Antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner and a library for obtaining said antigen-binding domain

Abstract

The present disclosure addresses the problem of providing: an antigen-binding molecule of which the antigen-binding activity is changed depending on the concentration of a low-molecular-weight compound specific to a target tissue; a polynucleotide encoding said antigen-binding molecule; a vector containing said polynucleotide; a cell carrying said vector; a library containing a plurality of said antigen-binding molecules that are different from each other; a pharmaceutical composition containing said antigen-binding molecule; a method for screening said antigen-binding molecule; and a method for producing the same. The inventors have found methylthioadenosine (MTA) to be a low-molecular-weight compound specific to a tumor tissue, have created an antigen-binding domain of which the binding activity to an antigen is changed depending on the concentration of MTA or created an antigen-binding molecule containing the antigen-binding domain, have further created 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, and have found that the problem can be solved by using the library. By using an antigen-binding molecule according to the present disclosure, various diseases (for example, cancer) caused by a target tissue (for example, a tumor tissue) can be treated in a target tissue-specific manner.

Description

[Technical Field]

[0001] The present disclosure relates to antigen-binding molecules containing antigen-binding domains whose antigen-binding activity changes in a methylthioadenosine (MTA)-dependent manner, methods for producing and screening for such antigen-binding domains or such antigen-binding molecules, libraries for obtaining such antigen-binding domains or such antigen-binding molecules and methods for designing such libraries, and pharmaceutical compositions containing such antigen-binding molecules. The present disclosure also relates to methods for designing libraries for efficiently obtaining antigen-binding domains whose antigen-binding activity changes in a small-molecule-dependent manner. Furthermore, the present disclosure also relates to antigen-binding molecules that specifically bind to MTA, and methods for measuring MTA concentrations and methods for diagnosing diseases using such antigen-binding molecules. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Among them, many IgG-type antibody drugs have been commercialized, and many antibody drugs are currently under development (Non-Patent Documents 1 and 2).

[0003] Approved antibody-based cancer therapeutics include Rituxan, which targets the CD20 antigen, cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC and other mechanisms. It is known that the cytotoxic activity through ADCC and other mechanisms depends on the number of antigens expressed on the target cells of the therapeutic antibody (Non-Patent Document 4). Therefore, a high expression level of the target antigen is preferable from the perspective of the efficacy of the therapeutic antibody. However, even if the expression level of the antigen is high, if the antigen is expressed in normal tissues, ADCC and other cytotoxic activities will be exerted against normal cells, resulting in serious side effects. Therefore, it is preferable that the antigen targeted by a therapeutic antibody as a cancer therapeutic drug is specifically expressed on cancer cells.

[0004] Following the success of antibody pharmaceuticals that exert cytotoxic activity through ADCC activity, improved second-generation antibody molecules have been reported that exert potent cytotoxic activity by, for example, enhancing ADCC activity by removing fucose from the N-glycosylation chains in the Fc region of native human IgG1 (Non-Patent Document 5) and enhancing ADCC activity by enhancing binding to FcγRIIIa through amino acid substitution in the Fc region of native human IgG1 (Non-Patent Document 6). As antibody pharmaceuticals that exert cytotoxic activity against cancer cells by mechanisms other than the above-mentioned NK cell-mediated ADCC activity, improved antibody molecules that exert even more potent cytotoxic activity have also been reported, such as antibody drug conjugates (ADCs) in which antibodies are conjugated with drugs that have potent cytotoxic activity (Non-Patent Document 7), and small molecule antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to the cancer cells (Non-Patent Document 8).

[0005] While these antibody molecules exhibit more potent cytotoxic activity, they can also exert cytotoxic activity against cancer cells with low antigen expression, they also exert cytotoxic activity against normal tissues with low antigen expression. In fact, compared to cetuximab, a natural human IgG1 directed against the EGFR antigen, EGFR-BiTE, a bispecific antibody directed against CD3 and EGFR, exerts potent cytotoxic activity against cancer cells by recruiting T cells to cancer cells, thereby exerting antitumor effects. However, because EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE was administered to cynomolgus monkeys (Non-Patent Document 9). Furthermore, bivatuzumab mertansine, an ADC consisting of mertansine conjugated to an antibody directed against CD44v6, which is highly expressed in cancer cells, has been shown to cause severe skin and liver toxicity in clinical trials, due to the CD44v6 expression in normal tissues (Non-Patent Document 10).

[0006] When using antibodies that can exert strong cytotoxic activity even against cancer cells with low antigen expression, the target antigen must 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 cytotoxic activity against cancer can be strengthened, side effects due to cytotoxicity against normal tissues can become a problem.

[0007] Recently, it has been shown that ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, prolongs overall survival in metastatic melanoma (Non-Patent Document 11). However, because ipilimumab systemically inhibits CTLA4, while tumor immunity is enhanced, it also causes serious side effects similar to autoimmune diseases 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, including those that improve effector function, antigen-binding ability, pharmacokinetics, and stability, or reduce the risk of immunogenicity (Non-Patent Document 13). However, few technologies have been reported that enable antibody drugs to specifically act on diseased tissues to resolve the side effects described above. For example, pH-dependent antibodies have been reported for lesion sites such as cancer tissues and inflammatory tissues, taking advantage of the acidic pH conditions in these diseased tissues (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 generate antibodies that detect and act on even small increases in hydrogen ion concentration, which are extremely small in molecular weight. Furthermore, normal tissues such as osteoclast bone resorption lacunae and tissues other than the target lesion may also have acidic pH, posing many challenges to the use of pH conditions as an environmental factor specific to the lesion site. On the other hand, a method has been reported in which antibodies that exhibit antigen-binding activity only upon cleavage by proteases expressed in lesion sites such as cancer tissues and inflammatory tissues (Patent Document 3). However, because antibody cleavage by proteases is irreversible, it has been thought that antibodies cleaved at the lesion site can return to normal tissue via the bloodstream and bind to antigens in normal tissues. Another issue is the cancer specificity of such proteases. To overcome this issue, antigen-binding molecules whose antigen-binding activity 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 extensive research to achieve the above-mentioned objectives, and have discovered methylthioadenosine (MTA) as a new small molecule compound specific to cancer tissues. They have also 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 treatments that involve administration of the antigen-binding molecule, and that the antigen-binding molecule is useful in the manufacture of medicaments for cancer treatment.

[0013] The present inventors also created methods for screening and producing antigen-binding domains whose antigen-binding activity changes in an MTA-dependent manner. Furthermore, the present inventors succeeded in creating a library that can be used to efficiently screen for antigen-binding domains whose antigen-binding activity changes in an MTA-dependent manner. The present inventors further succeeded in creating a library that can be used to screen for antigen-binding domains whose antigen-binding activity changes in an MTA- and / or small molecular weight compound other than MTA-dependent manner, and created methods for screening for and producing the antigen-binding domains whose antigen-binding activity changes in an MTA- and / or small molecular weight compound other than MTA-dependent manner. 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 such 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 differs 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 towards the antigen is substantially unaffected by adenosine. [A4] The antigen-binding molecule according to [A1] to [A3], wherein the antigen-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 towards 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 in an adenosine-dependent manner. [A7] The antigen-binding molecule according to [A1], [A2], or [A6], wherein the antigen-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 of 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 of any one of [A1] to [A9], wherein the antigen-binding molecule is an antibody. [A11] The antigen-binding molecule of any one of [A1] to [A10], which comprises an antibody Fc region. [A12] The antigen-binding molecule of [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 of [A15], wherein the amino acid residue that interacts with MTA interacts with MTA when the antigen-binding domain is bound to an antigen. [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 CDRs 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 W34, C35a, W47, F52, Y52e, and E101 in the heavy chain, and R32, S34, Y36, L46, Y49, S50, A89, G90, L91, and P96 (Kabat numbering) in the light chain. [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 located 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 position 34 of the heavy chain; M located at heavy chain position 35; C located at heavy chain position 35a; C located at position 50 of the heavy chain; 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 located 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 located 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 heavy chain position 52f; 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 located at position 54 of the heavy chain; G located at position 55 of the heavy chain; S located at heavy chain position 56; T located at heavy chain position 57; Y located at heavy chain position 58; Y located at heavy chain position 59; A located at heavy chain position 60; S located at heavy chain position 61; W located at heavy chain position 62; A located at heavy chain position 63; K located at position 64 of the heavy chain; G located at position 65 of the heavy chain; G located at position 95 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 96 of the heavy chain; G located 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 heavy chain position 100; 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 located 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 located at position 100c of the heavy chain; E located at heavy chain position 101; L located at heavy chain position 102; Q located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; E located 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 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 position 33 of the light chain; S located at position 34 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 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 located 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 located 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 located 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 located 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 position 94 of the light chain; N located at light chain position 95; I located at light chain position 95a; P located 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 specified 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 comprising 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 of 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 position 34 of the heavy chain; M located at heavy chain position 35; C located at heavy chain position 35a; C located at position 50 of the heavy chain; 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 position 53 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 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 heavy chain position 58; Y located at heavy chain position 59; A located at heavy chain position 60; S located at heavy chain position 61; W located at heavy chain position 62; V located at heavy chain position 63; N located at heavy chain position 64; G located at position 65 of the heavy chain; E located at position 95 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 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 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; S located at position 100 of the heavy chain; 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 located at heavy chain position 101; L located at heavy chain position 102; H located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; K located 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 position 33 of the light chain; A at position 34 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 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 located 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 position 54 of the light chain; A at position 55 of the light chain; S located at light chain position 56; Q located at position 89 of the light chain; G at position 90 of the light chain; T 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; 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 located 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 specified 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, and T100a, and light chain S91, Y95c, and N96 (Kabat numbering). [A26] The antigen-binding molecule of 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 position 29 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 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 position 32 of the heavy chain; 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 located at position 35 of the heavy chain; either D, I, or V at position 50 of the heavy chain; I located at heavy chain position 51; G located 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 located at heavy chain position 62; V located at heavy chain position 63; K located at position 64 of the heavy chain; A, F, or G at heavy chain position 65; G located at position 95 of the heavy chain; 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 located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; Q located 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 located at position 30 of the light chain; 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 position 33 of the light chain; S located at position 34 of the light chain; D at position 50 of the light chain; A located at light chain position 51; S located at light chain position 52; T at light chain position 53; L located at position 54 of the light chain; A at position 55 of the light chain; S located at light chain position 56; H located 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 located at light chain position 96; A or G at position 97 of the light chain; A, F, I, L, or V at position 98 of the light chain. [A27] The antigen-binding molecule of any one of [A1] to [A26], wherein the antigen is a molecule other than MTA, or a molecule other than MTA that is immunogenic in vivo in humans. [A28] The antigen-binding molecule of any one of [A1] to [A27], wherein the antigen is a peptide, polypeptide, or 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 to which the antigen-binding domain binds. [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 of [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 towards the second antigen in the presence of MTA differs from the binding activity of the second antigen-binding domain towards the second antigen in the absence of MTA. [A33] The antigen-binding molecule of any one of [A1] to [A32], wherein the antigen is a membrane-type molecule or a soluble molecule. [A34] The antigen-binding molecule according to [A33], wherein the antigen is a membrane molecule and is expressed in diseased 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 of [A40], which exhibits at least one cytotoxic activity selected from ADCC activity, ADCP activity, and CDC activity. [A42] The antigen-binding molecule according to any one of [A1] to [A39], which has agonistic activity against the antigen. [A43] The antigen-binding molecule of 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 cells are cancer cells. [A45] The antigen-binding molecule according to [A44], wherein the cancer cells are cancer cells in which the gene encoding MTAP is deleted or has reduced expression, 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 has reduced expression, or which have a mutation or splicing variant that reduces the enzyme activity. [A46] The antigen-binding molecule according to [A43], wherein the target cells are non-cancer cells present around cancer cells in which the gene encoding MTAP is deleted or has reduced expression, or which have 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 of any one of [A43] to

[47] , wherein the effector cell is a T cell. [A49] The antigen-binding molecule of [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 the enzyme 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 the antigen-binding molecule of 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 it from the culture supernatant. [G1] The antigen-binding molecule of any one of [A1] to [A54], which has a higher plasma retention and / or a lower plasma antigen accumulation capacity than 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 of [G1] and a pharmaceutically acceptable carrier. [G3] A method for producing an antigen-binding molecule that has higher plasma retention and / or lower plasma antigen accumulation capacity compared to a control antigen-binding molecule, the method comprising: (a) producing an antigen-binding molecule whose antigen-binding activity increases with increasing MTA concentration; 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 following exemplary embodiments. [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], which contains nucleic acids encoding multiple antibody variable region variants that have different sequences from each other and that have amino acids that are different from the amino acids at one or more amino acid positions in an unmodified antibody variable region that has binding activity to MTA, and / or multiple antibody variable region variants that have different sequences from each other and that have amino acids that are different from the amino acids at one or more amino acid positions in an unmodified antibody variable region that has binding activity to MTA. [B4] The library according to [B3], wherein the amino acid site in the modified antibody variable region containing 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 compared to the antibody variable region variant, and 3) Amino acid sites that are 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 containing 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, 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 exhibits a large rate of structural change upon MTA binding / unbinding. [B6] The library according to any one 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 shown in SEQ ID NO:52 and a light chain variable region shown in SEQ ID NO:53. [B8] The multiple antibody variable regions are selected from the group consisting of: 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 do not have to be the same amino acid. [B2] The library according to [B2]. [B9] The multiple antibody variable regions are selected from the group consisting of: H-CDR1 comprising XAXWMC (SEQ ID NO: 65); H-CDR2 containing CIFAX1XXYXXSGGSTYYASWAKG (SEQ ID NO: 71); H-CDR3 comprising GXGXXXGXXDEL (SEQ ID NO: 67); L-CDR1 comprising QSSEXVXXX1XLS (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, The library described in [B2], wherein X1 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T and V, and X or X1 present at different positions do not have to be the same amino acid. [B10] The multiple antibody variable regions are selected from the group consisting of: H-CDR1 comprising XXAXWMC (SEQ ID NO: 73); H-CDR2 containing CIFAX1XXYXXSGGSTYYASWAKG (SEQ ID NO: 71); H-CDR3 comprising GXGXXXGXXDEL (SEQ ID NO: 67); L-CDR1 comprising QSSEXVXXX1XLS (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, The library described in [B2], wherein X1 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T and V, and X or X1 present at different positions do not have to be the same amino acid. [B11] The multiple antibody variable regions are selected from the group consisting of: 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 do not have to be the same amino acid. [B2] The library according to [B2]. [B12] The library according to any one of [B3] to [B5], wherein the unmodified antibody variable region also has binding activity to adenosine. [B13] The library according to [B12], wherein the unmodified antibody variable regions also have binding activity to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, and / or ATP. [B14] The library according to 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 are selected from the group consisting of: H-CDR1 comprising X2X3X4X5G (SEQ ID NO: 80); X6IGX7X8X9X 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 containing DP (SEQ ID NO: 82); QSSQSVX 22 X 23 NNX 24 L-CDR1 containing 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 X2 is an amino acid selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is an amino acid selected from D, E, F, H, K, N, P, R and Y; X4 is an amino acid selected from A, I, P, T and V; X5 is an amino acid selected from A, E, F, H, I, K, L, M, N, Q, S, T, V, W and Y; X6 is an amino acid selected from D, I and V; X7 is an amino acid selected from A, D, E, G, I, K, Q and R; X8 is an amino acid selected from D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W and Y; X9 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, X 21 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 26is 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 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 is carried out by 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], comprising: [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 is carried out by the following steps (1) and (2): (1) contacting an antigen-binding domain displayed from the library described in [B15] with adenosine; and (2) selecting an antigen-binding domain that binds to adenosine in step (1); The library according to [B19] or [B20], comprising:

[0016] The present disclosure also encompasses the following exemplary embodiments. [C1] The following steps (a) and (b): (a) identifying an amino acid site in an antigen-binding domain having binding activity to MTA that satisfies at least one of the following (i) to (vi): (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 structure of the antigen-binding domain is compared between when bound to MTA and when not bound to MTA; (iii) amino acid sites not involved in binding to MTA; (iv) an amino acid site that does not significantly attenuate binding to MTA; (v) an amino acid site with diverse 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 form of the 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 comparing the structure of the antigen-binding domain variant containing the amino acid alteration when bound to MTA with the structure when not bound to MTA, 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 is compared between when bound to MTA and when not bound to MTA, the presence of the amino acid alteration does not inhibit the structural change of the antigen-binding domain variant; (3) The antigen-binding domain variants having the amino acid alterations do not exhibit significantly reduced MTA-binding activity compared to the unaltered antigen-binding domain; The manufacturing method described in [C1]. [C3] The production method according to any one of [C1] to [C2], wherein the antigen-binding domain having binding activity to MTA does not substantially bind to adenosine. [C4] The production 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, and / or 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 in an antigen-binding domain that has binding activity to a low molecular weight compound, which satisfies at least one of the following (i) or (ii): (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 comparing the structure of the antigen-binding domain when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound; (b) designing a library comprising nucleic acids encoding an unmodified form of the 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 when bound to the low molecular weight compound is compared with the structure when not bound to the low molecular weight compound, the rate of structural change at the amino acid site where the amino acid alteration 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 thereof not bound to the low molecular weight compound, the presence of the amino acid alteration does not inhibit the structural change of the antigen-binding domain variant; (3) The binding activity of the modified antigen-binding domain having the amino acid alterations to the small molecule compound is not significantly reduced 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 that satisfies 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 the rate of conformational change is large when comparing the structure of the antigen-binding domain when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound; (iii) an amino acid site that is not involved in binding to the small molecule compound; (iv) an amino acid site that does not significantly reduce the binding to the small molecule compound; (v) an amino acid site with diverse 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 form of the antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain that have different sequences and have amino acid modifications that satisfy at least one of the following (1) or (2) at one or more amino acid sites identified in step (a): (1) When the structure of the antigen-binding domain variant having the amino acid alteration when bound to the low molecular weight compound is compared with the structure when 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 thereof not bound to the low molecular weight compound, the presence of the amino acid alteration does not inhibit the structural change of the antigen-binding domain variant; A method for producing a library comprising: [C9] The method for producing 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 following exemplary embodiments. [D1] A method for screening antigen-binding molecules containing an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. [D2] The method described in [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 of any one of [D2] to [D3], comprising a step of selecting an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is higher than that in the presence of MTA at the second concentration. [D5] The method of any one of [D2] to [D3], comprising a step of selecting an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity in the presence of MTA at the first concentration is lower than the 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 containing 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 antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity is weaker than the standard confirmed 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 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 the antigen; (c) allowing the 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 the 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 containing antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) placing the antigen-binding molecule containing 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 containing 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 that 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 step (c) antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity is weaker than the criterion selected in 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); The method according to [D10] or [D11], comprising: [D13] The following steps (a) to (d): (a) contacting a library displaying antigen-binding molecules containing antigen-binding domains with an antigen in the presence of a first concentration of MTA; (b) selecting antigen-binding molecules 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 the 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], comprising comparing the antigen-binding activity of the antigen-binding domain in the presence of MTA with that in the absence of MTA. [D16] The method according to [D1] or [D15], which comprises a step of selecting antigen-binding domains whose antigen-binding activity in the presence and absence of MTA differs. [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 that 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 MTA; and (d) isolating antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity is weaker than the standard confirmed in step (b) in step (c); The method according to [D1], comprising: [D20] The method according to [D18] or [D19], wherein the ability of an antigen-binding molecule comprising an antigen-binding domain to bind to an MTA is confirmed before performing 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 the antigen; (c) allowing the antigen-binding molecule comprising an antigen-binding domain that does not bind to the antigen to bind to the antigen in the absence of MTA; and (d) isolating the 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 containing antigen-binding domains with an antigen in the presence of 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 containing antigen-binding domains with an antigen in the presence of MTA; (b) selecting an antigen-binding molecule comprising the antigen-binding domain that 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 step (c) antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity is weaker than the criterion selected in 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); wherein the library contacted with an antigen in the presence of MTA in step (a) displays antigen-binding molecules comprising the antigen-binding domains selected in steps (1) and (2). [D25] The following steps (a) to (d): (a) contacting a library displaying antigen-binding molecules containing antigen-binding domains with an antigen in the presence of an MTA; (b) selecting antigen-binding molecules 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 MTA; and (d) isolating the 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 antigen-binding molecules 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 following exemplary embodiments. [F1] A method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner. [F2] The method described in [F1], 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. [F3] The method according to [F1] or [F2], which comprises 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 of 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 of 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 of 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 the antigen-binding molecule comprising the antigen-binding domain isolated in step (c) has been 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 whose antigen-binding activity is weaker than the standard confirmed in step (b) in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding the 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 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 does not bind to the antigen in step (a); (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 the 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 the antigen-binding molecule comprising the antigen-binding domain isolated in step (c) has been 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 the antigen-binding domain that bound 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 domains selected 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 the 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] Before the step (a), the following steps (1) and (2) are carried out: (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], comprising: (a) contacting the library with the antigen in the presence of MTA at a first concentration with the library displaying the antigen-binding domains selected in 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 the 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 that in the absence of MTA. [F17] The method according to [F1] or [F16], which comprises a step of selecting antigen-binding domains whose antigen-binding activity in the presence and absence of MTA differs. [F18] The method according to [F1] or [F16], which comprises a step of selecting an antigen-binding domain whose antigen-binding activity in the presence of MTA is higher than that in the absence of MTA. [F19] The method of 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 the antigen-binding molecule comprising the antigen-binding domain isolated in step (c) has been 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 whose antigen-binding activity is weaker than the standard confirmed in step (b) in step (c); and (e) culturing cells into which a vector has been introduced to which a polynucleotide encoding the 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 step (a). [F23] 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 does not bind to the antigen in step (a); (c) allowing the antigen-binding domain that does not bind to the antigen to bind to the antigen in the absence 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 the 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 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 the antigen-binding molecule comprising the antigen-binding domain isolated in step (c) has been 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 MTA; (b) selecting the antigen-binding domain that bound 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 domains selected 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 the 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] Before the step (a), the following steps (1) and (2) are carried out: (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], comprising: [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) allowing the antigen-binding domain selected in step (b) to bind to an antigen in the absence 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 the 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 described in 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, a synthetic human antibody display library, 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 following exemplary embodiments. [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 does not substantially bind to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, and / or ATP. [E4] A method for measuring 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 either [E4] or [E5], in which the MTA concentration is measured using an antigen-antibody reaction. [E7] The measurement method according to any one of [E4] to [E5], which uses immunohistochemical staining. [E8] The measurement method according to any one of [E4] to [E5], wherein an imaging method is used. [E9] The measurement method according to 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 of any one of [E1] to [E3]. [E11] The method according to [E10], wherein the diagnosis is to determine the presence or absence of a disease or to predict the effectiveness 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] A method according to either [E12] or [E13], wherein the cancer is cancer tissue in which the gene encoding MTAP is deleted or underexpressed, or which has a mutation or splicing variant that reduces the enzyme activity. [E15] A diagnostic kit for a disease, comprising the antigen-binding molecule of 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. [Effects of the Invention]

[0020] The antigen-binding molecules disclosed herein, which contain an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner, and pharmaceutical compositions containing the same, do not act systemically in normal tissues or blood but act reversibly on cancer, thereby exerting medicinal effects while avoiding side effects and enabling cancer treatment. Furthermore, by using the library of the present disclosure comprising antigen-binding molecules comprising multiple antigen-binding domains with different sequences 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 explanation of the drawings]

[0021] [Figure 1] This figure shows the intracellular MTA concentration of each cell line. The vertical axis represents the intracellular MTA concentration, and the horizontal axis represents the cell line name and MTAP deficiency status. MTAP- indicates an MTAP-deficient cell line, and MTAP+ indicates an MTAP-non-deficient cell line. [Figure 2] This figure shows the MTA concentration in the culture medium when each cell line was cultured. The vertical axis represents the MTA concentration in the culture medium, and the horizontal axis represents the culture time, the name of the cell line, and the MTAP deficiency status. MTAP- indicates an MTAP-deficient cell line, and MTAP+ indicates an MTAP-non-deficient cell line. [Figure 3] This figure shows the MTA concentration in the culture medium when each cell line was cultured in medium to which MTA had been added beforehand. The vertical axis represents the MTA concentration in the culture medium, and the horizontal axis represents the culture time. Each spot represents a measured data point. The graph on the left shows the MTA concentration in the culture medium when MTAP-non-deficient HT-1376 cells were cultured, and the graph on the right shows the MTA concentration in the culture medium when MTAP-non-deficient SK-MES-1 cells were cultured. [Figure 4] This is a graph showing 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 represents a measured data point. [Figure 5]This figure shows the relationship between MTAP DNA amount and tissue MTA concentration in human clinical samples. 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 light-colored spots indicate samples in which the tissue MTA concentration was below the detection limit. The vertical axis represents the MTA concentration in the tissue, and the horizontal axis represents ΔCt, which is the Ct value of the MTAP gene minus the Ct value of the ΨX4 gene. [Figure 6] This figure shows 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, 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 represents a measured value, and empty spots represent data below the lower limit of quantitation for MTA. [Figure 7] This figure shows the amount of hIL-6R binding of C03H-BH076N17 / C03L-KT0 at different concentrations of MTA or adenosine, with the vertical axis representing the amount of hIL-6R binding per amount of solid-phase antibody, and the horizontal axis representing the concentration of MTA or adenosine. [Figure 8] This is a diagram showing the binding mode of SMB0002hFab and adenosine. In the diagram, 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 associated numbers indicate the distance between each amino acid residue and adenosine through hydrogen bonds, CH-π interactions, or π-π interactions. The units are shown in 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. 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), superimposed together. 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), superimposed together. In the figure, SMB0002hFab_2 is shown in gray, and the SMB0002hFab-adenosine complex is shown in black. [Figure 12]

[0039] Figure 1 shows the amount of binding to MTA of clones obtained after panning of a heavy chain variable region phage display library against MTA. The vertical axis shows absorbance by phage ELISA when MTA was not immobilized, and the horizontal axis shows absorbance when MTA was immobilized. [Figure 13] This figure shows the amount of binding to MTA of clones obtained after panning of a light chain variable region phage display library against MTA. The vertical axis shows the absorbance by phage ELISA when MTA was not immobilized, and the horizontal axis shows the absorbance when MTA was immobilized. [Figure 14] This figure shows the binding amounts of antibodies that bind to hIL-6R, hIL-6, and hIgA in an MTA-dependent manner, measured by SPR in the presence of MTA or adenosine. The vertical axis shows the binding amount (-100 to 200 RU), and the horizontal axis shows the reaction time (-100 to 1200 seconds, with 0 second being the start of the antigen reaction). [Figure 15] This is a diagram of the variable region and MTA extracted from the crystal structure of the complex between MTA0303Fab and MTA. In the diagram, the heavy chain of the antibody is shown in black, the light chain in gray, 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 diagram, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. Amino acid residues that form interactions with MTA are shown as stick models. The dashed lines and their associated numbers indicate the distances between each amino acid residue and MTA, including hydrogen bonds, CH-π interactions, π-π interactions, and sulfur-π interactions. The units are shown in Å. [Figure 17] This is a diagram showing the binding mode between MTA0303Fab and MTA. In the diagram, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. Amino acid residues that interact with MTA are shown as stick models. The dashed lines and their associated numbers indicate the hydrogen bond distances between the amino acid residues and 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 between MTA0330Fab and MTA. In the diagram, the heavy chain of the antibody is shown in black, the light chain in gray, 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 diagram, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. Amino acid residues that form interactions with MTA are shown as stick models. The dashed lines and their associated numbers indicate the distance between hydrogen bonds, CH-π interactions, or π-π interactions between each amino acid residue and MTA. The units are shown in Å. [Figure 20] This is a diagram of the crystal structure of the complex between 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 of the crystal structure of the complex between MTA0330Fab and MTA. In the diagram, 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 in gray. [Figure 23] Figure 1 shows an overlay of 1H-13C SOFAST-HMQC spectra of MTA0303Fab in the MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum in gray. [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 in gray. [Figure 25] Figure 1 shows an overlay of 1H-13C SOFAST-HMQC spectra of MTA0330Fab in the MTA-bound and unbound states, with the bound spectrum shown in black and the unbound spectrum in gray. [Figure 26] FIG. 1 shows that a small molecule switch antibody does not bind to an antigen in a normal environment where small molecules are not present, but binds to the antigen in target tissues where small molecules are present at high concentrations. [Figure 27] This is a diagram showing that a small molecule fulfills its switching function by being sandwiched between a small molecule antibody and an antigen complex. In the absence of the small molecule, the interaction between the antibody and antigen is insufficient and the antibody cannot bind to the antigen, but in the presence of the small molecule, it becomes sandwiched between the antibody and antigen, allowing the antibody to bind to the antigen. [Figure 28] Figure 1 shows the T cell activation ability of a bispecific antibody having an antigen-binding domain that binds to IL-6R and an antigen-binding domain that binds to CD3 in an MTA-dependent manner, tested in the presence or absence of MTA or ADO, using NFAT-RE-luc2-Jurkat cells. The X axis represents antibody concentration (μg / mL), and the Y axis represents 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 at 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. [Figure 30] This is a sensorgram showing the time course of binding of an anti-IL-6R antibody that binds to IL-6R in an MTA-dependent manner to the antigen (hIL-6R), as 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 INVENTION

[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, such as 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, and Val / V.

[0023] Amino acid modification To modify 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. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (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 an amber suppressor tRNA complementary to the UAG codon (amber codon), a type of stop codon, can also be suitably 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] Herein, expressions for amino acid modifications may be appropriately expressed by listing the one-letter or three-letter codes for the amino acid before and after the modification before and after a number representing a specific position. For example, the modification N100bL or Asn100bLeu, used when substituting an amino acid contained in an antibody variable region, represents a substitution of Asn at position 100b according to the Kabat numbering system with Leu. That is, the number represents the amino acid position according to the Kabat numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution. Similarly, the modification P238D or Pro238Asp, used when substituting an amino acid in the Fc region contained in an antibody constant region, represents a substitution of Pro at position 238 according to the EU numbering system with Asp. That is, the number indicates the position of the amino acid as expressed in 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 term "antigen" is not limited to a specific structure as long as it contains an epitope to which an antigen-binding domain binds. In one embodiment, the antigen is a peptide, polypeptide, or protein of four or more amino acids. Examples of 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, and 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, CMVUL, 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, サイトケラチン tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement defense factor (Decay accelerating factor)factor), 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 growth factor receptor 1 (IGFR), insulin-like growth factor receptor 2 (IGFR), insulin-like growth factor receptor 3 (IGFR), insulin-like growth factor receptor 4 (IGFR), insulin-like growth factor receptor 5 (IGFR), insulin-like growth factor receptor 6 (IGFR), insulin-like growth factor receptor 7 (IGFR), insulin-like growth factor receptor 8 (IGFR), insulin-like growth factor receptor 9 (IGFR), insulin-like growth factor receptor 1 ... 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, NC adherin, 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, 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-α 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-like receptor)receptor)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 Liquids 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, such as factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and antigens expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues. is preferred.

[0027] Although receptors are also listed as examples of antigens, these receptors present in soluble form in biological fluids can also be used as antigens to which antigen-binding molecules comprising an antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner can 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 the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968).

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

[0029] There is no limitation on the solution in which a soluble molecule exists, and the soluble molecule may exist in biological fluids, i.e., all fluids filling the vessels or the spaces between tissues and cells in a living body. In a non-limiting embodiment, a soluble molecule bound by an antigen-binding molecule of the present disclosure may exist in extracellular fluid. In vertebrates, extracellular fluid refers collectively to components in bone and cartilage, such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, spinal fluid, aspirate, or synovial fluid, as well as transcellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activity of cells, and fluids in the digestive tract and other body cavities), such as alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor).

[0030] When an antigen-binding molecule comprising an antigen-binding domain of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner 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 to a cytotoxic substance, as described below. Furthermore, preferred, non-limiting examples of antigen-binding molecules include antigen-binding molecules that have neutralizing activity instead of or in addition to the property of having cytotoxic activity or the property of binding or having the ability to bind to 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 the target antigen. Examples of such domains include the variable regions of the heavy and light chains of antibodies, a module called an A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectin (International Publication No. WO2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes, Affibody (International Publication No. WO1995 / 001937) using an IgG binding domain consisting of a 58-amino acid three-helix bundle of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeat (AR)) which are regions exposed on the molecular surface of ankyrin repeats (AR) with a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Suitable examples include lipocalin molecules such as lipocalin proteins (International Publication No. 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, found in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication No. WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish, are repeatedly stacked (International Publication No. WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present disclosure include antigen-binding domains comprising the variable regions of the heavy and light chains of an antibody. Preferred examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0032] antigen binding molecule In the present disclosure, the term "antigen-binding molecule containing an antigen-binding domain" is used in the broadest sense and specifically includes various molecular types as long as it contains an antigen-binding domain. An antigen-binding molecule may be a molecule consisting of only an antigen-binding domain, or a molecule containing an antigen-binding domain and other domains. For example, when an antigen-binding molecule is a molecule in which an antigen-binding domain and an Fc region are linked, examples include complete antibodies and antibody fragments. Antibodies may include single monoclonal antibodies (including agonist and antagonist antibodies), human antibodies, humanized antibodies, chimeric antibodies, etc. Scaffold molecules, in which existing stable α / β barrel protein structures or other three-dimensional structures are used as scaffolds, and only partial structures of these structures are compiled into libraries for constructing antigen-binding domains, may also be included in the antigen-binding molecules of the present disclosure.

[0033] antibody As used herein, an antibody refers to a natural or partially or fully synthetically produced immunoglobulin. Antibodies can be isolated from natural sources, such as plasma or serum, or from the culture supernatant of antibody-producing hybridoma cells. Alternatively, antibodies can be partially or fully synthesized using techniques such as genetic recombination. Examples of antibodies include immunoglobulin isotypes and their isotypic subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Antibodies of the present disclosure may include IgG1, IgG2, IgG3, and IgG4 of these isotypes. Multiple allotype sequences due to genetic polymorphisms for the human IgG1, IgG2, IgG3, and IgG4 constant regions 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 human IgG1 sequences, the amino acid sequence at positions 356-358 (EU numbering) may be DEL or EEM. Furthermore, 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 either of these sequences 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 Institutes of Health, Bethesda, Md., 1987 and 1991)). Herein, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids in the variable region are represented according to the Kabat numbering, and the amino acids in 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 heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

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

[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 antibody heavy chain constant region, spanning from the N-terminus of the hinge region of the papain cleavage site at approximately amino acid position 216 (EU numbering) to the hinge, CH2, and CH3 domains. The Fc region can be obtained from human IgG1, but is not limited to a specific IgG subclass. Preferred examples of the Fc region include Fc regions that have FcRn-binding activity in the acidic pH range, as described below. Preferred examples of the Fc region also include Fc regions that have Fcγ receptor-binding activity, as described below. Non-limiting examples of such Fc regions include the Fc regions of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), and IgG4 (SEQ ID NO: 8).

[0039] Low molecular weight antibody The antibodies used in the present disclosure are not limited to full-length antibody molecules, but may also be minibodies or modified versions thereof. Minibodies include antibody fragments in which a portion 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 antigen-binding activity. The minibodies of the present disclosure are not particularly limited as long as they are a portion 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, portions of VH and / or VL may be deleted as long as they have antigen-binding activity. The variable regions may also be chimerized or humanized. Specific examples of antibody fragments include Fab, Fab', F(ab')2, and Fv. 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 by constructing genes encoding these antibody fragments, introducing them into expression vectors, and then expressing them in appropriate host cells (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 are 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. Typically, the VL and VH in each polypeptide chain are linked by a linker that is so short, for example, about five residues, that they cannot bind to each other. Because the linker between the VL and VH encoded on the same polypeptide chain is too short to form a single-chain variable region fragment, they form a dimer, and thus diabodies have two antigen-binding sites.

[0042] scFv can be obtained by linking the heavy and light chain variable regions of an antibody. In this scFv, the heavy and light chain variable regions are linked via a linker, preferably a peptide linker (Huston et al. (Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883)). The heavy and light chain variable regions in an scFv may be derived from any of the antibodies described herein. The peptide linker linking the variable regions is not particularly limited, and can be, for example, any single-chain peptide consisting of about 3 to 25 residues, or a peptide linker as described below. The variable regions can be linked by PCR as described above. The DNA sequence encoding the heavy or light chain variable region of the antibody and the DNA sequence encoding the light or light chain variable region, all or part of the DNA encoding the desired amino acid sequence, are used as templates, and sequences corresponding to the sequences at both ends of the DNA sequence are then synthesized. DNA encoding the scFv can be amplified by PCR using a pair of primers having the sequences shown in Table 1. Subsequently, DNA encoding the peptide linker portion can be combined with a pair of primers having sequences designed so that both ends of the primer are linked to the H chain and L chain, respectively, to obtain DNA having the desired sequence. Once DNA encoding the scFv is prepared, expression vectors containing the DNA and recombinant cells transformed with the expression vectors can be obtained by standard methods, and the resulting recombinant cells can be cultured to express the DNA encoding the scFv, thereby obtaining the scFv.

[0043] sc(Fv)2 is a minibody made into a single chain by linking two VHs and two VLs with a linker or the like (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, preferred antibodies are those in which 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 limited to the above-mentioned 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 used to link the antibody variable regions may be the same as the linkers described above in the section on antigen-binding molecules. For example, particularly preferred embodiments of sc(Fv)2 in the present disclosure include the following sc(Fv)2: -[VH] peptide linker (15 amino acids) [VL] peptide linker (15 amino acids) [VH] peptide linker (15 amino acids) [VL] When four antibody variable regions are linked, three linkers are usually required, and the same linkers may be used for all of them, or different linkers may be used.

[0046] Such minibodies can be obtained by treating antibodies with enzymes such as papain or pepsin to generate antibody fragments, or by constructing DNA encoding these antibody fragments or minibodies, introducing it into an expression vector, and then expressing it 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; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0047] Furthermore, a non-limiting example of an 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 with a T cell signal domain is incorporated into a T cell, and a T cell into which such a chimeric antigen receptor has been incorporated, but is not limited to this.

[0048] Single Domain Antibodies A suitable example of the 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, exemplified by IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing of VH and VL, whereas single-domain antibodies are known to be able to exhibit antigen-binding activity solely through the domain structure of the single-domain antibody itself, without pairing with any other domain. 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 VHHs and shark VNARs, or antibody fragments comprising all or a portion of the VH domain or all or a portion of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments comprising all or a portion of the VH / VL domains of an antibody, include, but are not limited to, single-domain antibodies artificially created starting from a 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 genes capable of producing single-domain antibodies have been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which genes capable of producing single-domain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO 2015 / 143414 and U.S. Patent Publication No. US 2011 / 0123527 A1. Humanized single-domain antibodies can also be obtained by substituting human germline sequences or sequences similar thereto for the framework sequences of single-domain antibodies obtained from animals. Humanized single-domain antibodies (e.g., humanized VHHs) are also an embodiment of the single-domain antibodies of the present invention. A "humanized single-domain antibody" refers to a chimeric single-domain antibody comprising amino acid residues from non-human CDRs and human FRs. In one embodiment, a humanized single-domain antibody has all or substantially all CDRs corresponding to those of a non-human antibody, and all or substantially all FRs corresponding to those of a human antibody. In a humanized antibody, even if some of the residues in the FRs do not correspond to those of a human antibody, this is still considered 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 FRs must be changed to 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)).

[0053] Method for producing antibodies with desired binding activity to antigens Methods for producing antibodies that are independent of low-molecular-weight compounds, including MTA, and that have the desired binding activity toward antigens that are different molecules from the 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 examples below.

[0054] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. The monoclonal antibodies of the present invention also include "humanized antibodies" and "chimeric antibodies."

[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. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0056] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody production, can be obtained by expressing the IL-6R gene, whose nucleotide sequence is disclosed in SEQ ID NO: 2. Specifically, 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 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 in place of the IL-6R protein represented by SEQ ID NO: 1. Purified native 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 mammals. 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. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also be obtained by degrading the IL-6R protein using a protease. However, the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be any sequence 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 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 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0058] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein 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 prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in International Publication Nos. 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. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0060] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can also 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 immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the 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 the 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. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

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

[0064] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival 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 deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective 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 and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines 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, such as 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 conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0069] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0070] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 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, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0071] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0072] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient 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. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0073] Screening and monocloning of the desired antibody can be suitably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

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

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

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

[0077] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0078] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are 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 hybridoma cells that produce the anti-IL-6R antibody. To do this, total RNA is usually first extracted from the hybridoma. The following methods 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 similar. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be isolated from hybridomas. cDNA encoding antibody V regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, the 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 used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, which will be described later, can be introduced at both ends of the cDNA.

[0081] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether or not the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0082] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. 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 for PCR amplification of antibody genes. 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 recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

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

[0085] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibodies can be screened using the binding activity of the reconstituted immunoglobulins to IL-6R as an indicator. For example, when the goal is to obtain antibodies against IL-6R, it is more preferable that the antibodies bind to IL-6R specifically. 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) A step of selecting an antibody that binds to IL-6R-expressing cells.

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

[0087] Panning methods using phage vectors are also suitable for screening antibodies using binding activity as an index. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be enriched.

[0088] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. 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. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the 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 antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains 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 at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region. The two genes are digested with the same combination of restriction enzymes and fused in frame to construct a chimeric antibody expression vector.

[0089] To produce an anti-IL-6R monoclonal antibody, the 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 antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the Examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells are transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0090] For antibody gene expression, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. By co-transfecting the same host cells with vectors incorporating the H chain and L chain, antibody molecules comprising both H and L chains can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and 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. All of these expression systems can be applied to isolating the antigen-binding domains of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (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 cells (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. Callus cultured cells can be appropriately used for transformation of plant cells.

[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] Expression systems for antibody genes 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 used as appropriate. An expression vector containing the antibody gene of interest 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 host cells described above, 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 inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing a fusion gene with an antibody gene inserted therein is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the goat that received the embryo produces milk from which the desired antibody can be obtained as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats 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 molecules described herein are administered to humans, the antigen-binding domain of the antigen-binding molecule may be an antigen-binding domain derived from a recombinant antibody that has been artificially modified for purposes such as reducing heterologous antigenicity to humans. Examples of recombinant antibodies include humanized antibodies. These modified antibodies are produced appropriately using known methods.

[0097] Antibodies with the desired binding activity for a specific low molecular weight compound can be obtained by methods similar to those used to generate antibodies that bind to conventional protein antigens. One embodiment of a method for generating a sensitizing antigen used to obtain antibodies against a low molecular weight compound is to link a low molecular weight compound to mariculture keyhole limpet hemocyanin (KLH). A non-limiting example of a sensitizing antigen generated to obtain antibodies against MTA is 6'-MTA-keyhole limpet hemocyanin (6'-MTA-KLH). 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. Therefore, linking it to MTA is expected to enhance the production of antibodies against MTA. The low molecular weight compound linked to KLH is not limited to MTA, and a similar technique can be used to create sensitizing antigens for various synthetic low molecular weight compounds, 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; for example, one linked to biotin may be used as a 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 following exemplary embodiments. [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 the 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 antigen-binding molecules that bind to MTA using the biotinylated MTA described in [1] to [3].

[0098] Multispecific or multiparatopic antigen-binding molecules An antigen-binding molecule comprising at least two antigen-binding domains, wherein 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 the antigen molecule, is called a multispecific antigen-binding molecule from the viewpoint of the specificity of its reaction. When an antigen-binding molecule binds to two different epitopes via 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 via 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 structurally different from the first epitope are structurally different from each other. 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 the antigen molecule, is called a multiparatopic antigen-binding molecule from the perspective of its structural specificity. When an antigen-binding molecule binds to two different epitopes via two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a biparatopic antigen-binding molecule. Furthermore, when an antigen-binding molecule binds to three different epitopes via three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a tripleparatopic antigen-binding molecule.

[0100] Bispecific antibodies and methods for producing them Multivalent, multispecific or multiparatopic antigen-binding molecules comprising one or more antigen-binding domains and methods for their preparation 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 No. WO1996 / 034103 or WO1999 / 023221. The antigen-binding molecules of the present disclosure can be produced using the multispecific or multiparatopic antigen-binding molecules and methods for their preparation described therein.

[0101] As one embodiment of the above-mentioned multispecific or multiparatopic antigen-binding molecules and methods for preparing them, bispecific antibodies and methods for producing them are exemplified below. Bispecific antibodies are antibodies that contain two types of variable regions that specifically bind to different epitopes. IgG-type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein et al., Nature (1983) 305, 537-540).

[0102] When producing bispecific antibodies using recombinant techniques such as those described above in the antibody section, a method can be used in which genes encoding heavy chains containing two desired variable regions are introduced into cells and co-expressed. However, even considering the heavy chain combinations in such co-expression methods, the resulting mixture contains (i) a heavy chain combination in which one heavy chain containing a variable region that binds to a first epitope is paired with another heavy chain containing a variable region that binds to a second epitope, (ii) a heavy chain combination in which only heavy chains containing a variable region that binds to the first epitope are paired, and (iii) a heavy chain combination in which only heavy chains containing a variable region that binds to the second epitope are paired, in a molecular ratio of 2:1:1. It is difficult to purify antigen-binding molecules containing the desired heavy chain combination from a mixture of these three types of heavy chain combinations.

[0103] When producing bispecific antibodies using such recombinant techniques, bispecific antibodies containing heterogeneous heavy chain combinations can be preferentially secreted by modifying the CH3 domains of the heavy chains with appropriate amino acid substitutions. Specifically, the amino acid side chains in the CH3 domain of one heavy chain are replaced with larger side chains (knobs) and the amino acid side chains 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, promoting heterologous heavy chain formation and inhibiting homologous heavy chain formation (International Publication No. WO1996027011; Ridgway et al., Protein Engineering (1996) 9, 617-621; Merchant et al., Nat. Biotech. (1998) 16, 677-681).

[0104] Furthermore, techniques for producing bispecific antibodies are known that utilize methods for controlling the association of polypeptides or heteromultimers composed of polypeptides in the association of heavy chains. Specifically, a method can be employed to produce bispecific antibodies by modifying amino acid residues that form an interface within the heavy chain, thereby inhibiting the association of heavy chains with identical sequences and controlling the formation of two heavy chains with different sequences (International Publication No. WO 2006 / 106905). Such methods can also be employed when producing bispecific antibodies.

[0105] cancer As used herein, the term "cancer" generally refers to a malignant neoplasm, which may be metastatic or non-metastatic. Non-limiting examples of carcinomas originating from epithelial tissues, such as the digestive tract and skin, include brain tumors, 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 hematopoietic cancers 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, refers to any newly formed pathological tissue tumor. In this disclosure, a neoplasm results in the formation of a tumor, which is characterized in part by angiogenesis. Neoplasms 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 containing 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 as cancer tissue containing 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" means cancer tissue in which MTA is detected in larger amounts than in normal tissue, and non-limiting examples of normal tissue to be compared include normal tissue adjacent to the cancer tissue and tissue from a healthy individual. Furthermore, cancer tissues lacking methylthioadenosine phosphorylase (MTAP), which metabolizes MTA, or cancer tissues with reduced MTAP function are also examples of cancer tissues in which MTA accumulates. Specifically, cancer tissues in which the gene encoding MTAP is deleted or expression is reduced, cancer tissues expressing mutations or splicing variants that reduce MTAP activity, or cancer tissues with reduced MTAP enzymatic activity are examples of cancer tissues in which MTA accumulates. Reduced MTAP expression or function can be determined by comparison with normal tissues, and non-limiting examples of normal tissues to be compared include normal tissue adjacent to the cancer tissue and tissues from healthy individuals.

[0108] Cancer-associated fibroblast (CAF) In the present disclosure, "cancer-associated fibroblasts (CAFs)" refer to a heterogeneous population of cells of diverse origins, such as endothelial cells present in the periphery of cancer tissue. Non-limiting characteristics of CAFs include promoting cancer cell proliferation, angiogenesis, and vascular invasion, and creating a microenvironment favorable to cancer progression through control of immune responses. Furthermore, non-limiting examples of CAFs 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 macrophages present in cancer tissues and their surrounding areas. Examples include macrophage populations that form the cancer microenvironment together with fibroblasts and vascular endothelial cells. Non-limiting examples of TAM characteristics include the induction of neovascularization through the production of various angiogenic factors, such as the production of anti-inflammatory factors and the suppression of anti-tumor immunity by promoting the infiltration of regulatory T cells. Furthermore, non-limiting examples of TAMs include cells expressing markers 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 its 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. 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. 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" as used herein refers to a low molecular weight compound other than MTA that shares a partial structure 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 atom at the 5-position of adenosine. Further 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 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 "small molecular weight compound" as used in the present disclosure refers to naturally occurring or non-naturally occurring chemical substances other than "biopolymers" present in living organisms. Examples of small 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 small 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 can be a compound defined by qualitative cancer tissue specificity, such as being present in cancer tissue but not in non-cancerous tissue, or being absent in cancer tissue but present in non-cancerous tissue. In another embodiment, a cancer tissue-specific compound can be a compound defined by quantitative cancer tissue specificity, such as being present at a different concentration (e.g., higher or lower concentration) in cancer tissue compared to non-cancerous tissue. For example, a cancer tissue-specific compound can be differentially present at a given concentration. However, in general, cancer tissue-specific compounds will exhibit a cytotoxic effect 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 can be present at a concentration that is 50% or more times greater than the concentration of the non-cancer tissue, increasing to infinity (i.e., absent in non-cancer tissues), or generally at a concentration that is 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 statistically significant concentration (i.e., as determined using either Welch's t-test or Wilcoxon's rank sum test, p-value is less than 0.05 and / or q-value is less than 0.10). 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, cancer stromal cell-specific metabolites) produced by metabolic activity specific to cancer cells, immune cells, and stromal cells contained in cancer tissue, such as the following:

[0116] Cancer tissue-specific metabolites The term "metabolism" refers to chemical changes that occur within the tissues of an organism, and includes "anabolism" and "catabolism." Anabolism refers to the biosynthesis or accumulation of molecules, while catabolism refers to the breakdown of molecules. A "metabolite" is an intermediate or product resulting from metabolism. A "primary metabolite" refers to a metabolic product that is directly involved in the growth or reproduction process of a cell or organism, while a "secondary metabolite" refers to a product, such as an antibiotic or pigment, that results from metabolism that biosynthesizes a substance that is not directly involved in the growth or reproduction process or is not directly involved in the life processes common to cells or organisms. A metabolite can be a metabolic product of a "biopolymer" or a "small molecule." A "biopolymer" is a macromolecule 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 5,000 or more that form structures such as cells that make up the tissues of living organisms 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 composed of or containing amino acid analogs or non-amino acid groups.

[0117] In one non-limiting embodiment, the cancer tissue-specific metabolites described herein include cancer cell-specific small molecule metabolites (Eva Gottfried, Katrin Peter, and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132). Furthermore, metabolites produced at high levels by immune cells infiltrating cancer tissues and stromal cells (cancer stromal cells or cancer stromal fibroblasts (CAFs)) that support the survival and / or growth of cancer cells are also included. Examples of infiltrating immune cells include dendritic cells, suppressor dendritic cells, suppressor T cells, exhausted T cells, and myeloma-derived suppressor cells (MDSCs). Metabolites in the present invention also include compounds released from within cells to the extracellular space when cells present in cancer tissues (cancer cells, immune cells, and stromal cells) die due to apoptosis, necrosis, or other causes.

[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 conventional methods (e.g., 1741-1753), metabolic (metabolomic) analysis, centered on metabolic profiling, may be used as appropriate. That is, metabolic profiling may be performed 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, or the like, alone or in combination, to identify metabolites in a test sample.

[0119] These studies have revealed intratumor heterogeneity, which is structured by altered concentration gradients of metabolites (e.g., glucose or oxygen) and growth factors that enable cancer cells to grow under conditions of low oxygen tension (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 malignancy (Vizan et al. (Cancer Res. (2005) 65, 5512-5515)). Non-limiting examples of technical components of the metabolomics platform include 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 make it possible to identify cancer cell-specific metabolites in desired cancer tissues.

[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 dermatitis Spinal nerve damage in herniated discs and chronic lower back pain

[0121] Inflammatory tissue-specific metabolites Inflammatory tissue-specific metabolites are metabolites that are produced at high levels by immune cells infiltrating inflammatory tissues, and metabolites that are produced at high levels specifically by normal cells damaged in inflammatory tissues. Examples of infiltrating immune cells include effector T cells, mature dendritic cells, neutrophils, granulocytes (mast cells), and basophils. 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 non-naturally occurring chemical substances and their metabolites. One embodiment of the invention relates to non-naturally occurring chemical substances and their metabolites that accumulate in target tissues after administration from an exogenous source to the living body. Examples of non-naturally occurring 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). TH-302 is also known to be reduced to Br-IPM under hypoxic conditions, such as those 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, resulting in elevated 5-FU concentrations in the tumor (Desmoulin F. et al. Drug Metab Dispos. 2002). Therefore, antibodies using 5-FU as a switch may be able to selectively bind to target antigens only in tumor tissue. Furthermore, molecules other than metabolic enzymes, which are generated in tumor-specific hypoxic or acidic environments, may also be used as switches. For example, TH-302 (Duan JX, et al. J Med Chem. 2008) is metabolized to Br-IPM under hypoxic conditions. Therefore, antibodies using Br-IPM as a switch may be able to selectively bind to target antigens only in tumor tissue. For example, methods for administering non-natural compounds 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 small molecule 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 Non-limiting examples of the small molecule compounds or cancer tissue-specific compounds used in the present invention, particularly cancer cell-specific metabolites, include primary metabolites produced as a result of glucose metabolism, such as lactate, succinate, and citrate, which are present at higher concentrations in cancer tissues than in surrounding non-cancerous tissues. A 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, a characteristic of solid tumors.

[0126] In other words, it is believed that tumor cells have high expression of the M2 isoform of pyruvate kinase, which is required for glycolysis under anaerobic conditions, rather than the M1 isoform, and this expression is beneficial for tumor cell growth 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 with lactate dehydrogenase (LDH) under anaerobic conditions. This feedback inhibition promotes mitochondrial respiration (Krebs cycle) and inhibits cell proliferation, so upregulation of LDH, hexokinase, and glucose transporters (GLUT) is said to play an important role in tumor cell proliferation (Fantin et al. (Cancer Cell (2006) 9, 425-434)). Glucose is metabolized via glycolysis, and its final metabolic product, lactic acid, is co-transported with protons around the tumor, resulting in an acidic pH in the tissue surrounding the tumor. Lactic acid, the final product of glycolysis, and succinic acid and citric acid, which are produced by enhanced mitochondrial respiration, are known to accumulate in cancer tissue (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). Non-limiting examples of low-molecular-weight compounds, cancer tissue-specific compounds, and particularly cancer cell-specific metabolites used in the present invention include primary metabolites produced by glycolytic metabolism, such as lactic acid, succinic acid, and citric acid. It is also known that succinic acid, which is present at high concentrations within cells, leaks out of the cells upon cell death (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is thought that succinic acid concentrations are elevated 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, tumor cells, which require a continuous supply of essential and non-essential amino acids necessary for the biosynthesis of biopolymers under anaerobic conditions, are also known to exhibit altered amino acid metabolism. Glutamine, the most widely distributed amino acid in the body, acts as a nitrogen carrier containing two nitrogen atoms in its side chain. 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 characteristic 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 plasma glutamine levels but increased glutamate concentrations (Droge et al., Immunobiology (1987) 174, 473-479). Furthermore, in lung cancer tissue, 13 by 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 compounds and cancer tissue-specific compounds used in the present invention include alanine, glutamic acid, aspartic acid, and the like, which accumulate to high concentrations in cancer tissues through glutaminolysis or the like.

[0128] (3) Metabolites 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)). 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 these cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). IDO is also expressed in myeloid-derived suppressor cells (MDSCs) in cancer tissue, and these 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)). This suppression allows tumor cells to evade antitumor immune responses, and it has been proposed that glioma cell proliferation is promoted through an autocrine growth mechanism in which kynurenine acts as an endogenous ligand for the aryl hydrocarbon receptor (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, 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 small molecule compounds, cancer tissue-specific compounds, and particularly cancer cell-specific metabolites used in the present invention preferably include kynurenine and its metabolites, such as amino acid metabolites such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid.

[0133] [ka] kynurenic acid

[0134] (4) Arachidonic acid metabolites such as prostaglandin E2 Prostaglandin E2 (PGE2) (represented by the formula below) is a metabolite of arachidonic acid called a prostonoid, which includes 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 cyclooxygenases is altered in many cancer cells. Specifically, COX-1 is constitutively expressed in almost all tissues, whereas COX-2 is primarily found to be induced in tumors by certain inflammatory cytokines and oncogenes (Warner and Mitchell, supra). 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)). Furthermore, suppressor T cells infiltrating cancer tissue also produce prostaglandin E2 (Curr. Med. Chem. (2011) 18, 5217-5223). Low-molecular-weight compounds such as prostaglandins and leukotrienes, which are metabolites of arachidonic acid, are known to act as stimulatory factors that control the autocrine and / or paracrine growth of cancer (Nat. Rev. Cancer (2012) 12 (11)). 782-792). Suitable examples of the low molecular weight compounds, cancer tissue-specific compounds, and particularly cancer cell-specific metabolites and immune cell-specific metabolites infiltrating cancer tissues used in the present invention include, but are not limited to, arachidonic acid metabolites such as prostaglandin E2. In addition to prostaglandin E2, thromboxane A2 (TXA2) is also produced at elevated levels in cancer tissues such as colon cancer (J. Lab. Clin. Med. (1993) 122, 518-523), and is suitable as a non-limiting example of the arachidonic acid metabolite of the present invention.

[0135] [ka] Prostaglandin E2 (PGE2)

[0136] Furthermore, it is 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). Suitable examples of the low molecular weight compounds, inflammatory tissue-specific compounds, particularly inflammatory cell-specific metabolites and immune cell-specific metabolites that infiltrate into inflamed tissues used in the present invention include, but are not limited to, arachidonic acid metabolites 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 in normal tissue (PLoS One. (2008) 3, e2599). Multiple 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. Increased adenosine activity has been found in 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 the dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick and Willis (Biochem. J. (1989) 261, 541-550)).Furthermore, regulatory T cells infiltrating 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 via adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011), 18, 5217-23). ​​Non-limiting examples of low-molecular-weight compounds and cancer-tissue-specific compounds used in the present invention include ATP, ADP, AMP, adenosine, and the like, which accumulate at high concentrations in cancer tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in the accumulation of high concentrations 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). Furthermore, it is 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 concentrations are elevated 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). Elevated ATP concentrations have also been observed in fibrotic tissues of patients with pulmonary fibrosis (Am. J. Respir. Crit. Care Med. (2010) 182, 774-783). Suitable low-molecular-weight compounds and inflammatory tissue-specific compounds used in the present invention include, but are not limited to, ATP, ADP, AMP, adenosine, and the like, which accumulate at high concentrations in inflamed tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in the accumulation of high concentrations of inosine.

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

[0140] In recent years, it has also 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 at high concentrations in inflamed tissues due to the metabolism of purine nucleotides such as ATP.

[0141] (7) 1-Methylnicotinamide The enzyme nicotinamide N-methyltransferase is known to be overexpressed 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 overexpression 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 extracellularly by 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 is 1-methylnicotinamide, which accumulates at high concentrations in cancer tissues through the metabolism of nicotinamide. Small molecular weight compounds according to the present disclosure can interact with antigen-binding molecules. The amino acid residues in the antigen-binding molecule that can interact with small molecular weight compounds may be present in the antigen-binding domain of 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 small molecular weight compounds is exemplified by, but is not limited to, the antigen-binding domain.

[0142] Antigen-binding domain that specifically binds to an antigen As used herein, the term "antigen-binding domain that specifically binds to an antigen" refers to an antigen-binding domain that is specific for a particular epitope among multiple epitopes contained in a given antigen. Furthermore, when the epitopes bound by an antigen-binding domain are contained in multiple different antigens, an 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 refers to the binding activity of a specific binding molecule for a molecule other than the partner molecule being 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity for the partner molecule. When an antigen-binding domain is one whose antigen-binding activity changes depending on MTA or a small molecule other than MTA, the binding of the antigen-binding domain to the antigen is measured under conditions under which the antigen-binding activity of the antigen-binding domain is high (e.g., at a specific concentration of MTA or in the absence of MTA, at a specific concentration of a small molecule other than MTA, or in the absence of a small molecule 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 noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when the members of a binding pair reflect a monovalent 1:1 interaction, the binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of molecule X to its partner Y can generally be expressed as the 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 target tissue-specific compound concentration 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, e.g. 10 -9 M~10 -13 is the dissociation constant (KD) of the ATPase (M).

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

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

[0147] In one embodiment, antibody binding activity can be measured using, for example, the Octet RED96e system or Octet RED 384 system (Pall ForteBio), which employs Bio-Layer Interferometry (BLI) as the measurement principle. By using the system according to the supplier's instructions, qualitative analysis of binding characteristics and kinetic analysis of antigen-antibody reactions can be performed. In one non-limiting embodiment of a specific measurement method, an 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 changes in the amount of antibody-antigen binding. For example, when measuring the amount of binding between an antibody and an antigen in the presence of MTA, the binding reaction between the antibody and the analyte can be measured in a buffer containing 3000 nM 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, or 100 μM as the binding phase. The dissociation reaction between the antibody and the analyte can be measured by using the same buffer as the binding phase but without the analyte as the dissociation phase. Furthermore, by using a buffer containing the same concentration of analyte as the binding phase but without MTA as the dissociation phase, it is possible to observe over time whether the antibody-antigen binding in the presence of MTA reversibly dissociates in the absence of MTA.

[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-type molecule. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare) or a flow cytometer.

[0149] When measuring the antigen-binding activity of a test antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes in a low molecular weight compound-dependent manner, the interaction between the test antigen-binding molecule and the antigen can be measured in the presence or absence of a specific concentration of the low molecular weight compound. If an antigen-binding molecule that does not exhibit antigen-binding activity in the absence of a specific low-molecular-weight compound exhibits antigen-binding activity 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, if an antigen-binding molecule does not exhibit antigen-binding activity in the presence of a specific low-molecular-weight compound but exhibits antigen-binding activity in the absence of the low-molecular-weight compound, the binding activity can be evaluated in the absence of the low-molecular-weight compound. If the antigen-binding activity in the presence of a low concentration of a specific low-molecular-weight compound is higher than that 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, if the antigen-binding activity in the presence of a high concentration of a specific low-molecular-weight compound is lower than that 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 include, but are not limited to, ion concentration, ion composition, and temperature. Furthermore, it is of course possible to evaluate binding activity under conditions in which a plurality of different factors are combined.

[0150] epitope An epitope, which means an antigenic determinant present in an antigen, refers to 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 assessing the presence or absence of binding of the antigen-binding molecule.

[0151] An epitope can be defined by its structure. Alternatively, the 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 also be specified by the amino acid residues that constitute the epitope. Furthermore, when the epitope is a sugar chain, the epitope can also 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, typically comprising 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 comprising 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 the antibody that defines the epitope). Conformational epitopes may encompass 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, 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 the epitope is called the paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between them. The binding strength between this epitope and paratope is called affinity. The sum of the binding strengths when multiple antigens bind to multiple antigen-binding molecules is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding strengths act 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; however, methods for confirming epitope binding by test antigen-binding molecules containing antigen-binding domains for antigens 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 IL-6R antigen-binding domain recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region of IL-6R cDNA encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to the test antigen-binding molecule containing the IL-6R antigen-binding domain is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using an immobilized linear peptide as the 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 of binding of the antigen-binding molecule to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

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

[0158] Methods for measuring the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward 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). Specifically, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) 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 toward 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 toward IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule toward IL-6R-expressing cells to be compared.

[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 using a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade 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 trade names of Beckman Coulter)

[0161] For example, one suitable method for measuring the antigen-binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain is as follows: First, the test antigen-binding molecule is reacted with cells expressing IL-6R and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the desired concentration. For example, the antigen-binding molecule can 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 binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, the Geometric Mean value allows the binding activity of the test antigen-binding molecule, represented by the amount of binding of the test antigen-binding molecule, to be measured.

[0162] Antibodies that bind to the same epitope Whether a test antigen-binding molecule containing an IL-6R antigen-binding domain 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 can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0163] Specifically, in a 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 test antigen-binding molecule bound to 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 test antigen-binding molecules bound to the wells via the IL-6R protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0165] If a competitor 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 competitor antigen-binding molecule, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competitor 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 IL-6R antigen-binding domain binds has been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0167] For example, such binding activity can be measured by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. Alternatively, binding activity to the mutant peptide bound to a column can be measured by flowing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0168] Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with a suitable buffer to the desired concentration and used. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled 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). In other words, by obtaining the Geometric Mean value, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured.

[0169] In this method, "substantially no binding to mutant IL-6R-expressing cells" can be determined, for example, by the following method. First, test and control antigen-binding molecules bound to mutant IL-6R-expressing cells are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, 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 Formula 1 below.

[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) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant IL-6R-expressing cells, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. In this case, it is particularly preferred that the test antigen-binding molecules used to determine 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] A test antigen-binding molecule is deemed to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding molecule for the mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding molecule for the 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). When the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be determined to be identical.

[0173] When assessing whether test antigen-binding molecules containing antigen-binding domains whose antigen-binding activity changes in a small molecule-dependent manner compete with each other / bind to the same epitope, 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 the small molecule compound, and it is preferable to use the same concentration of the small molecule compound for all test antigen-binding molecules.

[0174] Antigen-binding domain whose antigen-binding activity changes depending on the small molecule compound As used herein, an "antigen-binding domain whose antigen-binding activity changes in a small molecular weight compound-dependent manner" refers to 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 In the present disclosure, an antigen-binding domain 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. Furthermore, the antigens to which these antigen-binding domains bind are antigens expressed in diseased tissues, more preferably antigens expressed in cancer tissues, and even more preferably antigens expressed 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 an 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 antigen-binding activity in the absence of MTA to that 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 greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of KD(in the absence of MTA) / KD(in the presence of MTA) is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be produced within the skills of a person skilled in the art. If 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 MTA is stronger than that in the absence of MTA includes an antigen-binding domain that does not substantially bind to an antigen in the absence of MTA.

[0178] As long as the antigen-binding activity of an antigen-binding domain of the present disclosure, which has 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 dissociation constant (KD) for the antigen in the presence of MTA to the KD in its absence, i.e., KD(in the presence of MTA) / KD(in the absence of MTA), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit 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, 1,000, or 10,000, as long as it can be produced by those skilled in the art. If no antigen-binding activity is observed in the presence of MTA, this upper limit becomes infinite. When the antigen-binding activity of an antigen-binding domain is weaker in the presence of MTA than in the absence of MTA, this includes an antigen-binding domain that does not substantially bind to an antigen in the presence of MTA.

[0179] Furthermore, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as another indicator of the ratio between the antigen-binding activity of an antigen-binding domain of the present disclosure (or an antigen-binding molecule comprising the domain) in the absence of MTA and the antigen-binding activity in the presence of MTA. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of the binding activity ratio, the ratio of kd (dissociation rate constant) for an antigen in the absence of MTA to kd (dissociation rate constant) in the presence of MTA, kd (in the absence of MTA) / kd (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. There are no particular upper limits to the value of Kd (in the absence of MTA) / kd (in the presence of MTA), and the value may be any value, such as 50, 100, or 200, as long as it is within the common technical knowledge of a person skilled in the art. In the absence of MTA, if no binding activity to the antigen 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, a condition in which 100 μM MTA is present can be considered to be the condition in the presence of MTA. Furthermore, as a non-limiting embodiment of the MTA concentration described as "in the presence of MTA" in the present disclosure, the concentrations exemplified below as thresholds for distinguishing between low and high concentrations of MTA 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 low concentration of MTA than in the presence of a high 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 that in the presence of a high concentration of MTA is not particularly limited. However, the ratio of the dissociation constant (KD) 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 preferably 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (in the presence of a low concentration of MTA) / KD (in the presence of a high concentration of MTA) value is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be achieved by those skilled in the art. If no antigen-binding activity is observed in the presence of a low concentration of MTA, this upper limit becomes an infinite value. An antigen-binding domain 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 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 an antigen-binding domain of the present disclosure, which exhibits weaker antigen-binding activity in the presence of a high concentration of MTA than 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 dissociation constant (KD) 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 greater, more preferably 10 or greater, and even more preferably 40 or greater. There is no particular upper limit to the value of KD(in the presence of a high concentration of MTA) / KD(in the presence of a low concentration of MTA), and any value, such as 400, 1,000, or 10,000, may be used as long as it is within the skill of a person skilled in the art. If 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 is weaker 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 high concentration of MTA.

[0184] Furthermore, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as another indicator of the ratio of antigen-binding activity of an antigen-binding domain of the present disclosure (or an antigen-binding molecule comprising said domain) in the presence of a low concentration of MTA to that in the presence of a high concentration of MTA. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of binding activity ratio, the ratio of the kd (dissociation rate constant) for an antigen in the presence of a low concentration of MTA to the kd (dissociation rate constant) 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 preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There are no particular upper limits to the value of Kd (in the presence of a low concentration of MTA) / kd (in the presence of a high concentration of MTA), and the value may be any value, such as 50, 100, or 200, as long as it can be produced within the technical common sense of a person skilled in the art. In the presence of low concentrations of MTA, if no binding activity to the antigen is observed, no dissociation occurs, and therefore this upper limit becomes infinite.

[0185] In addition to the method of measuring the KD value described above, methods for evaluating relative binding activity can also be used to evaluate binding activity. Non-limiting examples of such methods include commonly known techniques using a flow cytometer, ELISA, capillary electrophoresis, or liquid chromatography, but are not limited to these. Furthermore, even if the KD value cannot be directly calculated, for example, by comparing an antigen-binding molecule whose KD value has been calculated using Biacore with a reference molecule, it is possible to evaluate the relative binding activity of a test molecule, i.e., whether the binding activity is stronger or weaker than that of 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 can 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 can 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 100 times, at least 10 times, or at least 10 times the respective threshold. 3 times at least 10 4 times at least 10 5 times at least 10 6 The value can be appropriately set from 1 / 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 includes 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 share adenosine as a common skeleton in their molecules and are compounds similar to MTA. It is generally considered difficult to obtain an antigen-binding molecule of the present disclosure whose antigen-binding activity is substantially unaffected by the presence of these similar molecules and whose binding activity changes solely in an MTA-dependent manner. However, in the present disclosure, as exemplified in various Examples described below, a library was constructed to obtain antibodies that bind to an antibody in an MTA-dependent manner, and by screening such a library, it was possible to successfully obtain antigen-binding domains with excellent MTA-dependent specificity that bind to an antigen in an MTA-specific manner but do not exhibit antigen-binding dependent on low molecular weight compounds with a structure similar to MTA. As used herein, the phrase "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 times 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 altered 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 altered 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 an antigen-binding domain of the present disclosure whose antigen-binding activity is altered 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 antigen-binding activity changes depending on MTA, also changes depending on adenosine.Without being bound by any particular theory, it can be understood that the antigen-binding activity of the antigen-binding domain, whose binding activity changes depending on both MTA and adenosine molecules, changes by interacting with a common structure in both MTA and adenosine molecules.In this case, even if the concentration of MTA is reduced in the solvent used to evaluate 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 MTA concentration, the binding activity may be maintained or may be detected as being strong.Furthermore, even if the concentration of MTA is increased in the solvent used to evaluate binding activity, if the concentration of adenosine decreases beyond the degree of increase in binding activity due to the increase in MTA concentration, the binding activity may be maintained or may be detected as being 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 changes 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 all 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 an 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 of the present disclosure whose antigen-binding activity changes in an MTA-dependent manner 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 at least one amino acid selected from the group of amino acids listed 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 located 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 position 34 of the heavy chain; M located at heavy chain position 35; C located at heavy chain position 35a; C located at position 50 of the heavy chain; 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 located 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 located 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 heavy chain position 52f; 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 located at position 54 of the heavy chain; G located at position 55 of the heavy chain; S located at heavy chain position 56; T located at heavy chain position 57; Y located at heavy chain position 58; Y located at heavy chain position 59; A located at heavy chain position 60; S located at heavy chain position 61; W located at heavy chain position 62; A located at heavy chain position 63; K located at position 64 of the heavy chain; G located at position 65 of the heavy chain; G located at position 95 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 96 of the heavy chain; G located 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 heavy chain position 100; 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 located 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 located at position 100c of the heavy chain; E located at heavy chain position 101; L located at heavy chain position 102; Q located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; E located 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 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 position 33 of the light chain; S located at position 34 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 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 located 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 located 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 located 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 located 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 position 94 of the light chain; N located at light chain position 95; I located at light chain position 95a; P located 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 may include an antigen-binding molecule comprising 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 position 34 of the heavy chain; M located at heavy chain position 35; C located at heavy chain position 35a; C located at position 50 of the heavy chain; 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 position 53 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 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 heavy chain position 58; Y located at heavy chain position 59; A located at heavy chain position 60; S located at heavy chain position 61; W located at heavy chain position 62; V located at heavy chain position 63; N located at heavy chain position 64; G located at position 65 of the heavy chain; E located at position 95 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 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 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; S located at position 100 of the heavy chain; 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 located at heavy chain position 101; L located at heavy chain position 102; H located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; K located 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 position 33 of the light chain; A at position 34 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 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 located 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 position 54 of the light chain; A at position 55 of the light chain; S located at light chain position 56; Q located at position 89 of the light chain; G at position 90 of the light chain; T 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; 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 located 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 may include antigen-binding molecules comprising 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 position 29 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 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 position 32 of the heavy chain; 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 located at position 35 of the heavy chain; either D, I, or V at position 50 of the heavy chain; I located at heavy chain position 51; G located 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 located at heavy chain position 62; V located at heavy chain position 63; K located at position 64 of the heavy chain; A, F, or G at heavy chain position 65; G located at position 95 of the heavy chain; 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 located at position 24 of the light chain; S located at position 25 of the light chain; S located at position 26 of the light chain; Q located 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 located at position 30 of the light chain; 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 position 33 of the light chain; S located at position 34 of the light chain; D at position 50 of the light chain; A located at light chain position 51; S located at light chain position 52; T at light chain position 53; L located at position 54 of the light chain; A at position 55 of the light chain; S located at light chain position 56; H located 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 located at light chain position 96; A or G at position 97 of the light chain; A, F, I, L, or V at position 98 of the light chain.

[0194] The antigen-binding domain of the present disclosure may have amino acid residues that interact with MTA. The amino acid residues that interact with MTA may be present at the interface in the antigen-binding domain that directly interacts with the antigen, or may be present elsewhere. The "interface that directly interacts with the antigen" refers to the site where the antigen and antigen-binding molecule are in close proximity to each other in the structure of the complex between the antigen and the antigen-binding domain analyzed by techniques such as crystal structure analysis. The distance at which the antigen and antigen-binding molecule are in close proximity to each other is, for example, 4 to 6 Å. However, in the structure of the complex between the antigen and antigen-binding molecule, "the proximity of the antigen and antigen-binding molecule" refers to sites in the complex that are relatively close to each other 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 when the antigen-binding molecule is bound to the antigen and when the antigen is absent. 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 in which 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 in which 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 structural analysis using NMR, or amino acid mutagenesis.

[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 by 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 in the side chain or main chain of an amino acid forming the antigen-binding molecule and an atom of a small molecule 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 the antigen-binding molecule comprises 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 three-dimensional structure of a CDR loop or the like in the conformation that is required for MTA binding, or a state that satisfies both of these. The "state in which intermolecular interactions are formed" herein can be determined, for example, from crystal structure analysis of a complex between an MTA and an antigen-binding molecule, based on the interatomic distance between a non-hydrogen atom constituting the side chain or main chain of an amino acid constituting the antigen-binding molecule and a non-hydrogen atom constituting the MTA. For example, the interatomic distance is preferably, but not limited to, 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. 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 determined based 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 determined from the effect of introducing mutations into amino acid residues, such as Ala or Gly, on the activity of the small molecule compound, but is not limited to this. Regarding the "state of indirect influence" in this specification, whether or not the binding of MTA is indirectly influenced can be estimated by, for example, 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 between MTA and the antigen-binding molecule. More precisely, however, 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 by crystal structure analysis of a complex of an MTA, an antigen, and an antigen-binding molecule. Here, "interacting with MTA" refers to a state in which an atom in the side chain or main chain of an amino acid forming the antigen-binding molecule and an atom of a small molecule 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 the antigen-binding molecule comprises 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 three-dimensional structure of a CDR loop or the like to the conformation that MTA binds to in the presence of an antigen, or a state that satisfies both of these. The "state in which intermolecular interactions are formed" herein can be determined, for example, from crystal structure analysis of a complex of MTA, antigen, and antigen-binding molecule, based on the interatomic distance between non-hydrogen atoms constituting the side chains or main chains of amino acids that form the antigen-binding molecule in the presence of antigen and non-hydrogen atoms constituting MTA. For example, the interatomic distance is preferably, but not limited to, 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. 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 determined based 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 determined from the effect of introducing mutations into amino acid residues, such as Ala or Gly, on the activity of the small molecule compound, but is not limited to this. Regarding the "state of indirect influence" in this specification, whether or not the binding of MTA in the presence of an antigen is indirectly influenced can be estimated, for example, 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. More precisely, however, it can be determined from the effect on MTA activity of introducing a mutation into an amino acid residue, such as changing it to Ala or Gly.

[0199] In one 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 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. In one non-limiting embodiment, 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).

[0200] Another non-limiting example of an antigen-binding domain is an antibody variable region, in which the amino acid residues that interact with MTA are 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 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 comprising 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 example of an antigen-binding domain that interacts with MTA is an antigen-binding domain in which 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 heavy chain amino acid positions 33, 50, 52, 54, 56, 57, 58, 99, 100, 100a, 91, 95c, and 96, as specified by Kabat numbering, within the amino acid sequence of the antibody variable region. Furthermore, the antigen-binding domain is an antibody variable region, and the antibody variable region can be, for example, an antigen-binding domain comprising 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 molecules containing antigen-binding domains 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 comprised 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), and 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 is a chimeric polypeptide comprising 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. For example, a fusion protein can comprise a polypeptide comprising an amino acid sequence encoding at least a portion of an Fc region (e.g., a portion of the Fc region that confers binding to an Fcγ receptor and / or a portion of the Fc region that confers binding to FcRn). The amino acid sequences can reside in separate proteins that are carried together in the fusion protein, or they can normally reside in the same protein but are rearranged in a new arrangement in the fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis or by recombinant techniques in which a polynucleotide encoding the peptide regions in the desired relationship is created and expressed.

[0204] The domains in the antigen-binding molecules of the present disclosure can be linked directly by a polypeptide bond or via a linker. The linker can be any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (e.g., a linker disclosed in Holliger et al. (Protein Engineering (1996) 9 (3), 299-305)). In the present disclosure, however, 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. 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), with 15 amino acids being particularly preferred.

[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·Gly (SEQ ID NO: 26) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 21))n (Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 22))n [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 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), and the like. These crosslinkers are commercially available.

[0207] When multiple linkers are used to link each domain, all of the linkers may be of the same type, or different types of linkers may also 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. The ability to bind to each other via hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or combinations of these bonds may also be suitably utilized. For example, the affinity between antibody CH1 and CL may be utilized, or an Fc region derived from the aforementioned bispecific antibody may be used to associate heterologous Fc regions. Furthermore, disulfide bonds formed between domains may also be suitably utilized.

[0208] To link each domain via a peptide bond, polynucleotides encoding the domains are linked in-frame. Methods for linking polynucleotides in-frame include restriction fragment ligation, fusion PCR, and overlap PCR, and these methods can be used alone or in combination as appropriate to produce the antigen-binding molecules of the present disclosure. In the present disclosure, the terms "linked," "fused," "linked," and "fused" are used interchangeably. These terms refer to the linking of two or more elements or components, such as polypeptides, to form a single structure by any means, including the above-mentioned chemical conjugation or recombinant techniques. "In-frame fusion," when two or more elements or components are polypeptides, refers to the linking of two or more open reading frames to form a continuous, longer open reading frame while maintaining the correct reading frame of the polypeptide. When two Fab molecules are used as the antigen-binding domain, an antibody, which 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 with high plasma retention. In one aspect, the antigen-binding activity of the antigen-binding molecule increases as the MTA concentration increases. In certain embodiments, 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 any particular theory, the above-mentioned changes in plasma kinetics can be interpreted as follows: As the MTA concentration-dependent antigen-binding activity of the antigen-binding molecule increases, 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 decrease in antigen-dependent elimination (clearance) in most tissues in the body (tissues other than the target tissue) leads to high plasma retention of the antigen-binding molecule overall. Whether an antigen-binding molecule of the present invention has high plasma retention 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 higher plasma retention than 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 that depends on the MTA concentration. In certain embodiments, an antigen-binding molecule that does not have antigen-binding activity that depends on the compound concentration refers to an antigen-binding molecule whose antigen-binding activity in the presence and absence of MTA differs, for example, by 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 a comparative perspective, it is desirable that the antigen-binding activity of the antigen-binding molecule of the present disclosure and the control antigen-binding molecule in the presence of a sufficient amount of MTA is substantially equal to each other.

[0210] Here, the magnitude of antigen-dependent elimination of antigen-binding molecules detected in vivo is thought to vary depending on the quantitative balance between antigens and antigen-binding molecules present in plasma. Generally, the more antigens / fewer antigen-binding molecules present in plasma, the easier it is to detect antigen-dependent elimination of antigen-binding molecules; conversely, the less antigens / more antigen-binding molecules present in plasma, the harder it is to detect antigen-dependent elimination of antigen-binding molecules. The antigen-binding molecules of the present disclosure do not need to exhibit high plasma retention under all conditions; they only need to exhibit high plasma retention under appropriate conditions that allow sufficient antigen-dependent elimination to 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 with low plasma antigen accumulation capacity. In a further aspect, the antigen-binding activity of the antigen-binding molecule increases as the MTA concentration increases. In a specific 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 any particular theory, the above-mentioned changes in plasma kinetics can be interpreted as follows: As the MTA concentration-dependent antigen-binding activity of an antigen-binding molecule increases, 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 antigen-antibody complexes 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, antigen clearance decreases and the antigen concentration in plasma increases (antigen accumulation). A decrease in the ability to form antigen-antibody complexes in most tissues in the body (tissues other than target tissues) leads to overall low antigen accumulation (in other words, low antigen accumulation capacity of the antigen-binding molecule). Whether an antigen-binding molecule of the present invention has low plasma antigen accumulation capacity 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 lower plasma antigen accumulation capacity than the control antigen-binding molecule. In one embodiment, the control antigen-binding molecule does not have antigen-binding activity that depends on the MTA concentration. In a specific embodiment, an antigen-binding molecule that does not have antigen-binding activity that depends on the MTA concentration refers to an antigen-binding molecule whose antigen-binding activity in the presence and absence of the compound differs, for example, by 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 is substantially equal to each other in the presence of a sufficient amount of a compound.

[0212] Here, the amount of antigen-antibody complexes formed in vivo is thought to depend on the amounts of antigens and antibodies present in plasma. Generally, it is thought that the amount of antigen-antibody complexes formed increases as the amount of antigen and antibody in plasma increases, and conversely, the amount of antigen-antibody complexes formed decreases as the amount of antigen and antibody in plasma decreases. The antigen-binding molecules of the present invention do not need to exhibit low plasma antigen accumulation capacity under all conditions, but only need to exhibit low plasma antigen accumulation capacity under appropriate conditions under which sufficient antigen-antibody complexes are formed. When the amount of antigen in plasma is low, the amount of antigen should be increased by some artificial means before evaluating the plasma antigen accumulation capacity. Good too.

[0213] Fcγ receptor (FcγR) Fcγ receptors (also referred to as FcγR) refer to receptors capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and refer to any member of a family of proteins substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131, i.e., FcγRIIa (H) and FcγRIIa (R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIIIa (including allotypes V158 and F158, i.e., FcγRIIIa (V) and FcγRIIIa (V)). These include, but are not limited to, FcγRIII (CD16), including FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms or allotypes. Suitable 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 shown in SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIa are shown in SEQ ID NOs: 15 (BC033678.1) and 16 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are shown in SEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (database accession numbers such as RefSeq are shown in parentheses). Whether 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 formats, as well as by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and 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 composed of an α chain that binds to the Fc region of IgG and a common γ chain with ITAMs that transduce 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 cytoplasmic domain. These receptors are expressed on many immune cells, including macrophages, mast cells, and antigen-presenting cells. Binding of these receptors to the Fc region of IgG transmits activation signals that promote the phagocytic activity of macrophages, the production of inflammatory cytokines, mast cell degranulation, and enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are referred to herein as activating Fcγ receptors.

[0215] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which 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 suppression of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic activity and the ability to produce inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are referred to herein as inhibitory Fcγ receptors.

[0216] Binding activity of the Fc region to FcγR As described above, examples of Fc regions contained in antigen-binding molecules of the present disclosure include Fc regions that have binding activity to Fcγ receptors. Non-limiting examples of such Fc regions include the Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), and IgG4 (SEQ ID NO: 8). Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the FACS and ELISA formats described above, as well as ALPHA screens (Amplified Luminescent Proximity Homogeneous Assays) and BIACORE methods utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0217] The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. If the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

[0218] For example, donor beads are bound to antigen-binding molecules containing biotin-labeled Fc regions, and acceptor beads are bound to Fcγ receptors tagged with glutathione S-transferase (GST). In the absence of competing antigen-binding molecules containing Fc region variants, antigen-binding molecules with native Fc regions interact with Fcγ receptors, generating a signal at 520-620 nm. Antigen-binding molecules containing untagged Fc region variants compete with the interaction between antigen-binding molecules with native Fc regions and Fcγ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar is known. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in frame in an operably linked vector in cells, and purifying the gene using a glutathione column. The resulting signals are suitably analyzed by fitting them 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) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is passed over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of shift (i.e., 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 (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and affinity (KD) can be calculated from the ratio of these constants. Inhibition assays are also suitable for use with 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 engineered Fc region 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), Fc regions that have altered FcγR binding and higher Fcγ receptor-binding activity than that of the Fc region of native human IgG may also be used as appropriate. As used herein, "Fc region of native human IgG" refers to an Fc region in which the sugar chain attached 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 Fc regions with altered FcγR binding can be prepared by modifying the amino acids of the Fc region of native human IgG. Whether the FcγR-binding activity of an Fc region with altered FcγR binding is higher than that of the Fc region of native human IgG can be determined appropriately using the method described above in the section on binding activity.

[0221] In the present disclosure, "amino acid modification" or "amino acid modification" of an Fc region includes modifying the amino acid sequence to a different amino acid sequence from that 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 human Fcγ receptors in the neutral pH range. Furthermore, Fc regions that have been further modified using an already modified Fc region can also be suitably used as the Fc region of the present disclosure. The starting Fc region can refer to the polypeptide itself, a composition containing the starting Fc region, or the amino acid sequence encoding the starting Fc region. The starting Fc region can include known Fc regions produced by recombinant means, as outlined in the antibody section. The source of the starting Fc region can be obtained from any non-human animal organism or human. Preferably, the organism is selected from the group consisting of 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 IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting Fc region. Similarly, as used herein, 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 known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, International Publication Nos. WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0222] Examples of modifications include one or more mutations, such as substitution of amino acid residues different from those of the starting Fc region, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the starting Fc region. Preferably, the amino acid sequence of the modified Fc region comprises at least a portion of a non-naturally occurring Fc region. Such variants necessarily have less than 100% sequence identity or similarity with the starting Fc region. In a preferred embodiment, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100%, and most preferably about 95% to less than 100%, to the amino acid sequence of the starting Fc region. In one non-limiting embodiment of the present disclosure, there is at least one amino acid difference between the starting Fc region and the FcγR-binding modified Fc region of the present disclosure. The amino acid differences between the starting Fc region and the altered FcγR binding Fc regions of the present disclosure can also be preferably identified by the differences in specific amino acids at the amino acid residue positions specified by the EU numbering system described above. Methods for creating such variants are exemplified in the section "Amino acid modifications."

[0223] The antigen-binding molecules of the present disclosure include FcγR-binding modified Fc regions (FcγR-binding modified Fc regions) that have higher Fcγ receptor-binding activity than that of native human IgG Fc regions and can be obtained by any method, specifically by modifying the amino acids of a human IgG immunoglobulin used as the starting Fc region. Preferred IgG immunoglobulin Fc regions for modification include, for exampl...

Claims

1. i) The binding activity to the antigen changes depending on the concentration of 5'-Methylthioadenosine (MTA), and ii) has no binding activity to the antigen in the absence of MTA and in the presence of 100 μM adenosine; an antigen-binding molecule comprising an antigen-binding domain, The antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); The antigen is a molecule other than MTA. Antigen-binding molecules.

2. The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule is an antibody.

3. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding molecule is a bispecific antibody.

4. The antigen-binding molecule according to claim 1 , wherein the antigen is a membrane-type molecule and is expressed in cancer tissue.

5. The antigen-binding molecule of claim 3 , wherein the bispecific antibody comprises an antigen-binding domain that binds to CD3 and an antigen-binding domain that binds to an antigen expressed on a cancer cell.

6. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 5.

7. A method for producing the antigen-binding molecule according to any one of claims 1 to 5, comprising: Providing a vector to which a polynucleotide encoding the antigen-binding molecule according to any one of claims 1 to 5 is operably linked; Providing a cell into which the vector has been introduced; Culturing the cells; and recovering the antigen-binding molecule from the culture medium of the cells; A method comprising:

Images