing them are also described in non-patent literature such as Conrath et al. (J.Biol.Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302) and Kontermann RE (2011) Bispecific Antibodies (Springer-Verlag), as well as in patent literature such as International Publication WO1996 / 034103 or WO1999 / 023221. The antigen-binding molecules of the present disclosure can be prepared by using the multispecific or multiparatopic antigen-binding molecules and methods for preparing them described therein.

[0101] As an embodiment of the above-mentioned multispecific or multiparatopic antigen-binding molecule and a method for preparing the same, a bispecific antibody and a method for producing the same are exemplified below. A bispecific antibody is an antibody that contains two types of variable regions that specifically bind to different epitopes. An IgG-type bispecific antibody can be secreted by a hybrid hybridoma (quadroma) generated by fusing two types of hybridomas that produce IgG antibodies (Milstein et al. (Nature (1983) 305, 537-540)).

[0102] When producing a bispecific antibody using a recombinant technique as described above in the section on antibodies, a method of introducing genes encoding heavy chains containing two types of variable regions of interest into cells and co-expressing them can be adopted. However, even if only considering the combination of heavy chains in such a co-expression method, (i) a combination of heavy chains in which a heavy chain containing a variable region binding to a first epitope is paired with a heavy chain containing a variable region binding to a second epitope, (ii) a combination of heavy chains in which only heavy chains containing a variable region binding to a first epitope are paired, and (iii) a combination of heavy chains in which only heavy chains containing a variable region binding to a second epitope are paired, is obtained in a mixture in a molecular ratio of 2:1:1. It is difficult to purify an antigen-binding molecule containing the desired combination of heavy chains from a mixture of these three types of heavy chain combinations.

[0103] When producing bispecific antibodies using such recombinant techniques, bispecific antibodies containing a heterogeneous combination of heavy chains can be preferentially secreted by modifying the CH3 domains constituting the heavy chains with appropriate amino acid substitutions. Specifically, the amino acid side chains present in the CH3 domain of one heavy chain are replaced with larger side chains (knobs) and the amino acid side chains present in the CH3 domain of the other heavy chain are replaced with smaller side chains (holes) so that the knobs can be positioned in the holes, thereby promoting the formation of heterologous heavy chains and inhibiting the formation of homologous heavy chains (International Publication WO1996027011, Ridgway et al. (Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat. Biotech. (1998) 16, 677-681)).

[0104] Also known is a technique for producing a bispecific antibody by utilizing a method for controlling the association of polypeptides or the association of heterogeneous multimers constituted by polypeptides in the association of heavy chains. That is, a method for controlling the association of heavy chains having the same sequence by modifying amino acid residues that form an interface within the heavy chains, thereby inhibiting the association of heavy chains having the same sequence, so that two heavy chains having different sequences are formed, can be employed in the production of a bispecific antibody (International Publication WO2006 / 106905). Such a method can also be employed in the production of a bispecific antibody.

[0105] cancer In this specification, the term "cancer" is generally used to refer to malignant neoplasms, which may be metastatic or non-metastatic. For example, non-limiting examples of carcinomas originating from epithelial tissues such as the digestive tract and skin include brain cancer, skin cancer, head and neck cancer, esophageal cancer, lung cancer, stomach cancer, duodenal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, pancreatic cancer, liver cancer, colorectal cancer, colon cancer, bladder cancer, and ovarian cancer. Non-limiting examples of sarcomas originating from non-epithelial tissues (stroma) such as muscle include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Further, non-limiting examples of hematologic cancers of hematopoietic origin include malignant lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, leukemias, including acute or chronic myelocytic leukemia, and acute or chronic lymphatic leukemia, and multiple myeloma. The term "neoplasm" as used broadly herein means any newly formed pathological tissue tumor. In the present disclosure, a neoplasm results in the formation of a tumor, which is characterized in part by angiogenesis. A neoplasm can be benign, e.g., hemangioma, glioma, teratoma, etc., or malignant, e.g., carcinoma, sarcoma, glioma, astrocytoma, neuroblastoma, retinoblastoma, etc.

[0106] The term "cancer tissue" refers to tissue that contains at least one cancer cell. Thus, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such that cancer tissue contains cancer cells and blood vessels. As used herein, a tumor mass refers to a foci of tumor tissue. The term "tumor" is generally used to refer to benign or malignant neoplasms.

[0107] In the present disclosure, "cancer tissue in which MTA accumulates" refers to cancer tissue in which MTA is detected in larger amounts than in normal tissue, and non-limiting examples of normal tissues to be compared include normal tissues adjacent to the cancer tissue and tissues of healthy individuals. In addition, cancer tissues in which methylthioadenosine phosphorylase (MTAP), which metabolizes MTA, is deficient or in which the function of MTAP is reduced are also examples of cancer tissues in which MTA accumulates. Specifically, cancer tissues in which the gene encoding MTAP is deficient or expression is reduced, cancer tissues in which a mutation or splicing variant that reduces the activity of MTAP is expressed, or cancer tissues in which the enzyme activity of MTAP is reduced are examples of cancer tissues in which MTA accumulates. The reduction in the expression or function of MTAP can be determined by comparing with normal tissues, and non-limiting examples of normal tissues to be compared include normal tissues adjacent to cancer tissues and tissues of healthy individuals.

[0108] Cancer-associated fibroblast (CAF) In the present disclosure, "cancer-associated fibroblast (CAF)" refers to a heterogeneous population of various origins, such as endothelial cells present in the periphery of cancer tissue. Non-limiting characteristics of CAF include promoting cancer cell proliferation, promoting angiogenesis, promoting vascular invasion of cancer cells, and constructing a microenvironment favorable for cancer progression through control of immune response, etc. Furthermore, non-limiting examples of CAF include cells expressing markers selected from α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), tenascin-C (TN-C), periostin (POSTN), NG2 chondroitin sulfate proteoglycan (NG2), platelet derived growth factor receptor (PDGFR), vimentin, desmin, fibroblast specific protein-1 (FSP1), and fibronectin.

[0109] Tumor-associated macrophage(TAM) In the present disclosure, "tumor-associated macrophage (TAM)" refers to a macrophage present in cancer tissue and its surrounding area. For example, a macrophage population that forms a cancer microenvironment together with fibroblasts and vascular endothelial cells is exemplified. A non-limiting feature of TAM is that it induces neovascularization by producing various angiogenic factors, such as suppressing anti-tumor immunity by promoting the production of anti-inflammatory factors and the infiltration of regulatory T cells. Furthermore, a non-limiting embodiment of TAM is a cell expressing a marker selected from CD163, CD204, IL-10, TGF-β, and Prastaglandin E2.

[0110] Effector cells In the present disclosure, "effector cells" refers to T cells (CD4 + (Helper lymphocytes) T cells and / or CD8 +Although the term may be used in the broadest sense to include leukocytes such as (cytotoxic) T cells), polymorphonuclear leukocytes (neutrophils, eosinophils, basophils, mast cells), monocytes, macrophages, histiocytes or natural killer cells (NK cells), NK-like T cells, Kupffer cells, Langerhans cells, or lymphokine-activated killer cells (LAK cells), B lymphocytes, or antigen-presenting cells such as dendritic cells or macrophages, examples of suitable effector cells include CD8 + (Cytotoxic) T cells, NK cells, or macrophages are included. Any membrane-type molecule expressed on the cell membrane of effector cells can be used as an antigen to which at least one antigen-binding domain contained in the antigen-binding molecule of the present disclosure binds, and suitable membrane-type molecules include, but are not limited to, polypeptides constituting TCR, CD3, CD2, CD28, CD44, CD16, CD32, CD64, or NKG2D or NK cell activating ligands.

[0111] Methylthioadenosine (MTA) The term "MTA" as used herein refers to methylthioadenosine, specifically, the compound represented by the following chemical formula: [ka] MTA (CAS number: 2457-80-9)

[0112] MTA Analogues The term "MTA analog" used herein refers to a low molecular weight compound other than MTA that has a partial structure in common with MTA. Examples of MTA analogs include low molecular weight compounds that have adenosine as a common backbone in the molecule, and low molecular weight compounds that have a side chain containing a sulfur atom or an oxygen atom at the carbon at the 5th position of adenosine. Furthermore, examples of MTA analogs include, but are not limited to, metabolites of the polyamine biosynthetic pathway such as S-adenosylmethionine (SAM) and S-adenosylhomocysteine ​​(SAH, S-(5'-Adenosyl)-L-homocysteine) (Stevens et al. (J Chromatogr A. 2010 May 7;1217(19):3282-8)). [ka] SAM [ka] SAH In addition, non-limiting examples of MTA analogs include adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). [ka] Adenosine [ka] Adenosine monophosphate (AMP) [ka] Adenosine diphosphate (ADP) [ka] Adenosine triphosphate (ATP)

[0113] low molecular compound The term "low molecular weight compound" as used in the present disclosure refers to a naturally occurring or non-naturally occurring chemical substance other than the "biopolymer" present in a living body. Examples of low molecular weight compounds include, but are not limited to, naturally occurring or artificially synthesized compounds with a molecular weight of 10,000 or less, preferably compounds with a molecular weight of 1,000 or less. Non-limiting examples of low molecular weight compounds include cancer tissue-specific compounds, inflamed tissue-specific compounds, and non-natural compounds.

[0114] Cancer tissue-specific compounds As used herein, the term "cancer tissue-specific compound" refers to a compound that is differentially present in cancer tissue compared to non-cancerous tissue.

[0115] For example, in some embodiments, a cancer tissue-specific compound may be a compound that is defined by a qualitative cancer tissue specificity, such as being present in cancer tissue but not in non-cancerous tissue, or being present in non-cancerous tissue but not in cancer tissue. In another embodiment, a cancer tissue-specific compound may be a compound that is defined by a quantitative cancer tissue specificity, such as being present in cancer tissue at a different concentration (e.g., higher or lower concentration) compared to non-cancerous tissue. For example, a cancer tissue-specific compound is differentially present at a given concentration. However, in general, a cancer tissue specific compound will have a specific activity of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 5-fold, at least 10 ... 3 times at least 10 4 times at least 10 5 times at least 10 6The cancer tissue specific compound may be present at a concentration that is increased by 100% or more, up to infinity (i.e., absent in non-cancer tissue), or generally at a concentration that is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% (i.e., representing absence). The cancer tissue specific compound is preferably differentially present at a concentration that is statistically significant (i.e., p-value is less than 0.05 and / or q-value is less than 0.10, as determined using either Welch's t-test or Wilcoxon's rank sum test). Non-limiting examples of cancer tissue-specific compounds include compounds that are cancer tissue-specific metabolites (cancer tissue-specific metabolites; cancer cell-specific metabolites, immune cell-specific metabolites infiltrating cancer tissue, and cancer stromal cell-specific metabolites) produced by metabolic activity specific to cancer cells, immune cells, and stromal cells contained in cancer tissue, as described below.

[0116] Cancer tissue-specific metabolites The term "metabolism" refers to chemical changes occurring in the tissues of an organism, including "anabolism" and "catabolism." Anabolism refers to the biosynthesis or accumulation of molecules, whereas catabolism refers to the breakdown of molecules. A "metabolite" is an intermediate or product resulting from the metabolism of a substance. A "primary metabolite" refers to a metabolic product that is directly involved in the growth or reproduction process of a cell or organism, whereas a "secondary metabolite" refers to a product, such as an antibiotic or a pigment, that is the result of metabolism that synthesizes a substance that is not directly involved in the growth or reproduction process or is not directly involved in the life phenomena common to cells or organisms. A metabolite can be a metabolic product of a "biopolymer" or a "small molecule." A "biopolymer" is a polymer that is composed of one or more types of repeating units. Biopolymers are generally found in biological systems and include molecules with a molecular weight of approximately 5000 or more that form structures such as cells that make up the tissues of an organism and the intercellular and intertissue matrices attached to them, particularly polysaccharides (carbohydrates, etc.), peptides (this term is used to include polypeptides and proteins), and polynucleotides, as well as analogs thereof, such as compounds that are composed of or contain amino acid analogs or non-amino acid groups.

[0117] A non-limiting example of a cancer tissue-specific metabolite described herein is preferably a cancer cell-specific low molecular weight metabolite (Eva Gottfried, Katrin Peter and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132). Furthermore, the metabolites include metabolites that are produced in high amounts by immune cells infiltrating into cancer tissues and metabolites that are produced in high amounts by stromal cells (cancer stromal cells or cancer stromal fibroblasts (CAFs)) that support the survival and / or growth of cancer cells. Examples of infiltrating immune cells include dendritic cells, inhibitory dendritic cells, inhibitory T cells, exhausted T cells, myeloma derived suppressor cells (MDSCs), and the like. In addition, the metabolites in the present invention also include compounds that are released from inside the cells to outside the cells when cells (cancer cells, immune cells, stromal cells) present in cancer tissues die due to apoptosis, necrosis, or the like.

[0118] To identify cancer cell-specific metabolites, analyses at the transcriptome level (e.g., Dhanasekaran et al. (Nature (2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. USA (2004) 101, 811-816, or Perou et al. (Nature (2000) 406, 747-752) are exemplified) or at the proteome level (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15, Hood et al. (Mol. Cell. Proteomics (2005) 4, In addition to the above-mentioned methods, metabolic (metabolomic) analysis centered on metabolic profiling may be appropriately used. That is, metabolic profiling using high pressure liquid chromatography (HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol. Recognit. (1997) 10, 182-187), mass spectrometry (Gates and Sweeley (Clin. Chem. (1978) 24, 1663-1673) (GC / MS and LC / MS)), ELISA, etc., alone and / or in combination, may be appropriately used to identify metabolites in a test sample.

[0119] These studies have revealed intratumoral heterogeneity constituted by altered concentration gradients of metabolites (e.g., glucose or oxygen) and growth factors that enable cancer cells to grow under low oxygen tension conditions (Dang and Semenza (Trends Biochem. Sci. (1999) 24, 68-72)). These studies have also used cell line models to understand changes in energy utilization pathways due to different degrees of tumor aggressiveness (Vizan et al. (Cancer Res. (2005) 65, 5512-5515). A non-limiting embodiment of the technical components of the metabolomics platform includes sample extraction, separation, detection, spectroscopic analysis, data normalization, delineation of class-specific metabolites, pathway mapping, confirmation, and functional characterization of candidate metabolites as described in Lawton et al. (Pharmacogenomics (2008) 9, 383). These methods allow the identification of cancer cell-specific metabolites in the desired cancer tissue.

[0120] Inflamed tissue-specific compounds As used herein, the term "inflammatory tissue-specific compound" refers to a compound that is differentially present in inflamed tissue compared to non-inflamed tissue. As used herein, "inflamed tissue" includes, for example, the following: -Joints in rheumatoid arthritis and osteoarthritis Lungs (alveoli) in bronchial asthma and COPD Digestive tract in inflammatory bowel disease, Crohn's disease and ulcerative colitis Fibrotic tissue in liver, kidney and lung fibrosis -Tissues undergoing rejection in organ transplants -Blood vessels and heart (myocardium) in arteriosclerosis and heart failure Visceral fat in metabolic syndrome Skin tissue in atopic dermatitis and other skin conditions -Spinal nerve damage in herniated discs and chronic lower back pain

[0121] Inflammatory tissue-specific metabolites The inflammatory tissue-specific metabolites are metabolites that are produced in high amounts by immune cells infiltrating into inflammatory tissues, and metabolites that are produced in high amounts by normal cells specifically damaged in inflammatory tissues. Examples of infiltrating immune cells include effector T cells, mature dendritic cells, neutrophils, granular cells (mast cells), basophils, and the like. The metabolites in the present invention also include compounds that are released from inside to outside the cells when cells (immune cells, normal cells) present in inflammatory tissues die due to apoptosis, necrosis, or the like.

[0122] The term "non-natural compound" as used herein refers to a non-naturally occurring chemical substance and its metabolite. One embodiment of the invention is a non-naturally occurring chemical substance and its metabolite that accumulate in target tissues after being administered from outside the body to the body. Examples of non-natural compounds include (1) capecitabine (Xeloda) and its metabolite 5-FU (fluorouracil), and (2) TH-302 and bromoisophosphamide mustard (Br-IPM). 5-FU is a metabolite of capecitabine (Xeloda), and is known to be metabolized by the cancer tissue-specific metabolic enzymes cytidine deaminase and thymidine phosphorylase (Desmoulin F. et al. Drug Metab Dispos. 2002). It is also known that TH-302 is converted to Br-IPM by reduction under hypoxic conditions, such as around cancer tissue (Duan JX, et al. J Med Chem. 2008). For example, when capecitabine (xeloda) is administered, it is metabolized to 5-FU by cancer-specific metabolic enzymes, etc., and the concentration of 5-FU increases in the cancer local area (Desmoulin F. et al. Drug Metab Dispos. 2002). Therefore, it is considered that an antibody that uses 5-FU as a switch can selectively bind to a target antigen only in the cancer local area. In addition to metabolic enzymes, it is also considered possible to use molecules that are generated in a cancer-specific hypoxic or acidic environment as a switch. For example, TH-302 (Duan JX, et al. J Med Chem. 2008) is metabolized to Br-IPM under hypoxic conditions, so it is considered that an antibody that uses Br-IPM as a switch can selectively bind to a target antigen only in the cancer local area. For example, methods for administering a non-natural compound to a living body include known administration methods such as oral administration, ocular administration, transdermal administration, intranasal administration, intravenous administration, and pulmonary administration, but are not limited to these.

[0123] Non-limiting examples of low molecular weight compounds according to the present disclosure include MTA, SAM, SAH, adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and the like.

[0124] Further non-limiting examples of small molecule compounds according to the present disclosure include the following compounds:

[0125] (1) Primary metabolic products of glycolysis or the Krebs cycle, such as lactate, succinate, and citrate A non-limiting embodiment of the low molecular weight compound or cancer tissue-specific compound used in the present invention, particularly the cancer cell-specific metabolite, is preferably a primary metabolite generated as a result of glucose metabolism, such as lactate, succinate, or citrate, which is present at higher concentrations in cancer tissue than in surrounding non-cancerous tissue. The glycolytic phenotype, characterized by the upregulation of glycolytic (Embden-Myerhof pathway) enzymes such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH), has long been known as the Warburg effect, which is a characteristic of solid tumors.

[0126] In other words, it is believed that the high expression of pyruvate kinase M2 isoform, which is necessary for glycolysis under anaerobic conditions, rather than M1 isoform, in tumor cells is favorable for the growth of tumor cells in vivo (Christofk et al. (Nature (2008) 452, 230-233)). Pyruvate produced by pyruvate kinase is feedback inhibited by lactate, which is produced as a result of an equilibrium reaction by lactate dehydrogenase (LDH) under anaerobic conditions. This feedback inhibition promotes mitochondrial respiration (Krebs cycle) and inhibits cell proliferation, so it is said that upregulation of LDH, hexokinase, and glucose transporters (GLUTs) plays an important role in tumor cell proliferation (Fantin et al. (Cancer Cell (2006) 9, 425-434)). Glucose is metabolized in glycolysis, and its final metabolic product, lactic acid, is co-transported with protons around the tumor, which is said to cause the pH of the tissue surrounding the tumor to change to an acidic condition. It is known that lactic acid, the final product of glycolysis, and succinic acid and citric acid produced by promoting respiration in mitochondria, accumulate in cancer tissue (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). As a non-limiting embodiment of the low-molecular-weight compound, cancer tissue-specific compound, and particularly cancer cell-specific metabolite used in the present invention, preferred examples include lactic acid, succinic acid, citric acid, and the like, which are primary metabolic products produced by the metabolism of the glycolysis. It is also known that succinic acid, which is present in high concentrations within cells, leaks out of the cells due to cell death (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is thought that the concentration of succinic acid increases in cancer tissues where cell death occurs frequently.

[0127] (2) Amino acids such as alanine, glutamic acid, and aspartic acid In addition to the glucose metabolism described above, it is known that amino acid metabolism is also altered in tumor cells, which require a continuous supply of essential and non-essential amino acids necessary for the biosynthesis of biopolymers under anaerobic conditions. Glutamine is the most widely distributed amino acid in the body, containing two nitrogen atoms in its side chain and acting as a nitrogen carrier. Tumor cells with an increased rate of glutamine uptake into cells are said to function as glutamine traps. This increased uptake of glutamine and its conversion to glutamate and lactate is called "glutaminolysis" and is believed to be a hallmark of transformed (tumor) cells (Mazurek and Eigenbrodt, Anticancer Res. (2003) 23, 1149-1154, and Mazurek et al., J. Cell. Physiol. (1999) 181, 136-146). As a result, cancer patients show decreased levels of glutamine in plasma while increasing glutamate concentrations (Droge et al., Immunobiology (1987) 174, 473-479). And in lung cancer tissue, 13 Metabolic studies of C-radiolabeled glucose 13 C-labeled succinic acid, 13 C-labeled alanine, 13 C-labeled glutamic acid, and 13 A correlation was observed between the concentrations of C-labeled citric acid. Non-limiting examples of the low molecular weight compound and cancer tissue-specific compound used in the present invention include alanine, glutamic acid, aspartic acid, and the like, which accumulate at high concentrations in cancer tissues due to the degradation of glutamine or the like.

[0128] (3) Metabolic products of amino acids such as kynurenine Indoleamine 2,3-dioxygenase (IDO) is a tryptophan metabolic enzyme that is highly expressed in many cancers, including melanoma, colon cancer, and kidney cancer (Uyttenhove et al. (Nat. Med. (2003) 9, 1269-127), and two isoforms are known to exist (Lob et al. (CancerImmunol. Immunother. (2009) 58, 153-157)). IDO catalyzes the conversion of tryptophan to kynurenine (represented by the formula below) and is the first enzyme in the de novo pathway of nicotinamide nucleotide (NAD). In gliomas that do not express IDO, kynurenine is produced from tryptophan by tryptophan 2,3-dioxygenase (TDO) in the liver (Opitz et al. (Nature (2011) 478, 7368, 197-203)). IDO is also expressed in dendritic cells infiltrating cancer tissue, and dendritic cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). IDO is also expressed in myeloid-derived suppressor cells (MDSCs) in cancer tissue, and MDSCs also produce kynurenine (Yu et al. (J. Immunol. (2013) 190, 3783-3797)). [ka] Kynurenine

[0129] Kynurenine is known to suppress allogeneic T cell responses (Frumento et al., J. Exp. Med. (2002) 196, 459-468), and it has been proposed that this suppression allows tumor cells to evade antitumor immune responses, and that glioma cell proliferation is promoted through an autocrine proliferation mechanism in which kynurenine acts as an endogenous ligand for the aryl hydrocarbon receptor expressed in gliomas (Opitz et al., supra). Kynurenine is converted to anthranilic acid (represented by the formula below) by kynurenidase and to 3-hydroxykynurenine (represented by the formula below) by kynurenine 3-hydroxylase. Both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, which is a precursor of NAD.

[0130] [ka] Anthranilic Acid

[0131] [ka] 3-Hydroxykynurenine

[0132] Kynurenine is converted to kynurenic acid (represented by the following formula) by kynurenine aminotransferase. Non-limiting examples of the low molecular weight compounds, cancer tissue-specific compounds, and particularly cancer cell-specific metabolites used in the present invention include kynurenine and its metabolites, such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, which are amino acid metabolites.

[0133] [ka] Kynurenic acid

[0134] (4) Prostaglandin E2 and other metabolic products of arachidonic acid Prostaglandin E2 (PGE2) (represented by the formula below) is a metabolite of arachidonic acid called plastonoids, including prostaglandins and thromboxanes, synthesized by cyclooxygenase (COX)-1 / 2 (Warner and Mitchell (FASEB J. (2004) 18, 790-804)). PGE2 promotes the proliferation of colon cancer cells and inhibits their apoptosis (Sheng et al. (Cancer Res. (1998) 58, 362-366)). It is known that the expression of cyclooxygenase is altered in many cancer cells. That is, COX-1 is constitutively expressed in almost all tissues, whereas COX-2 is mainly found to be induced by certain inflammatory cytokines and oncogenes in tumors (Warner and Mitchell (ibid.)). It has also been reported that overexpression of COX-2 is associated with poor prognosis in breast cancer (Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)) and rapid disease progression in ovarian cancer (Denker et al. (Mod. Pathol. (2006) 19, 1261-1269)). Inhibitory T cells infiltrating cancer tissue also produce prostaglandin E2 (Curr. Med. Chem. (2011) 18, 5217-5223). It is known that low molecular weight compounds such as prostaglandins and leukotrienes, which are metabolites of arachidonic acid, act as stimulatory factors that control the autocrine and / or paracrine growth of cancer (Nat. Rev. Cancer (2012) 12 (11) 782-792). As a non-limiting embodiment of the low molecular weight compound, cancer tissue-specific compound, particularly cancer cell-specific metabolites and immune cell-specific metabolites infiltrating cancer tissues used in the present invention, preferred examples include arachidonic acid metabolites such as prostaglandin E2. In addition to prostaglandin E2, thromboxane A2 (TXA2) is produced at increased levels in cancer tissues such as colon cancer (J. Lab. Clin. Med. (1993) 122, 518-523), and is a preferred embodiment of the arachidonic acid metabolite of the present invention.

[0135] [ka] Prostaglandin E2 (PGE2)

[0136] It is also known that PGE2 concentrations are high in rheumatoid arthritis and osteoarthritis (Eur. J. Clin. Pharmacol. (1994) 46, 3-7., Clin. Exp. Rheumatol. (1999) 17, 151-160, Am. J. Vet. Res. (2004) 65, 1269-1275.). As a non-limiting embodiment of the low molecular weight compound, inflammatory tissue-specific compound, particularly inflammatory cell-specific metabolites and immune cell-specific metabolites that infiltrate into inflammatory tissues, used in the present invention, preferred examples include metabolites of arachidonic acid such as prostaglandin E2.

[0137] (5) Nucleosides containing a purine ring structure, such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) It is known that when cancer cells die, a large amount of ATP leaks out of the cell. Therefore, the ATP concentration in cancer tissue is significantly higher than that in normal tissue (PLoS One. (2008) 3, e2599). Several types of cells release adenine nucleotides in the form of ATP, ADP, and AMP. Adenosine is metabolized by cell surface extracellular enzymes such as eco-5'-nucleotidase (CD73) (Resta and Thompson (Immunol. Rev. (1998) 161, 95-109) and Sadej et al. (Melanoma Res. (2006) 16, 213-222). Adenosine is a purine nucleoside that is constitutively present in the extracellular environment at low concentrations, but a marked increase in extracellular adenosine concentrations has been reported in hypoxic tissues found in solid tumors (Blay and Hoskin (Cancer Res. (1997) 57, 2602-2605). CD73 is expressed on the surface of tumor and immune cells (Kobie et al. (J. Immunol. (2006) 177, 6780-6786) and breast cancer (Canbolat et al. (Breast Cancer Res. Treat. (1996) 37, 189-193), gastric cancer (Durak et al. (Cancer Lett. (1994) 84, 199-202), pancreatic cancer (Flocke and Mannherz (Biochim. Biophys. Acta (1991) 1076, 273-281), and glioblastoma (Bardot et al. (Br. J. Cancer (1994) 70, 212-218)). It has been proposed that the accumulation of adenosine in cancer tissues may be due to increased intracellular adenosine production resulting from dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick and Willis (Biochem. J. (1989) 261, 541-550).Furthermore, inhibitory T cells infiltrating into cancer tissues also express ATPase and produce adenosine (Proc. Natl. Acad. Sci. (2006) 103 (35), 13132-13137, Curr. Med. Chem. (2011) 18, 5217-5223). The produced adenosine is thought to create an immunosuppressive environment in cancer tissues through adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011), 18, 5217-23). ​​As a non-limiting embodiment of the low molecular weight compound and cancer tissue-specific compound used in the present invention, ATP, ADP, AMP, adenosine, etc., which accumulate at high concentrations in cancer tissues due to the metabolism of purine nucleotides such as ATP, are preferably mentioned. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in high accumulation of inosine.

[0138] It is also known that ATP concentrations are high in alveoli where inflammation due to bronchial asthma is occurring (Nat. Med. (2007) 13, 913-919). It is also known that ATP concentrations are high in alveoli where inflammation due to COPD is occurring (Am. J. Respir. Crit. Care Med. (2010) 181, 928-934). It has also been observed that adenosine concentrations are high in the synovial fluid of patients with rheumatoid arthritis (Journal of Pharmaceutical and Biomedical Analysis (2004) 36 877-882). It is also known that ATP concentrations are high in tissues where rejection is occurring due to GVHD (Nat. Med. (2010) 16, 1434-1438). It is also known that adenosine concentration is increased in fibrotic tissues in the lungs, liver, and kidneys (FASEB J. (2008) 22, 2263-2272, J. Immunol. (2006) 176, 4449-4458, J. Am. Soc. Nephrol. (2011) 22 (5), 890-901, PLoS ONE J. (2010) 5 (2), e9242). It has also been observed that ATP concentration is increased in fibrotic tissues of patients with pulmonary fibrosis (Am. J. Respir. Crit. Care Med. (2010) 182, 774-783). As a non-limiting embodiment of the low molecular weight compound and inflammatory tissue-specific compound used in the present invention, ATP, ADP, AMP, adenosine, etc., which are accumulated at high concentrations in inflammatory tissues by metabolism of purine nucleotides such as ATP, are preferred. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in high accumulation of inosine.

[0139] (6)Uric acid Uric acid is a product of the metabolic pathway of purine nucleosides in vivo, and is released outside cells, such as in blood or interstitial space. In recent years, it has been revealed that uric acid is released from dead cells present at lesion sites, such as cancer tissues (Nat. Med. (2007) 13, 851-856). As a non-limiting embodiment of the low molecular weight compound and cancer tissue-specific compound used in the present invention, uric acid, which accumulates in high concentrations in cancer tissues due to the metabolism of such purine nucleotides such as ATP, is also preferably exemplified.

[0140] In recent years, it has been revealed that uric acid released from cells undergoing necrosis promotes inflammatory responses (J. Clin. Invest. (2010) 120 (6), 1939-1949). A non-limiting example of the low molecular weight compound and inflamed tissue-specific compound used in the present invention is uric acid, which accumulates to high concentrations in inflammatory tissues due to the metabolism of purine nucleotides such as ATP.

[0141] (7) 1-Methylnicotinamide It is known that the enzyme nicotinamide N-methyltransferase is highly expressed in several human cancer tissues. When this enzyme produces the stable metabolite 1-methylnicotinamide from nicotinamide, it consumes the methyl group of the methyl donor S-adenosylmethionine (SAM). It has been proposed that high expression of nicotinamide N-methyltransferase contributes to tumorigenesis through a mechanism that impairs DNA methylation ability due to a decrease in SAM concentration in cancer cells (Ulanovskaya et al. (Nat. Chem. Biol. (2013) 9 (5) 300-306)). 1-methylnicotinamide, a stable metabolic product of this enzyme, is known to be secreted outside the cells of cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). A non-limiting example of the low molecular weight compound and cancer tissue-specific compound used in the present invention preferably includes 1-methylnicotinamide, which accumulates at high concentrations in cancer tissues through the metabolism of nicotinamide. The low molecular weight compound in the present disclosure can interact with an antigen-binding molecule. The amino acid residue in the antigen-binding molecule that can interact with a low molecular weight compound may be present in the antigen-binding domain in the antigen-binding molecule, or may be present in a site other than the antigen-binding domain. The site in the antigen-binding molecule that interacts with a low molecular weight compound is exemplified by, but is not limited to, the antigen-binding domain.

[0142] Antigen-binding domain that specifically binds to an antigen In the present specification, the term "antigen-binding domain that specifically binds to an antigen" is used when the antigen-binding domain is specific to a specific epitope among multiple epitopes contained in a certain antigen. In addition, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope. Here, "substantially no binding" is determined according to the method described in the above section on binding activity, and means that the binding activity of the specific binding molecule to a molecule other than the partner is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the partner molecule. When the antigen-binding domain is an antigen-binding domain whose binding activity to an antigen changes depending on MTA or a low molecular weight compound other than MTA, the binding of the antigen-binding domain to an antigen is measured under conditions under which the binding activity of the antigen-binding domain to the antigen is high (for example, under a specific concentration of MTA or in the absence of MTA, under a specific concentration of a low molecular weight compound other than MTA or in the absence of a low molecular weight compound other than MTA).

[0143] Antigen-binding activity and method for confirming antigen-binding activity The term "binding activity" refers to the strength of the total non-covalent interaction between one or more binding sites of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Here, "binding activity" is not strictly limited to 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when the members of a binding pair reflect a monovalent 1:1 interaction, the binding activity refers to the inherent binding affinity ("affinity"). When the members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of a molecule X to its partner Y can generally be expressed by a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand". Binding activity can be measured by conventional methods known in the art, including those described herein. Conditions other than the concentration of the target tissue-specific compound can be appropriately determined by those skilled in the art. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0144] Binding activity of antigen-binding molecules In certain embodiments, the antigen-binding molecule provided herein is an antibody, and the binding activity of the antibody is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 is the dissociation constant (KD) of the ATPase (M).

[0145] In one embodiment, the binding activity of the antibody is measured by a ligand capture method using, for example, a BIACORE® T200 or BIACORE® 4000 (GE Healthcare, Uppsala, Sweden) that uses surface plasmon resonance analysis as a measurement principle. The BIACORE® Control Software is used to operate the instrument. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, an anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with a 10 mM sodium acetate solution of appropriate pH and injected at an appropriate flow rate and injection time. The binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween (registered trademark)-20) as the measurement buffer, at a flow rate of 10-30 μL / min, and at a measurement temperature of preferably 25°C or 37°C. When the measurement is performed by having the ligand capture molecule capture an antibody as a ligand, the antibody is injected to capture a desired amount, and then a serial dilution (analyte) of the antigen and / or Fc receptor prepared using the measurement buffer is injected. When the measurement is performed by having the ligand capture molecule capture an antigen and / or Fc receptor as a ligand, the antigen and / or Fc receptor is injected to capture a desired amount, and then a serial dilution (analyte) of the antibody prepared using the measurement buffer is injected.

[0146] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetics parameter calculation is performed by simultaneously fitting the sensorgrams of binding and dissociation using a 1:1 binding model, and the binding rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. As another parameter of binding activity, the "amount of analyte bound per unit amount of ligand" can also be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of ligand captured (RU).

[0147] In one embodiment, the binding activity of the antibody can be measured by, for example, the Octet RED96e system or the Octet RED 384 system (Pall ForteBio) using the Bio-Layer Interferometry (BLI) method as the measurement principle. By using the system according to the supplier's instructions, qualitative binding characteristic analysis and kinetic analysis of the antigen-antibody reaction can be performed. As a non-limiting embodiment of a specific measurement method, the antibody is immobilized on a Protein A (ProA) biosensor (Pall ForteBio), and then the antigen is allowed to interact with the antibody as an analyte, thereby measuring the change in the amount of binding between the antibody and the antigen. For example, when measuring the amount of binding between an antibody that binds to an antigen in the presence of MTA, it is possible to measure the binding reaction between the antibody and the analyte in a buffer containing 3000 nM of analyte diluted with 20 mM ACES, 150 mM NaCl, 0.05% (w / v) Tween 20, pH 7.4 to which MTA has been added at final concentrations of 0, 10, and 100 μM as the binding phase, and it is possible to measure the dissociation reaction between the antibody and the analyte by using a buffer similar to the binding phase that does not contain analyte as the dissociation phase. Furthermore, it is possible to observe over time that the binding between the antibody and the antigen in the presence of MTA reversibly dissociates in the absence of MTA by using a buffer that contains the same concentration of analyte as the binding phase but does not contain MTA as the dissociation phase.

[0148] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble molecule, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane molecule. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), a flow cytometer, etc.

[0149] When measuring the antigen-binding activity of a test antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity changes depending on a low molecular weight compound, the interaction between the test antigen-binding molecule and the antigen can be carried out in the presence or absence of a specific concentration of a low molecular weight compound. When an antigen-binding molecule that does not exhibit antigen-binding activity in the absence of a specific low molecular weight compound exhibits binding activity to the antigen in the presence of the low molecular weight compound, the binding activity can be evaluated in the presence of the low molecular weight compound. Similarly, when an antigen-binding molecule does not exhibit binding activity to the antigen in the presence of a specific low molecular weight compound and exhibits binding activity to the antigen in the absence of the low molecular weight compound, the binding activity can be evaluated in the absence of the low molecular weight compound. When the antigen-binding activity in the presence of a specific low molecular weight compound at a low concentration is higher than the antigen-binding activity in the presence of a high concentration of the low molecular weight compound, the binding activity can be evaluated in the presence of a high concentration of the low molecular weight compound. Similarly, when the antigen-binding activity in the presence of a specific low molecular weight compound at a high concentration is lower than the antigen-binding activity in the presence of a low concentration of the low molecular weight compound, the binding activity can be evaluated in the presence of a low concentration of the low molecular weight compound. Conditions that can affect the binding activity of an antigen and an antigen-binding molecule are not limited to the presence / absence of a low molecular weight compound or the concentration of the low molecular weight compound, and examples thereof include, but are not limited to, ion concentration, ion composition, and temperature. Furthermore, it is of course possible to evaluate the binding activity under conditions in which a plurality of different factors as described above are combined.

[0150] Epitope An epitope, which means an antigenic determinant present in an antigen, means a site on an antigen to which an antigen-binding domain in an antigen-binding molecule disclosed herein binds. The site on an antigen to which an antigen-binding molecule of the present disclosure binds can be defined by evaluating the presence or absence of binding of the antigen-binding molecule.

[0151] An epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity to an antigen in an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that constitute the epitope. When the epitope is a sugar chain, the epitope can be specified by a specific sugar chain structure.

[0152] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope. A linear epitope typically comprises at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0153] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids that comprise the epitope is not the single defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by an antibody that defines the epitope). Conformational epitopes may include an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (Ed.).

[0154] The structure of the antigen-binding domain that binds to an epitope is called a paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between the epitope and paratope. The binding strength between the epitope and paratope is called affinity. The sum of the binding strengths when multiple antigens and multiple antigen-binding molecules bind is called avidity. When an antibody or the like that contains multiple antigen-binding domains (i.e., is multivalent) binds to multiple epitopes, the binding strengths work synergistically, so avidity is higher than affinity.

[0155] Methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for IL-6R are exemplified below, but methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for an antigen other than IL-6R can also be appropriately carried out in accordance with the following examples.

[0156] For example, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For the above purpose, a linear peptide consisting of an amino acid sequence constituting the extracellular domain of IL-6R is synthesized. The peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region in the cDNA of IL-6R that encodes an amino acid sequence corresponding to the extracellular domain. Next, the binding activity between the linear peptide consisting of the amino acid sequence constituting the extracellular domain and the test antigen-binding molecule containing an antigen-binding domain for IL-6R is evaluated. For example, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding molecule to IL-6R-expressing cells. The binding activity of the antigen-binding molecule to the linear peptide can be determined by these tests.

[0157] Furthermore, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a conformational epitope can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. Examples of such cases include when a test antigen-binding molecule containing an antigen-binding domain for IL-6R strongly binds to IL-6R-expressing cells upon contact with the cells, while the antigen-binding molecule does not substantially bind to a linear peptide consisting of an amino acid sequence constituting the extracellular domain of immobilized IL-6R. Here, "not substantially binding" refers to a binding activity that is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.

[0158] Methods for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to IL-6R-expressing cells include, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, the binding activity can be evaluated by the principle of ELISA or FACS (fluorescence activated cell sorting) using IL-6R-expressing cells as antigens.

[0159] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain towards IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody-binding titer towards IL-6R-expressing cells is determined to compare the binding activity of the test antigen-binding molecule towards IL-6R-expressing cells.

[0160] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected by a flow cytometer. Known examples of flow cytometers include the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All of these are product names of BD Biosciences.) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are product names of Beckman Coulter)

[0161] For example, the following method is an example of a suitable method for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to an antigen. First, the test antigen-binding molecule is stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule that has been reacted with cells expressing IL-6R. The test antigen-binding molecule is diluted with a suitable buffer solution to prepare the antigen-binding molecule at a desired concentration for use. For example, the antigen-binding molecule may be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). That is, the binding activity of the test antigen-binding molecule represented by the amount of binding of the test antigen-binding molecule can be measured by obtaining the Geometric Mean value.

[0162] Antibodies that bind to the same epitope Whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules is detected by cross-blocking assays, etc. For example, competitive ELISA assays are preferred cross-blocking assays.

[0163] Specifically, in the cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to the IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to wells coated with IL-6R protein.

[0164] The amount of the test antigen-binding molecule bound to the well via the IL-6R protein can be easily measured by labeling the antigen-binding molecule in advance. For example, a biotin-labeled antigen-binding molecule can be measured by using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly called competitive ELISA assays. The antigen-binding molecule can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.

[0165] If a competing antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of a candidate competing antigen-binding molecule association, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competing antigen-binding molecule or competes for binding to the same epitope.

[0166] When the structure of the epitope to which a test antigen-binding molecule containing an antigen-binding domain for IL-6R binds has been identified, whether the test antigen-binding molecule and a control antigen-binding molecule share an epitope can be assessed by comparing the binding activity of both antigen-binding molecules against a peptide in which an amino acid mutation has been introduced into the peptide constituting the epitope.

[0167] Such binding activity can be measured, for example, by comparing the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced in the above-mentioned ELISA format. As a method other than ELISA, the binding activity to the mutant peptide bound to a column can also be measured by flowing the test antigen-binding molecule and the control antigen-binding molecule down the column and then quantifying the antigen-binding molecule eluted in the eluate. Methods of adsorbing a mutant peptide to a column, for example as a fusion peptide with GST, are known.

[0168] In addition, when the identified epitope is a conformational epitope, the fact that the test antigen-binding molecule and the control antigen-binding molecule share the epitope can be evaluated by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to a cell suspension in which these cells are suspended in an appropriate buffer such as PBS. Then, an FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added to the cell suspension that has been appropriately washed with a buffer. The fluorescence intensity and cell number of the cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test antigen-binding molecule and the control antigen-binding molecule are used at the desired concentration by appropriately diluting them with a suitable buffer. For example, they are used at any concentration between 10 μg / ml and 10 ng / ml. The amount of the labeled antibody bound to the cell is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the geometric mean value, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, represented by the amount of bound labeled antibody, can be measured.

[0169] In this method, for example, "not substantially binding to mutant IL-6R-expressing cells" can be determined by the following method. First, test antigen-binding molecules and control antigen-binding molecules bound to cells expressing mutant IL-6R are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the polypeptide complex according to the following formula 1:

[0170] (Formula 1) ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0171] The Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value) reflecting the binding amount of the test antigen-binding molecule to mutant IL-6R-expressing cells obtained by the analysis is compared with the ΔGeo-Mean comparison value reflecting the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. In this case, it is particularly preferable that the test antigen-binding molecules used when determining the ΔGeo-Mean comparison values ​​for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. An antigen-binding molecule previously confirmed to recognize an epitope in IL-6R is used as a control antigen-binding molecule.

[0172] If the ΔGeo-Mean comparison value of the test antigen-binding molecule for mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% lower than the ΔGeo-Mean comparison value of the test antigen-binding molecule for IL-6R-expressing cells, the antigen-binding molecule is deemed to "not substantially bind to mutant IL-6R-expressing cells." The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). If the comparison values ​​are substantially equivalent, the epitopes of the test antigen-binding molecule and the control antigen-binding molecule can be evaluated to be identical.

[0173] When assessing whether test antigen-binding molecules containing an antigen-binding domain whose antigen-binding activity changes depending on a small molecular weight compound compete with each other / bind to the same epitope, the interaction between the test antigen-binding molecule and the antigen can be carried out at a specific concentration of the small molecular weight compound or in the absence of the small molecular weight compound, and it is preferable to use the same concentration condition of the small molecular weight compound between the test antigen-binding molecules.

[0174] Antigen-binding domain whose antigen-binding activity changes depending on low-molecular-weight compounds As used herein, an "antigen-binding domain whose antigen-binding activity changes in a small molecular weight compound-dependent manner" means an antigen-binding domain whose binding activity towards an antigen, which is a molecule different from the small molecular weight compound, changes in the presence of different concentrations of the small molecular weight compound.

[0175] Antigen-binding domain whose binding activity to antigen changes in an MTA-dependent manner The antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, is an antigen-binding domain whose binding activity to an antigen, which is a molecule different from MTA, differs under different MTA concentrations. The antigens to which these antigen-binding domains bind may be membrane-type molecules or soluble molecules. The antigens to which these antigen-binding domains bind are antigens expressed in diseased tissues, more preferably in cancer tissues, and even more preferably in cancer tissues where MTA accumulates. The antigens expressed in cancer tissues may be antigens expressed in cancer cells, or may be antigens expressed in cancer stromal cells or immune tissues in cancer tissues.

[0176] Non-limiting examples of antigen-binding domains whose antigen-binding activity changes in an MTA-dependent manner include antigen-binding domains whose antigen-binding activity in the presence of MTA is stronger than that in the absence of MTA, and antigen-binding domains whose antigen-binding activity in the presence of MTA is weaker than that in the absence of MTA.

[0177] As long as the antigen-binding activity of the antigen-binding domain of the present disclosure, which has stronger antigen-binding activity in the presence of MTA than in the absence of MTA, is weaker than the antigen-binding activity in the presence of MTA, the ratio of the antigen-binding activity in the absence of MTA to the antigen-binding activity in the presence of MTA is not particularly limited, but preferably the ratio of the KD (Dissociation constant) for the antigen in the absence of MTA to the KD in the presence of MTA, KD (in the absence of MTA) / KD (in the presence of MTA), is 2 or more, more preferably the KD (in the absence of MTA) / KD (in the presence of MTA) is 10 or more, and even more preferably the KD (in the absence of MTA) / KD (in the presence of MTA) is 40 or more. The upper limit of the KD (in the absence of MTA) / KD (in the presence of MTA) is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by a person skilled in the art. When no antigen-binding activity is observed in the absence of MTA, this upper limit becomes infinite. An antigen-binding domain whose antigen-binding activity in the presence of an MTA is stronger than its antigen-binding activity in the absence of an MTA includes an antigen-binding domain that does not substantially bind to an antigen in the absence of an MTA.

[0178] As long as the antigen-binding activity of the antigen-binding domain of the present disclosure, which has a weaker antigen-binding activity in the presence of MTA than in the absence of MTA, is stronger than the antigen-binding activity in the presence of MTA, the ratio of the antigen-binding activity in the absence of MTA to the antigen-binding activity in the presence of MTA is not particularly limited, but preferably the ratio of the KD (dissociation constant) for the antigen in the presence of MTA to the KD in the absence of MTA, KD (in the presence of MTA) / KD (in the absence of MTA), is 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD (in the presence of MTA) / KD (in the absence of MTA) is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by the technology of a person skilled in the art. When no antigen-binding activity is observed in the presence of MTA, this upper limit becomes an infinite value. An antigen-binding domain whose antigen-binding activity in the presence of MTA is weaker than its antigen-binding activity in the absence of MTA includes an antigen-binding domain that does not substantially bind to an antigen in the presence of MTA.

[0179] In addition, as another indicator showing the ratio of the antigen-binding activity of the antigen-binding domain of the present disclosure (or an antigen-binding molecule containing the domain) in the absence of MTA to that in the presence of MTA, for example, the dissociation rate constant kd (Dissociation rate constant) can also be suitably used. When kd (Dissociation rate constant) is used instead of KD (Dissociation constant) as an indicator showing the ratio of binding activity, the value of kd (in the absence of MTA) / kd (in the presence of MTA), which is the ratio of kd (Dissociation rate constant) for the antigen in the absence of MTA to kd (Dissociation rate constant) in the presence of MTA, is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (in the absence of MTA) / kd (in the presence of MTA) is not particularly limited, and may be any value, such as 50, 100, 200, etc., as long as it can be produced within the technical common sense of a person skilled in the art. In the absence of MTA, when no antigen-binding activity is observed, no dissociation occurs, and therefore this upper limit becomes infinite.

[0180] The condition in the presence of MTA can be set to an appropriate MTA concentration, and as a non-limiting example, the condition in the presence of 100 μM MTA can be considered as the condition in the presence of MTA. In addition, as a non-limiting embodiment of the MTA concentration described as "in the presence of MTA" in the present disclosure, the concentration exemplified as a threshold value for distinguishing between low and high concentrations of MTA described below can be applied.

[0181] Non-limiting examples of antigen-binding domains whose antigen-binding activity changes in an MTA-dependent manner include antigen-binding domains whose antigen-binding activity in the presence of a high concentration of MTA is stronger than that in the presence of a low concentration of MTA, and antigen-binding domains whose antigen-binding activity in the presence of a high concentration of MTA is weaker than that in the presence of a low concentration of MTA.

[0182] As long as the antigen-binding activity of an antigen-binding domain of the present disclosure, which has stronger antigen-binding activity in the presence of a high concentration of MTA than in the presence of a low concentration of MTA, is weaker than the antigen-binding activity in the presence of a high concentration of MTA, the ratio of the antigen-binding activity in the presence of a low concentration of MTA to the antigen-binding activity in the presence of a high concentration of MTA is not particularly limited, but preferably the ratio of the KD (Dissociation constant) for the antigen in the presence of a low concentration of MTA to the KD in the presence of a high concentration of MTA, KD(in the presence of a low concentration of MTA) / KD(in the presence of a high concentration of MTA), is 2 or more, more preferably the KD(in the presence of a low concentration of MTA) / KD(in the presence of a high concentration of MTA) is 10 or more, and even more preferably the KD(in the presence of a low concentration of MTA) / KD(in the presence of a high concentration of MTA) is 40 or more. The upper limit of the KD (in the presence of low concentration MTA) / KD (in the presence of high concentration MTA) value is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by those skilled in the art. When no antigen-binding activity is observed in the presence of low concentration MTA, this upper limit becomes an infinite value. An antigen-binding domain that has stronger antigen-binding activity in the presence of a high concentration of MTA than in the presence of a low concentration of MTA includes an antigen-binding domain that does not substantially bind to an antigen in the presence of a low concentration of MTA.

[0183] As long as the antigen-binding activity of the antigen-binding domain of the present disclosure, which is weaker than the antigen-binding activity in the presence of a high concentration of MTA in the presence of a low concentration of MTA, is stronger than the antigen-binding activity in the presence of a high concentration of MTA, the ratio of the antigen-binding activity in the presence of a low concentration of MTA to the antigen-binding activity in the presence of a high concentration of MTA is not particularly limited, but preferably the ratio of the KD (Dissociation constant) for the antigen in the presence of a high concentration of MTA to the KD in the presence of a low concentration of MTA, KD (in the presence of a high concentration of MTA) / KD (in the presence of a low concentration of MTA), is 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD (in the presence of a high concentration of MTA) / KD (in the presence of a low concentration of MTA) is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by the techniques of a person skilled in the art. When no antigen-binding activity is observed in the presence of a high concentration of MTA, this upper limit becomes infinite. An antigen-binding domain whose antigen-binding activity in the presence of a high concentration of MTA is weaker than that in the presence of a low concentration of MTA includes an antigen-binding domain that does not substantially bind to an antigen in the presence of a high concentration of MTA.

[0184] In addition, as another indicator showing the ratio of the antigen-binding activity of the antigen-binding domain of the present disclosure (or an antigen-binding molecule containing the domain) in the presence of a low concentration of MTA to that in the presence of a high concentration of MTA, for example, the dissociation rate constant kd (Dissociation rate constant) can also be suitably used. When kd (Dissociation rate constant) is used instead of KD (Dissociation constant) as an indicator showing the ratio of binding activity, the value of kd (in the presence of low concentration of MTA) / kd (in the presence of high concentration of MTA), which is the ratio of kd (Dissociation rate constant) for an antigen in the presence of low concentration of MTA to kd (Dissociation rate constant) in the presence of high concentration of MTA, is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (in the presence of low concentration of MTA) / kd (in the presence of high concentration of MTA) is not particularly limited, and may be any value such as 50, 100, 200, etc., as long as it can be produced within the technical common sense of a person skilled in the art. In the presence of a low concentration of MTA, when no antigen-binding activity is observed, no dissociation occurs, and therefore this upper limit becomes infinite.

[0185] As a method for evaluating binding activity, in addition to the above-mentioned method for measuring KD value, a method for evaluating relative binding activity can also be used. As a non-limiting example of such a method, a method using a flow cytometer, ELISA, capillary electrophoresis, liquid chromatography, etc. is generally known, but is not limited to these. In addition, even if the KD value cannot be calculated directly, it is possible to evaluate the relative binding activity of the test molecule, that is, the binding activity of the test molecule is stronger or weaker than that of the reference molecule, by comparing the antigen-binding molecule whose KD value has been calculated by Biacore with the reference molecule.

[0186] In a non-limiting embodiment of the threshold for distinguishing between low and high concentrations of MTA, the low concentration condition may be appropriately set from the threshold values ​​of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, or 0 M. Depending on the set threshold, the high concentration condition may be at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 10 ... 3 times at least 10 4 times at least 10 5 times at least 10 6 The value can be appropriately set from 1 to 2 times.

[0187] A non-limiting embodiment of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, is an antigen-binding domain whose antigen-binding activity is substantially unaffected by one or more small molecule compounds selected from adenosine, S-(5'-adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP. Here, adenosine, S-(5'-adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP have adenosine as a common backbone in the molecule and are compounds similar to MTA. It is generally considered difficult to obtain an antigen-binding molecule in which the antigen-binding activity of the antigen-binding domain of the present disclosure is substantially unaffected by the presence of these similar molecules and whose binding activity changes only depending on MTA. However, in the present disclosure, a library was constructed to obtain antibodies that bind to an antibody in an MTA-dependent manner, as exemplified in various Examples described below, and by screening such a library, it was possible to successfully obtain an antigen-binding domain with excellent MTA-dependent specificity that binds to an antigen in an MTA-specific dependent manner and does not bind to an antigen in a dependent manner on a low molecular weight compound having a structure similar to MTA. As used herein, "antigen-binding activity is substantially unaffected by a small molecular weight compound" means that the antigen-binding activity of an antigen-binding domain in the presence of the small molecular weight compound is at least 0.5- to 2-fold higher than that in the absence of the small molecular weight compound.

[0188] In one non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is altered in an MTA-dependent manner, is substantially unaffected by adenosine. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is altered in an MTA-dependent manner, is substantially unaffected by S-(5'-Adenosyl)-L-homocysteine ​​(SAH). In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is altered in an MTA-dependent manner, is substantially unaffected by SAM. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is altered in an MTA-dependent manner, is substantially unaffected by adenosine, AMP, ADP, or ATP. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is changed in an MTA-dependent manner, is substantially unaffected by adenosine or S-(5'-Adenosyl)-L-homocysteine ​​(SAH). In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is changed in an MTA-dependent manner, is substantially unaffected by adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), or SAM. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is changed in an MTA-dependent manner, is substantially unaffected by adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, or ATP.

[0189] A non-limiting embodiment of the antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner includes an antigen-binding domain whose antigen-binding activity also changes depending on one or more small molecular weight compounds selected from adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP. In a non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose binding activity to an antigen changes depending on MTA, also changes depending on adenosine. The antigen-binding domain, whose binding activity changes depending on both MTA and adenosine molecules, can be understood to change its binding activity to an antigen by interacting with a common structure in both MTA and adenosine molecules, without being bound by a particular theory. In this case, even if the concentration of MTA is reduced in the solvent used to evaluate the binding activity, if the concentration of adenosine is present at a concentration that exceeds the degree of decrease in binding activity due to the decrease in the concentration of MTA, the binding activity may be maintained or detected strongly. In addition, even if the concentration of MTA is increased in the solvent used to evaluate the binding activity, if the concentration of adenosine is reduced beyond the degree of increase in binding activity due to the increase in the concentration of MTA, the binding activity may be maintained or detected low. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, also changes in an S-(5'-Adenosyl)-L-homocysteine ​​(SAH)-dependent manner. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity is altered in an MTA-dependent manner, also changes in a SAM-dependent manner. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, changes depending on any of adenosine, AMP, ADP, and ATP. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, changes depending on both adenosine and S-(5'-Adenosyl)-L-homocysteine ​​(SAH). In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, changes depending on all of adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), and SAM. In another non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, whose antigen-binding activity changes in an MTA-dependent manner, changes depending on any of adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP.

[0190] A non-limiting example of an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner in the present disclosure is an antigen-binding domain comprising an antibody variable region and / or a single-domain antibody.

[0191] In one non-limiting embodiment, the antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner is an antibody variable region, and the antibody variable region may include an antigen-binding molecule that contains at least one amino acid selected from the group of amino acids below (Kabat numbering): any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; A at heavy chain position 32; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 33 of the heavy chain; W located at heavy chain position 34; M located at heavy chain position 35; C located at heavy chain position 35a; C at heavy chain position 50; I located at heavy chain position 51; F located at heavy chain position 52; A located at heavy chain position 52a; any of A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at position 52b of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52c of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52d of the heavy chain; Y located at heavy chain position 52e; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52f of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52g of the heavy chain; S located at heavy chain position 53; G at heavy chain position 54; G at heavy chain position 55; S located at heavy chain position 56; T located at heavy chain position 57; Y at heavy chain position 58; Y at heavy chain position 59; A at heavy chain position 60; S located at heavy chain position 61; W at heavy chain position 62; A at heavy chain position 63; K located at heavy chain position 64; G at heavy chain position 65; G at heavy chain position 95; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 96 of the heavy chain; G at heavy chain position 97; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 99 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100 of the heavy chain; G located at position 100a of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100b of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 100c of the heavy chain; E located at heavy chain position 101; L located at heavy chain position 102; Q at position 24 of the light chain; S at position 25 of the light chain; S at light chain position 26; E located at light chain position 27; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at light chain position 27a; V at light chain position 28; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the light chain; any of A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at position 31 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the light chain; L located at light chain position 33; S at light chain position 34; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 49 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 50 of the light chain; A at light chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the light chain; T at light chain position 53; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 54 of the light chain; P at light chain position 55; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 56 of the light chain; A at light chain position 89; G at position 90 of the light chain; L located at light chain position 91; Y at light chain position 92; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 93 of the light chain; G at light chain position 94; N at light chain position 95; I located at light chain position 95a; P at light chain position 96; A at position 97 of the light chain.

[0192] In another non-limiting embodiment, the antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner is an antibody variable region, and the antibody variable region can be exemplified by an antigen-binding molecule that contains at least one amino acid selected from the group of amino acids below (Kabat numbering): any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 33 of the heavy chain; W located at heavy chain position 34; M located at heavy chain position 35; C located at heavy chain position 35a; C at heavy chain position 50; I located at heavy chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the heavy chain; S located at heavy chain position 52a; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at heavy chain position 53; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 54 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 55 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 56 of the heavy chain; T located at heavy chain position 57; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 58 of the heavy chain; Y at heavy chain position 59; A at heavy chain position 60; S located at heavy chain position 61; W at heavy chain position 62; V located at heavy chain position 63; N at heavy chain position 64; G at heavy chain position 65; E located at heavy chain position 95; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 96 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 97 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 99 of the heavy chain; S located at heavy chain position 100; G located at position 100a of the heavy chain; A located at position 100b of the heavy chain; L located at position 100c of the heavy chain; N at heavy chain position 101; L located at heavy chain position 102; H at light chain position 24; S at position 25 of the light chain; S at light chain position 26; K at position 27 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at light chain position 27a; V located at light chain position 27b; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 28 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 31 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 32 of the light chain; L located at light chain position 33; A at light chain position 34; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 49 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 50 of the light chain; A at light chain position 51; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 52 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 53 of the light chain; L located at light chain position 54; A at light chain position 55; S at light chain position 56; Q at light chain position 89; G at position 90 of the light chain; T at light chain position 91; Y at light chain position 92; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 93 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 94 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 95 of the light chain; any of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 95a of the light chain; F located at light chain position 95b; Y located at light chain position 95c; F at light chain position 96; A at position 97 of the light chain.

[0193] In a further non-limiting embodiment, the antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner is an antibody variable region, and the antibody variable region can be exemplified by an antigen-binding molecule that contains at least one amino acid selected from the group of amino acids below (Kabat numbering): any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y at position 26 of the heavy chain; any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 28 of the heavy chain; either A or L at heavy chain position 29; any of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at position 30 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y at position 31 of the heavy chain; any of D, E, F, H, N, P, R, or Y at heavy chain position 32; any of A, I, P, T, or V at position 33 of the heavy chain; any of A, E, F, H, I, K, L, M, N, Q, S, T, V, W or Y at position 34 of the heavy chain; G at position 35 of the heavy chain; either D, I or V at heavy chain position 50; I located at heavy chain position 51; G at heavy chain position 52; any of A, D, E, G, I, K, Q, or R at position 53 of the heavy chain; any of D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, or Y at position 54 of the heavy chain; any of A, D, E, F, G, or H at position 55 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y at position 56 of the heavy chain; any of A, D, E, G, H, I, K, L, N, P, Q, R, S, T, or V at position 57 of the heavy chain; W located at heavy chain position 58; any of A, D, E, F, G, H, I, K, L, Q, R, S, T, V, W, or Y at position 59 of the heavy chain; P located at heavy chain position 60; any of A, F, Q, R, S, T, V, W, or Y at position 61 of the heavy chain; W at heavy chain position 62; V located at heavy chain position 63; K located at heavy chain position 64; A, F, or G at heavy chain position 65; G at heavy chain position 95; any of A, E, F, G, H, K, L, Q, R, S, T, W, or Y at position 96 of the heavy chain; any of A, F, H, K, N, W, or Y at position 97 of the heavy chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 98 of the heavy chain; any of A, D, E, G, H, Q, or S at position 99 of the heavy chain; F or Y at heavy chain position 100; N, T or V at position 100a of the heavy chain N located at position 100b of the heavy chain; A located at position 100c of the heavy chain; F or W located at position 100d of the heavy chain; D located at heavy chain position 101; P located at heavy chain position 102; Q at position 24 of the light chain; S at position 25 of the light chain; S at light chain position 26; Q at position 27 of the light chain; S located at light chain position 27e; V located at light chain position 27f; any of A, E, F, H, I, K, L, N, R, S, T, V, W, or Y at position 28 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 29 of the light chain; N at light chain position 30; N at light chain position 31; any of A, E, F, G, H, S, or Y at position 32 of the light chain; L located at light chain position 33; S at light chain position 34; D at position 50 of the light chain; A at light chain position 51; S at light chain position 52; T at light chain position 53; L located at light chain position 54; A at light chain position 55; S at light chain position 56; H at light chain position 89; G at position 90 of the light chain; either A, S, or T at position 91 of the light chain; any of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at position 92 of the light chain; any of A, D, E, F, G, H, L, N, Q, R, S, T, V, or Y at position 93 of the light chain; any of A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 94 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95 of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, V, W, or Y at position 95a of the light chain; any of A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95b of the light chain; any of A, F, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 95c of the light chain; D located at light chain position 96d; N at light chain position 96; A or G at light chain position 97; A, F, I, L or V at position 98 of the light chain.

[0194] The antigen-binding domain in the present disclosure may have amino acid residues that interact with MTA. The amino acid residues that interact with MTA may be present at an interface in the antigen-binding domain that directly interacts with the antigen, or may be present in other parts. The "interface that directly interacts with the antigen" refers to a site where the antigen and the antigen-binding molecule are close to each other in the structure of a complex of the antigen and the antigen-binding domain analyzed by a method such as crystal structure analysis. The distance at which the antigen and the antigen-binding molecule are close to each other can be 4 to 6 Å, but in the structure of the complex of the antigen and the antigen-binding molecule, "the antigen and the antigen-binding molecule are close to each other" refers to a site that is relatively close in the complex, and is not limited to the distance exemplified above. The amino acid residues that interact with MTA may be residues that interact with MTA when the antigen-binding molecule is bound to the antigen, or may be residues that interact with MTA in the absence of the antigen. Furthermore, the amino acid residues that interact with MTA may be residues that interact with MTA both in the state where the antigen-binding molecule is bound to the antigen and in the absence of the antigen. Furthermore, the amino acid residue that interacts with MTA may be a single residue or multiple residues in the antigen-binding domain.

[0195] In an embodiment where the antigen-binding domain comprises an antibody heavy chain variable region and an antibody light chain variable region, the amino acids that interact with MTA may be present in the CDRs or FRs of the antibody variable regions. In an embodiment where the antigen-binding domain comprises a single domain antibody, the amino acids that interact with MTA may be present in the CDRs or FRs of the single domain antibody.

[0196] The amino acid residues in the antigen-binding domain that interact with MTA can be identified by techniques such as crystal structure analysis of a binary complex of MTA and an antigen-binding molecule containing the antigen-binding domain, or a ternary complex of MTA, antigen, and an antigen-binding molecule containing the antigen-binding domain, three-dimensional structure analysis using NMR, or amino acid mutation introduction.

[0197] In a non-limiting embodiment of the present disclosure, amino acid residues of an antigen-binding molecule that interact with MTA can be identified from crystal structure analysis of a complex between an MTA and an antigen-binding molecule. Here, "interacting with MTA" refers to a state in which an atom of a side chain or main chain of an amino acid forming an antigen-binding molecule and an atom of a low molecular weight compound form an antigen-binding molecule-MTA interaction at a distance that can affect MTA binding activity, or a state in which, in an embodiment in which the antigen-binding domain in an antigen-binding molecule includes an antibody heavy chain variable region and an antibody light chain variable region, a certain amino acid residue contributes to MTA binding, including an indirect effect such as stabilizing the conformation of the three-dimensional structure of a CDR loop or the like to the conformation at the time of MTA binding, or a state that satisfies both of these. The "state in which an intermolecular interaction is formed" in this specification can be determined based on the interatomic distance between a non-hydrogen atom constituting the side chain or main chain of an amino acid constituting an antigen-binding molecule and a non-hydrogen atom constituting an MTA, for example, from crystal structure analysis of a complex between an MTA and an antigen-binding molecule. For example, the interatomic distance is preferably within 3.0 Å, 3.2 Å, 3.4 Å, 3.6 Å, 3.8 Å, 4.0 Å, 4.2 Å, 4.4 Å, 4.6 Å, 4.8 Å, or 5.0 Å, but is not limited thereto. More preferably, the interatomic distance is within 3.6 Å, 3.8 Å, 4.0 Å, or 4.2 Å. More specifically, the possibility of direct interaction can be judged based on the information on the interatomic distances in the three-dimensional structure, the type of intermolecular interaction formed, and the type of atoms. More precisely, it can be judged from the effect of introducing a mutation into an amino acid residue, such as changing it to Ala or Gly, on the activity of the low-molecular compound, but is not limited to this. Regarding the "indirectly affecting state" in this specification, for example, whether or not the binding of MTA is indirectly affected can be estimated by detailed analysis of the conformation of each amino acid residue and the state of intermolecular interactions with surrounding residues from the three-dimensional structure of a complex between MTA and an antigen-binding molecule, but more accurately, it can be determined from the effect on the activity of MTA of introducing a mutation into an amino acid residue, such as modifying it to Ala or Gly.

[0198] In a non-limiting embodiment of the present disclosure, amino acid residues of an antigen-binding molecule that interact with MTA can be identified from crystal structure analysis of a complex of MTA, antigen, and antigen-binding molecule. Here, "interacting with MTA" refers to a state in which an atom of a side chain or main chain of an amino acid forming an antigen-binding molecule and an atom of a low molecular weight compound form an antigen-binding molecule-MTA interaction at a distance that can affect MTA binding activity in the presence of an antigen, or a state in which, in an embodiment in which the antigen-binding domain in an antigen-binding molecule includes an antibody heavy chain variable region and an antibody light chain variable region, a certain amino acid residue contributes to MTA binding, including an indirect effect such as stabilizing the conformation of a CDR loop or the like to the conformation when MTA binds in the presence of an antigen, or a state that satisfies both of these. The "state in which an intermolecular interaction is formed" in this specification can be determined, for example, from a crystal structure analysis of a complex of MTA, antigen, and antigen-binding molecule, based on the interatomic distance between a non-hydrogen atom constituting the side chain or main chain of an amino acid that forms an antigen-binding molecule in the presence of an antigen and a non-hydrogen atom constituting MTA. For example, the interatomic distance is preferably within 3.0 Å, 3.2 Å, 3.4 Å, 3.6 Å, 3.8 Å, 4.0 Å, 4.2 Å, 4.4 Å, 4.6 Å, 4.8 Å, or 5.0 Å, but is not limited thereto. More preferably, the interatomic distance is within 3.6 Å, 3.8 Å, 4.0 Å, or 4.2 Å. More specifically, the possibility of direct interaction can be judged based on the information on the interatomic distances in the three-dimensional structure, the type of intermolecular interaction formed, and the type of atoms. More precisely, it can be judged from the effect of introducing a mutation into an amino acid residue, such as changing it to Ala or Gly, on the activity of the low-molecular compound, but is not limited to this. Regarding the "indirectly influencing state" in this specification, for example, whether or not the binding of MTA in the presence of an antigen is indirectly affected can be estimated by detailed analysis of the conformation of each amino acid residue and the state of intermolecular interactions with surrounding residues from the three-dimensional structure of the complex of MTA, antigen, and antigen-binding molecule, but more accurately, it can be determined from the effect on the activity of MTA of introducing a mutation into an amino acid residue, such as modifying it to Ala or Gly.

[0199] In a non-limiting embodiment, the antigen-binding domain is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid positions selected from the group consisting of amino acid positions 34, 35a, 47, 52, 52e, and 101 in the heavy chain and 32, 34, 36, 46, 49, 50, 89, 90, 91, and 96 in the light chain, as specified by Kabat numbering, in the amino acid sequence of the antibody variable region. In one non-limiting embodiment, the antigen-binding domain is an antibody variable region, and the antibody variable region includes an antigen-binding domain containing at least one or more amino acids selected from heavy chain W34, C35a, W47, F52, Y52e, and E101, and light chain R32, S34, Y36, L46, Y49, S50, A89, G90, L91, and P96 (Kabat numbering).

[0200] Another non-limiting example of an antigen-binding domain is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid positions selected from the group consisting of amino acid positions 34, 47, 50, 58, 95, 98, 99, and 100a in the heavy chain and 28, 91, 95b, 95c, and 96 in the light chain, as specified by Kabat numbering, in the amino acid sequence of the antibody variable region. In a non-limiting embodiment, the antigen-binding domain is an antibody variable region, and the antibody variable region includes an antigen-binding domain containing at least one amino acid selected from heavy chain W34, W47, C50, Y58, E95, F98, G99, and G100a, and light chain Y28, T91, F95b, Y95c, and F96 (Kabat numbering).

[0201] Another non-limiting embodiment of the antigen-binding domain that interacts with MTA is an antigen-binding domain that is an antibody variable region, and the amino acid residues that interact with MTA are amino acid residues located at at least one or more amino acid sites selected from the group consisting of amino acid sites 33, 50, 52, 54, 56, 57, 58, 99, 100, 100a, 91, 95c, and 96 in the heavy chain as specified by Kabat numbering in the amino acid sequence of the antibody variable region. Furthermore, the antigen-binding domain is an antibody variable region, and examples of the antibody variable region include an antigen-binding domain containing at least one amino acid selected from heavy chain A33, I50, G52, D54, S56, T57, W58, G99, Y100, and T100a, and light chain S91, Y95c, and N96 (Kabat numbering).

[0202] Antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner In a non-limiting embodiment, the antigen-binding molecule of the present disclosure comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner is a molecule comprising an antibody Fc region. The antibody Fc region contained in the antigen-binding molecule of the present disclosure may be a native Fc region or a modified Fc region. Examples of native Fc regions include the Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).

[0203] The antigen binding molecule of the present disclosure can comprise at least a portion of an Fc region that mediates binding to an Fcγ receptor and / or binding to FcRn. For example, in one non-limiting embodiment, the antigen binding molecule can be an antibody or an Fc fusion protein. A fusion protein refers to a chimeric polypeptide that comprises a polypeptide comprising a first amino acid sequence linked to a polypeptide having a second amino acid sequence to which it is not naturally linked in nature. For example, a fusion protein can comprise a polypeptide comprising an amino acid sequence that encodes at least a portion of an Fc region (e.g., a portion of an Fc region that confers binding to an Fcγ receptor and / or a portion of an Fc region that confers binding to an FcRn). The amino acid sequences can be present in separate proteins that are carried together in the fusion protein, or they can normally be present in the same protein, but are put into a new rearrangement in the fusion polypeptide. Fusion proteins can be made, for example, by chemical synthesis or by recombinant techniques that create and express a polynucleotide in which peptide regions are encoded in the desired relationship.

[0204] The domains in the antigen-binding molecule of the present disclosure may be linked directly by a polypeptide bond or via a linker. As the linker, any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (for example, a linker disclosed in Holliger et al. (Protein Engineering (1996) 9 (3), 299-305)) may be used, but in the present disclosure, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose, but the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids.

[0205] For example, for a peptide linker: Ser Gly Ser Gly Gly Ser Ser Gly Gly Gly Gly Gly Ser (SEQ ID NO: 19) Ser Gly Gly Gly (SEQ ID NO: 20) Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 21) Ser Gly Gly Gly Gly (SEQ ID NO: 22) Gly Gly Gly Gly Gly Ser (SEQ ID NO: 23) Ser Gly Gly Gly Gly Gly (SEQ ID NO: 24) Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 25) Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 26) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 21)) (Ser Gly Gly Gly Gly (SEQ ID NO: 22)) [n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0206] The synthetic chemical linker (chemical crosslinker) is a crosslinker that is commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), etc., and these crosslinkers are commercially available.

[0207] When multiple linkers are used to link each domain, all of the same type of linkers may be used, or different types of linkers may be used. In addition to the linkers exemplified above, linkers having peptide tags such as His tags, HA tags, myc tags, and FLAG tags may also be used as appropriate. In addition, the property of binding to each other by hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or combinations of these bonds may also be preferably used. For example, the affinity between CH1 and CL of an antibody may be used, or an Fc region originating from the bispecific antibody described above may be used when associating hetero Fc regions. In addition, disulfide bonds formed between domains may also be preferably used.

[0208] In order to link each domain by a peptide bond, polynucleotides encoding the domains are linked in frame. Methods for linking polynucleotides in frame include known techniques such as restriction fragment ligation, fusion PCR, and overlap PCR, and these techniques may be used alone or in combination as appropriate for the preparation of the antigen-binding molecules of the present disclosure. In the present disclosure, the terms "linked", "fused", "linked" or "fused" are used interchangeably. These terms refer to linking two or more elements or components such as polypeptides to form a single structure by any means, including the above-mentioned chemical bonding means or recombinant techniques. In-frame fusion refers to linking two or more open reading frame units to form a continuous longer open reading frame so as to maintain the correct reading frame of the polypeptide when two or more elements or components are polypeptides. When two Fab molecules are used as the antigen-binding domain, an antibody that is an antigen-binding molecule of the present disclosure in which the antigen-binding domain and a constant region including an Fc region are linked in frame by peptide bonds without a linker can be used as a suitable antigen-binding molecule of the present disclosure.

[0209] In another aspect, the present disclosure provides an antigen-binding molecule having high plasma retention. In one aspect, the antigen-binding activity of the antigen-binding molecule increases as the concentration of MTA increases. In one embodiment, the antigen-binding molecule has higher antigen-binding activity in target tissues than in non-target tissues. In some aspects, the antigen-binding molecule is an antibody. Without being bound by a particular theory, the above-mentioned change in kinetics in plasma can be interpreted as follows. As the antigen-binding activity of the antigen-binding molecule increases depending on the concentration of MTA, the antigen-binding ability of the antigen-binding molecule in tissues other than the target tissue decreases. As a result, the antigen-dependent elimination (clearance) of the antigen-binding molecule in tissues other than the target tissue decreases. The fact that the antigen-dependent elimination (clearance) decreases in most tissues in the body (tissues other than the target tissue) leads to high plasma retention of the antigen-binding molecule in a comprehensive view. The antigen-binding molecule of the present invention can be judged to have high plasma retention by relative comparison with a control antigen-binding molecule. In some embodiments, an antigen-binding molecule whose antigen-binding activity increases with increasing MTA concentration has high plasma retention compared to a control antigen-binding molecule. In one embodiment, the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of MTA. In a specific embodiment, an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of a compound means an antigen-binding molecule whose difference in antigen-binding activity between the presence and absence of MTA is, for example, less than 2 times, less than 1.8 times, less than 1.5 times, less than 1.3 times, less than 1.2 times, or less than 1.1 times. From the viewpoint of comparison, it is desirable that the antigen-binding activity of the antigen-binding molecule of the present disclosure and the antigen-binding molecule of the control are substantially equal to each other in the presence of a sufficient amount of MTA.

[0210] Here, the magnitude of antigen-dependent disappearance of antigen-binding molecules detected in vivo is considered to vary depending on the quantitative balance between antigens and antigen-binding molecules present in plasma. In general, the more antigens / the fewer antigen-binding molecules present in plasma, the easier it is to detect antigen-dependent disappearance of antigen-binding molecules, and conversely, the less antigens / the more antigen-binding molecules present in plasma, the harder it is to detect antigen-dependent disappearance of antigen-binding molecules. The antigen-binding molecules of the present disclosure do not need to exhibit high plasma retention under all conditions, but only need to exhibit high plasma retention under appropriate conditions in which sufficient antigen-dependent disappearance can be detected. When the amount of antigen in plasma is low, the amount of antigen may be increased by some artificial means before evaluating plasma retention.

[0211] In another aspect, the present invention provides an antigen-binding molecule having low antigen accumulation in plasma. In a further aspect, the antigen-binding activity of the antigen-binding molecule increases as the concentration of MTA increases. In a particular embodiment, MTA is a target tissue-specific compound. In a further embodiment, the antigen-binding molecule has higher antigen-binding activity in target tissues than in non-target tissues. In some aspects, the antigen-binding molecule is an antibody. Without being bound by a particular theory, the above-mentioned change in dynamics in plasma can be interpreted as follows. As the antigen-binding activity of the antigen-binding molecule increases depending on the concentration of MTA, the antigen-binding ability of the antigen-binding molecule in tissues other than the target tissue decreases. As a result, the ability of the antigen-binding molecule to form an antigen-antibody complex in tissues other than the target tissue decreases. It is generally known that when an antigen-binding molecule such as an antibody binds to an antigen, the clearance of the antigen decreases and the antigen concentration in plasma increases (antigen accumulates). In most tissues in the body (tissues other than target tissues), the reduced ability to form antigen-antibody complexes leads to low antigen accumulation (in other words, low antigen accumulation ability of antigen-binding molecules) in a comprehensive sense. Whether an antigen-binding molecule of the present invention has low antigen accumulation ability in plasma can be determined by relative comparison with a control antigen-binding molecule. In some embodiments, an antigen-binding molecule whose antigen-binding activity increases with increasing MTA concentration has a low antigen accumulation ability in plasma compared to a control antigen-binding molecule. In one embodiment, the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the MTA concentration. In a specific embodiment, an antigen-binding molecule that does not have antigen-binding activity dependent on the MTA concentration refers to an antigen-binding molecule whose difference in antigen-binding activity between the presence and absence of the compound is, for example, less than 2-fold, less than 1.8-fold, less than 1.5-fold, less than 1.3-fold, less than 1.2-fold, or less than 1.1-fold. From the standpoint of comparison, it is desirable that the antigen-binding activity of an antigen-binding molecule of the present invention and a control antigen-binding molecule are substantially equivalent to each other in the presence of a sufficient amount of a compound.

[0212] Here, it is believed that the amount of antigen-antibody complexes formed in vivo depends on the amounts of antigen and antibody present in plasma. In general, it is believed that the more the amount of antigen and antibody in plasma increases, the more the amount of antigen-antibody complexes formed increases, and conversely, the more the amount of antigen-antibody complexes formed decreases as the amount of antigen and antibody in plasma decreases. The antigen-binding molecule of the present invention does not need to exhibit low plasma antigen accumulation ability under all conditions, but only needs to exhibit low plasma antigen accumulation ability under appropriate conditions in which sufficient antigen-antibody complexes are formed. When the amount of antigen in plasma is low, it is necessary to increase the amount of antigen by some artificial means and then evaluate the antigen accumulation ability in plasma. Good too.

[0213] Fcγ receptor (FcγR) Fcγ receptor (also written as FcγR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and refers to any member of the family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131, i.e., FcγRIIa (H) and FcγRIIa (R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and isoforms FcγRIIIa (including allotypes V158 and F158, i.e., FcγRIIIa (V) and FcγRIIIa (V). FcγR includes, but is not limited to, FcγRIII (CD16), including FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and any undiscovered human FcγRs or FcγR isoforms or allotypes. FcγR may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), and any undiscovered mouse FcγRs or FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI are shown in SEQ ID NOs: 9 (NM_000566.3) and 10 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of human FcγRIIa (allotype H131) are shown in SEQ ID NOs: 11 (BC020823.1) and 12 (AAH20823.1), respectively (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 12 is substituted with Arg). The polynucleotide sequence and amino acid sequence of FcγRIIb are shown in SEQ ID NOs: 12 (BC020823.1) and 12 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIa are set forth in SEQ ID NOs: 15 (BC033678.1) and 16 (AAH33678.1), and the polynucleotide and amino acid sequences of FcγRIIIb are set forth in SEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (database accession numbers such as RefSeq are indicated in parentheses). Whether or not an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the above-described FACS or ELISA format, as well as by the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) or the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0214] FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), are associated with an α chain that binds to the Fc region of IgG and a common γ chain that has ITAMs that transmit activation signals intracellularly. On the other hand, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its own cytoplasmic domain. These receptors are expressed on many immune cells, such as macrophages, mast cells, and antigen-presenting cells. The activation signals transmitted by the binding of these receptors to the Fc region of IgG promote the phagocytic ability of macrophages, the production of inflammatory cytokines, the degranulation of mast cells, and the enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are referred to as activating Fcγ receptors in this specification.

[0215] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM that transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses activation signals from the BCR, resulting in the inhibition of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic ability and the ability to produce inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are referred to as inhibitory Fcγ receptors in this specification.

[0216] Binding activity of the Fc region to FcγR As described above, examples of the Fc region contained in the antigen-binding molecule of the present disclosure include Fc regions having binding activity to Fcγ receptors. Non-limiting examples of such Fc regions include Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8). Whether or not an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) or the BIACORE method using the surface plasmon resonance (SPR) phenomenon, in addition to the FACS and ELISA formats described above (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0217] The ALPHA screen is carried out by ALPHA technology using two beads, donor and acceptor, based on the following principle: A luminescence signal is detected only when a molecule bound to a donor bead biologically interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

[0218] For example, an antigen-binding molecule containing a biotin-labeled Fc region is bound to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a competing antigen-binding molecule containing an Fc region variant, an antigen-binding molecule having a native Fc region and an Fcγ receptor interact to generate a signal at 520-620 nm. An antigen-binding molecule containing an untagged Fc region variant competes with the interaction between an antigen-binding molecule having a native Fc region and an Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of the competition. It is known to biotinylate an antigen-binding molecule such as an antibody using Sulfo-NHS-biotin or the like. The method of tagging Fcγ receptor with GST may be appropriately selected by expressing the fusion gene, which is an in-frame fusion of a polynucleotide encoding Fcγ receptor and a polynucleotide encoding GST, in a vector operably linked to the vector, and purifying the fusion gene using a glutathione column. The obtained signal is suitably analyzed by fitting it to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0219] One of the substances (ligand) for which interaction is to be observed is fixed on the gold film of a sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass. When the other substance (analyte) for which interaction is to be observed is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond is dissociated, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be calculated from the curve of the sensorgram, and the affinity (KD) can be calculated from the ratio of these constants. Inhibition measurement methods are also preferably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0220] Fcγ receptor (FcγR) binding modified Fc region As the Fc region included in the present disclosure, in addition to the Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8), FcγR-binding modified Fc regions having higher binding activity to Fcγ receptors than that of the Fc region of native human IgG may also be used as appropriate. In this specification, "native human IgG Fc region" refers to an Fc region in which the sugar chain bound to position 297 (EU numbering) of the Fc region of human IgG1, IgG2, IgG3, or IgG4 exemplified by SEQ ID NO: 5, 6, 7, or 8 is a fucose-containing sugar chain. Such an FcγR-binding modified Fc region can be prepared by modifying the amino acids of the Fc region of native human IgG. Whether or not the binding activity of an FcγR-binding modified Fc region to FcγR is higher than that of the Fc region of native human IgG may be appropriately determined using the method described in the above section on binding activity.

[0221] In the present disclosure, "amino acid modification" or "amino acid modification" of an Fc region includes modification to an amino acid sequence different from the amino acid sequence of the starting Fc region. Any Fc region can be used as the starting Fc region as long as the modified variant of the starting Fc region can bind to a human Fcγ receptor in a neutral pH range. In addition, an Fc region that has already been modified and further modified can also be suitably used as the Fc region of the present disclosure. The starting Fc region may refer to the polypeptide itself, a composition containing the starting Fc region, or an amino acid sequence encoding the starting Fc region. The starting Fc region may include known Fc regions produced by recombination as outlined in the section on antibodies. The source of the starting Fc region may be obtained from any organism of a non-human animal or a human, but is not limited thereto. Preferably, the any organism is an organism selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cows, horses, camels, and non-human primates. In another embodiment, the starting Fc region can also be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Preferably, the starting Fc region can be obtained from human IgG1, but is not limited to a particular class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting Fc region. Similarly, in the present specification, it is meant that the Fc region of any class or subclass of IgG from any of the above-mentioned organisms can preferably be used as the starting Fc region. Examples of naturally occurring IgG variants or engineered forms are described in the known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460...

Claims

Claim 1. An antigen-binding molecule comprising a plurality of antigen-binding domains having different sequences from each other, and / or a library mainly composed of a nucleic acid encoding an antigen-binding molecule comprising a plurality of antigen-binding domains having different sequences from each other, wherein: the antigen-binding domain is an antigen-binding domain that interacts with 5'-methylthioadenosine (MTA); the antigen to which the antigen-binding domain binds is a molecule other than MTA; the antigen-binding domain is an antibody variable region; the antibody variable region is an antibody variable region variant having an amino acid different from the amino acid located at one or more amino acid sites in the unmodified antibody variable region having binding activity to MTA; the unmodified antibody variable region does not substantially bind to adenosine or / and S-(5'-Adenosyl)-L-homocysteine (SAH), the library. Claim 2. The library according to claim 1, wherein the unmodified antibody variable region is any of the following: a) an antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 46 and a light chain variable region represented by SEQ ID NO: 47; b) an antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 50 and a light chain variable region represented by SEQ ID NO: 51; c) an antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 48 and a light chain variable region represented by SEQ ID NO: 49; d) an antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 52 and a light chain variable region represented by SEQ ID NO:

53. Claim 3. The library according to claim 1 or 2, wherein the antigen-binding molecule is an antibody. Claim 4. The following steps (a) and (b): (a) In an unmodified antigen-binding domain having binding activity to MTA, a step of identifying an amino acid site satisfying at least one of the following (i) to (vi): (i) an amino acid site exposed on the surface of the unmodified antigen-binding domain; (ii) an amino acid site located in a region having a large structural change rate when comparing the structure when the unmodified antigen-binding domain binds to MTA and the structure when it does not bind to MTA; (iii) an amino acid site not involved in the binding to MTA; (iv) an amino acid site that does not significantly attenuate the binding to MTA; (v) an amino acid site having diversity in amino acid occurrence frequency in the animal species to which the unmodified antigen-binding domain belongs; or (vi) Amino acid sites that are not important for the formation of the canonical structure; (b) In the unmodified antigen-binding domain, a library is designed that contains a nucleic acid encoding the unmodified antigen-binding domain and nucleic acids encoding a plurality of variants of the unmodified antigen-binding domain that have different sequences from each other and have amino acid modifications at one or more amino acid sites identified in step (a), wherein the amino acid modification satisfies at least one of the following (1) to (3): (1) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to MTA with the structure when not bound to MTA, the rate of structural change of the amino acid site where the amino acid modification is located is large; (2) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to MTA with the structure when not bound to MTA, the structural change of the antigen-binding domain variant is not inhibited by the presence of the amino acid modification; (3) The antigen-binding domain variant having the amino acid modification does not have a significantly reduced binding activity to MTA compared to the unmodified antigen-binding domain, comprising, the unmodified antigen-binding domain does not substantially bind to adenosine or / and S-(5'-Adenosyl)-L-homocysteine (SAH), A method for producing a library, wherein the unmodified antigen-binding domain and the modified antigen-binding domain are an unmodified antibody variable region and a modified antibody variable region, respectively.

5. The method according to claim 4, further comprising the step of selecting an antigen-binding domain variant that binds to MTA.

6. The method according to claim 4 or 5, wherein the unmodified antigen-binding domain is any of the following: a) An antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 46 and a light chain variable region represented by SEQ ID NO: 47; b) An antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 50 and a light chain variable region represented by SEQ ID NO: 51; c) An antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 48 and a light chain variable region represented by SEQ ID NO: 49; d) An antibody variable region comprising a heavy chain variable region represented by SEQ ID NO: 5

Images