Antigen-binding molecules containing antigen-binding domains whose binding activity to antigens changes in an MTA-dependent manner, and libraries for obtaining such antigen-binding domains.

Abstract

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

Description

[Technical Field]

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

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

[0003] To date, antibody drugs such as rituxan for the CD20 antigen, cetuximab for the EGFR antigen, and herceptin for the HER2 antigen have been approved as cancer treatments (Non-Patent Literature 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC, etc. It is known that this cytotoxic activity by ADCC, etc. depends on the number of antigens expressed on the target cells of the therapeutic antibody (Non-Patent Literature 4), so a high expression level of the target antigen is preferable from the viewpoint 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 tissue, cytotoxic activity by ADCC, etc. will be exerted against normal cells as well, which poses a major problem in terms of side effects. Therefore, it is preferable that the antigen targeted by therapeutic antibodies as cancer treatments is specifically expressed on cancer cells.

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

[0005] Antibody molecules that exhibit such potent cytotoxic activity can exert cytotoxic activity even against cancer cells that do not express the antigen in large quantities, but they also exert similar cytotoxic activity against normal tissues that do not express the antigen in large quantities. In fact, compared to cetuximab, a natural human IgG1 against the EGFR antigen, EGFR-BiTE, a bispecific antibody against CD3 and EGFR, can exert potent cytotoxic activity against cancer cells and exert an antitumor effect by recruiting T cells to cancer cells. On the other hand, since EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE is administered to cynomolgus monkeys (Non-Patent Literature 9). Furthermore, bivatuzumab mertansine, an ADC in which mertansine is conjugated to an antibody against CD44v6, which is highly expressed in cancer cells, has been shown to cause serious skin and hepatotoxicity in clinical practice because CD44v6 is also expressed in normal tissues (Non-Patent Literature 10).

[0006] When using antibodies that can exert potent cytotoxic activity even against cancer cells with low antigen expression, the target antigen needs to be expressed in a highly cancer-specific manner. However, as evidenced by the fact that HER2, the target antigen of Herceptin, and EGFR, the target antigen of cetuximab, are also expressed in normal tissues, the number of cancer antigens that are expressed in a highly cancer-specific manner is thought to be limited. Therefore, while it is possible to enhance cytotoxic activity against cancer, side effects due to cytotoxicity in normal tissues may become a problem.

[0007] Recently, iprimumab, which enhances tumor immunity by inhibiting CTLA4, a component that contributes to immunosuppression in cancer, has been shown to prolong overall survival in metastatic melanoma (Non-Patent Literature 11). However, because iprimumab inhibits CTLA4 systemically, while tumor immunity is enhanced, it is problematic that it can cause serious autoimmune disease-like side effects due to systemic immune activation (Non-Patent Literature 12).

[0008] Various technologies have been developed that can be applied to second-generation antibody drugs, and technologies that improve effector function, antigen binding ability, pharmacokinetics, and stability, or reduce immunogenicity risk have been reported (Non-Patent Literature 13). However, there have been very few reports of technologies that enable antibody drugs to act specifically on diseased tissue to solve the above-mentioned side effects. For example, for lesion sites such as cancerous tissue and inflammatory tissue, pH-dependent antibodies that utilize the fact that the pH in these diseased tissues is acidic have been reported (Patent Literature 1, 2). However, the decrease in pH (i.e., increase in hydrogen ion concentration) in cancerous tissue and inflammatory tissue compared to normal tissue is slight, and it is difficult to produce antibodies that act by detecting a slight increase in hydrogen ion concentration, which has an extremely small molecular weight. At the same time, the pH may also be acidic in normal tissue such as osteoclast bone resorption areas and tissues other than the target lesion, so it was thought that there are still many challenges in utilizing pH conditions as an environmental factor specific to the lesion site. On the other hand, a method has been reported for producing antibodies that only exhibit antigen-binding activity when cleaved by proteases expressed in lesion sites such as cancerous tissue and inflammatory tissue (Patent Literature 3). However, since antibody cleavage by proteases is irreversible, a problem arises because antibodies cleaved at the lesion site can return to normal tissue via the bloodstream and bind to antigens in normal tissue as well. Furthermore, the cancer specificity of such proteases is also considered a problem. To overcome these challenges, antigen-binding molecules have been reported in which the binding activity to antigens changes depending on the concentration of disease tissue-specific compounds (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

[0011] This disclosure aims to discover new small molecule compounds that are specifically present or produced in diseased tissues, to provide antigen-binding molecules (small molecule compound switch antigen-binding molecules) whose binding to target antigens is controlled in a manner dependent on such small molecule compounds, 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 inventors diligently conducted research to achieve the above objectives and discovered methylthioadenosine (MTA) as a low-molecular-weight compound specific to cancer tissue. Furthermore, they created an antigen-binding molecule containing an antigen-binding domain whose binding activity to the antigen changes in an MTA-dependent manner. The inventors also found that this antigen-binding molecule or a pharmaceutical composition containing this antigen-binding molecule is useful for cancer treatment, as well as useful in cancer treatment involving the administration of this antigen-binding molecule, and useful in the manufacture of pharmaceuticals for cancer treatment.

[0013] The inventors have also created a method for screening and producing antigen-binding domains whose binding activity to antigens changes in an MTA-dependent manner. Furthermore, the inventors have succeeded in creating a library that can efficiently screen antigen-binding domains whose binding activity to antigens changes in an MTA-dependent manner. The inventors have further succeeded in creating a library that can screen antigen-binding domains whose binding activity to antigens changes in an MTA and / or other small molecule compound-dependent manner, and have also created a method for screening and producing the aforementioned antigen-binding domains whose binding activity to antigens changes in an MTA and / or other small molecule compound-dependent manner. Furthermore, the inventors have also succeeded in obtaining an antigen-binding molecule that specifically binds to MTA itself.

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

[47] , wherein the effector cell is a T cell. [A49] The antigen-binding molecule described in [A48], wherein the antigen expressed in the effector cells is a T cell receptor (TCR) complex. [A50] An antigen-binding molecule according to either [A48] or [A49], wherein the antigen expressed in the effector cells is CD3. [A51] An antigen-binding molecule described in any one of [A48] to [A50] that activates effector cells to induce cytotoxic activity against target cells. [A52] An antigen-binding molecule having TDCC activity, as described in any one of [A48] to [A51]. An 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 toward the antigen. [A54] An antigen-binding molecule according to 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. A pharmaceutical composition containing an antigen-binding molecule described in any one of [A1] to [A54] [A55]. [A56] A pharmaceutical composition for the treatment of cancer containing an antigen-binding molecule as an active ingredient, as described in any one of [A1] to [A55]. [A57] The pharmaceutical composition according to [A56], wherein the cancer is a cancer in which MTA is accumulated in the tissue. [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 deficient or has reduced expression, or has a mutation or splicing variant that reduces enzyme activity. [A59] The cancer is a cancer in which the activity of MTAP is deficient or reduced, the pharmaceutical composition according to any one of [A56] to [A58]. A method for producing an antigen-binding molecule described in any one of [A1] to [A54] [A60]. [A61] A polynucleotide encoding an antigen-binding molecule as described in any one of [A1] through [A52]. [A62] [A61] Vectors containing polynucleotides Cells containing the vectors described in [A63] and [A62]. Antigen-binding molecules recovered from the culture supernatant after culturing the cells described in [A64] and [A63]. [G1] An antigen-binding molecule according to any one of [A1] to [A54], having high plasma retention and / or low plasma antigen accumulation ability compared to a control antigen-binding molecule that does not bind to MTA in a concentration-dependent manner. A pharmaceutical preparation comprising the antigen-binding molecule described in [G2] [G1] and a pharmaceutically acceptable carrier. [G3] A method for producing an antigen-binding molecule having high plasma retention and / or low plasma antigen accumulation capacity compared to a control antigen-binding molecule, comprising: (a) producing an antigen-binding molecule whose antigen-binding activity increases as the concentration of MTA increases; and (b) measuring the plasma retention and / or plasma antigen accumulation capacity of the antigen-binding molecule produced in (a).

[0015] This disclosure also includes the embodiments described below as illustrative examples. [B1] A library comprising nucleic acids encoding antigen-binding molecules containing multiple antigen-binding domains having different sequences from each other, and / or antigen-binding molecules containing multiple antigen-binding domains having different sequences from each other, wherein the library mainly consists of antigen-binding molecules containing antigen-binding domains having amino acid residues that interact with MTA, and / or nucleic acids encoding the said antigen-binding molecules. [B2] The library according to [B1], wherein the antigen-binding domain is an antibody variable region. [B3] A library according to [B2] comprising: a plurality of antibody variable region variants having amino acids different from those located at one or more amino acid sites in an unmodified antibody variable region having binding activity to MTA, and having different sequences from each other; and / or a nucleic acid encoding a plurality of antibody variable region variants having amino acids different from those located at one or more amino acid sites in an unmodified antibody variable region having binding activity to MTA, and having different sequences from each other. [B4] The amino acid sites in the modified antibody variable region that have different amino acids from those in the unmodified antibody variable region are one or more amino acid sites selected from the group of amino acid sites listed below, as described in [B3]: 1) The amino acid site in the unmodified antibody variable region that does not participate in binding to MTA, 2) Amino acid sites that do not significantly reduce the binding of the antibody variable region variant to the 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 the antigen. [B5] The amino acid sites in the modified antibody variable region that have different amino acids from those in the unmodified antibody variable region are one or more amino acid sites selected from the group of amino acid sites listed below, as described in [B3]: 1) The amino acid site in the unmodified antibody variable region that does not participate in binding to MTA, 2) Amino acid sites that do not significantly reduce the binding of the antibody variable region variant to MTA compared to the antibody variable region variant, 3) The amino acid site corresponding to the amino acid site exposed on the surface of the unmodified antibody variable region, and 4) The amino acid site corresponding to the amino acid site located in the region where the structural change rate is large when MTA is bound or unbound in the unmodified antibody variable region. [B6] The unmodified antibody variable region is substantially incompatible with adenosine and / or S-(5'-Adenosyl)-L-homocysteine ​​(SAH). The library according to any one of [B3] to [B5]. [B7] The unmodified antibody variable region is one of the following, from any one of the libraries listed in [B3] to [B6]: a) The antibody variable region, which includes the heavy chain variable region shown in SEQ ID NO: 46 and the light chain variable region shown in SEQ ID NO: 47; b) The antibody variable region, which includes the heavy chain variable region indicated by SEQ ID NO: 50 and the light chain variable region indicated by SEQ ID NO: 51; c) Antibody variable region including the heavy chain variable region shown in SEQ ID NO: 48 and the light chain variable region shown in SEQ ID NO: 49; d) The antibody variable region, which includes the heavy chain variable region shown in SEQ ID NO: 52 and the light chain variable region shown in SEQ ID NO: 53. [B8] The aforementioned multiple antibody variable regions are as follows: H-CDR1 containing XAXWMC (Sequence ID: 65); H-CDR2 containing CIFAXXXYXXSGGSTYYASWAKG (Sequence ID: 66); H-CDR3 containing GXGXXXGXXDEL (Sequence ID: 67); L-CDR1 containing QSSEXVXXXXLS (Sequence ID: 68); L-CDR2 containing XAXTXPX (SEQ ID NO: 69); and L-CDR3 containing AGLYXGNIPA (SEQ ID NO: 70); The antibody variable region includes X, where X refers to any amino acid, and X at different positions does not have to be the same type of amino acid; see the library described in [B2]. [B9] The aforementioned multiple antibody variable regions are as follows: H-CDR1 containing XAXWMC (Sequence ID: 65); H-CDR2 containing CIFAX1XXYXXSGGSTYYASWAKG (Sequence ID: 71); H-CDR3 containing GXGXXXGXXDEL (Sequence ID: 67); L-CDR1 containing QSSEXVXXX1XLS (Sequence ID: 72); L-CDR2 containing XAXTXPX (SEQ ID NO: 69); and L-CDR3 containing AGLYXGNIPA (SEQ ID NO: 70); This is an antibody variable region that includes, X is any amino acid. 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 located at different positions do not have to be the same type of amino acid. (See the library in [B2].) [B10] The aforementioned multiple antibody variable regions are as follows: H-CDR1 containing XXAXWMC (Sequence ID: 73); H-CDR2 containing CIFAX1XXYXXSGGSTYYASWAKG (Sequence ID: 71); H-CDR3 containing GXGXXXGXXDEL (Sequence ID: 67); L-CDR1 containing QSSEXVXXX1XLS (Sequence ID: 72); L-CDR2 containing XAXTXPX (SEQ ID NO: 69); and L-CDR3 containing AGLYXGNIPA (SEQ ID NO: 70); This is an antibody variable region that includes, X is any amino acid. 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 located at different positions do not have to be the same type of amino acid. (See the library in [B2].) [B11] The aforementioned multiple antibody variable regions are as follows: H-CDR1 containing XXXWMC (Sequence ID: 74); H-CDR2 containing CIXSXXXXTXYASWVNG (Sequence ID: 75); H-CDR3 containing EXXXXSGALNL (Sequence ID: 76); L-CDR1 containing HSSKXVXXXXXLA (Sequence ID: 77); L-CDR2 containing XAXXLAS (Sequence ID: 78); and L-CDR3 containing QGTYXXXXFYFA (Sequence ID: 79); The antibody variable region includes X, where X refers to any amino acid, and X at different positions does not have to be the same type of amino acid; see the library described in [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 described in [B12], in which the unmodified antibody variable region also has binding activity to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, and / or ATP. [B14] The library described in any one of [B3] to [B5] or [B12], wherein the unmodified antibody variable region is an antibody variable region having a heavy chain variable region indicated by SEQ ID NO: 31 and a light chain variable region indicated by SEQ ID NO: 32. [B15] The aforementioned multiple antibody variable regions are as follows: H-CDR1 containing X2X3X4X5G (SEQ ID NO: 80); X6IGX7X8X9X 10 X 11 WX 12 PX 13 WVKX 14 (SEQ ID NO: 81) containing H-CDR2; GX 15 X 16 X 17 X 18 X 19 X 20 NAX 21 (SEQ ID NO: 82) containing H-CDR3; QSSQSVX 22 X 23 NNX 24 (SEQ ID NO: 83) containing L-CDR1; (SEQ ID NO: 84) containing DASTLAS L-CDR2; and HGX 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 DNX 33 (SEQ ID NO: 85) containing L-CDR3; An antibody variable region containing, 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 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y. X 11 The amino acids are selected from A, D, E, G, H, I, K, L, N, P, Q, R, S, T, and V. X 12 These are amino acids selected from A, D, E, F, G, H, I, K, L, Q, R, S, T, V, W, and Y. X 13 These are amino acids selected from A, F, Q, R, S, T, V, W, and Y. X 14 These are amino acids selected from A, F, and G. X 15 These are amino acids selected from A, E, F, G, H, K, L, Q, R, S, T, W, and Y. X 16 These are amino acids selected from F, H, K, N, W, and Y. X 17 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, and Y. X 18 These are amino acids selected from A, D, E, G, H, Q, and S. X 19 These are amino acids selected from F and Y. X 20 These are amino acids selected from N, T, and V. X 21 These are amino acids selected from F and W. X 22 These are amino acids selected from A, E, F, H, I, K, L, N, R, S, T, V, W, and Y. X 23 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, and Y. X 24 These are amino acids selected from A, E, F, G, H, S, and Y. X 25 These are amino acids selected from A, S, and T. X 26These are amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y. X 27 These are amino acids selected from A, D, E, F, G, H, L, N, Q, R, S, T, V, and Y. X 28 These are amino acids selected from A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, and Y. X 29 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y. X 30 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, V, W, and Y. X 31 These are amino acids selected from A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, and Y. X 32 These are amino acids selected from A, F, H, I, K, L, N, P, Q, R, S, T, V, W, and Y. X 33 The amino acids are selected from A and G, as described in the library in [B2]. [B16] A library enriched with nucleic acids encoding antigen-binding molecules containing antigen-binding domains that bind to MTA, from the libraries described in [B15]. [B17] The library described in [B16] wherein the binding of the antigen-binding domain to the MTA is binding to the MTA in the absence of the antigen. [B18] The concentration is carried out in the following steps (1) to (2): (1) A step of contacting an antigen-binding domain displayed from the library described in [B15] with the MTA, and (2) A step of selecting the antigen-binding domain that has been bound to the MTA in step (1), The libraries listed in [B16] or [B17], including the following. [B19] A library enriched with nucleic acids encoding antigen-binding domains that bind to adenosine, derived from the libraries described in [B15]. [B20] The library described in [B19] wherein the binding of the antigen-binding domain to adenosine is in the absence of the antigen. [B21] The concentration is carried out in the following steps (1) to (2): (1) A step of contacting an antigen-binding domain displayed from the library described in [B15] with adenosine, and (2) A step of selecting the antigen-binding domain that has been bound to adenosine in step (1), The libraries listed in [B19] or [B20] include the following.

[0016] This disclosure also includes the embodiments described below as illustrative examples. [C1] The following steps (a) and (b): (a) A step of 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) Amino acid sites located in regions with a large rate of structural change when comparing the structure when the antigen-binding domain is bound to the MTA with the structure when it is not bound to the MTA; (iii) Amino acid sites not involved in binding with MTA; (iv) Amino acid sites that do not significantly reduce binding to MTA; (v) an amino acid site with diverse amino acid occurrence 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) A step of designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain, each having an amino acid modification at one or more amino acid sites identified in step (a), and having different sequences from each other. A method for manufacturing a library that includes [this library]. [C2] The amino acid modification in step (b) satisfies at least one of the following conditions (1) to (3): (1) When comparing the structure of an antigen-binding domain variant having the amino acid modification when bound to an MTA with the structure when not bound to an MTA, the structural change rate at the amino acid site where the modified amino acid is located is large; (2) When comparing the structure of an antigen-binding domain variant having the amino acid modification when bound to an MTA with the structure when not bound to an MTA, the presence of the modified amino acid does not inhibit the structural change of the antigen-binding domain variant; (3) The antigen-binding domain modified product having the amino acid modification does not exhibit significantly reduced binding activity to MTA compared to the unmodified antigen-binding domain product; The manufacturing method described in [C1]. [C3] The method for producing an antigen-binding domain having binding activity to the MTA substantially does not bind to adenosine, as described in any one of [C1] to [C2]. [C4] The antigen-binding domain having binding activity to the MTA does not bind to (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, and / or ATP. [C3] The method for producing the antigen-binding domain according to the present invention. [C5] A library manufactured by one of the manufacturing methods described in [C1] through [C4]. [C6] The following steps (a) and (b): (a) A step of identifying an amino acid site in an antigen-binding domain having binding activity to a small molecule compound that satisfies at least one of the following conditions (i) or (ii): (i) an amino acid site exposed on the surface of the antigen-binding domain; (ii) Amino acid sites located in regions with a large rate of structural change when comparing the structure when the antigen-binding domain is bound to the low molecular weight compound with the structure when it is not bound to the low molecular weight compound; (b) A step of designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain, each having an amino acid modification at one or more amino acid sites identified in step (a), and having different sequences from each other. A method for manufacturing a library that includes [this library]. [C7] The amino acid modification in step (b) satisfies at least one of the following (1) to (3): (1) When comparing the structure of an antigen-binding domain variant having the amino acid modification when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound, the structural change rate of the amino acid site where the modified amino acid is located is large; (2) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound, the presence of the modified amino acid does not inhibit the structural change of the antigen-binding domain variant; (3) The antigen-binding domain modified product having the amino acid modification does not exhibit significantly reduced binding activity to the low-molecular-weight compound compared to the unmodified antigen-binding domain product; The manufacturing method described in [C5]. [C8] The following steps (a) and (b): (a) A step of identifying an amino acid site in an antigen-binding domain that interacts with a small molecule compound, satisfying at least one of the following conditions (i) to (iv): (i) an amino acid site exposed on the surface of the antigen-binding domain; (ii) Amino acid sites located in regions with a large rate of structural change when comparing the structure when the antigen-binding domain is bound to the low molecular weight compound with the structure when it is not bound to the low molecular weight compound; (iii) Amino acid sites that are not involved in binding with the low molecular weight compound; (iv) Amino acid sites that do not significantly weaken the binding to the low molecular weight compound; (v) an amino acid site with diverse amino acid occurrence 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) A step of designing a library comprising nucleic acids encoding an unmodified antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain, each having a different sequence from the others, and each variant having an amino acid modification satisfying at least one of the following (1) or (2) at one or more amino acid sites identified in step (a): (1) When comparing the structure of an antigen-binding domain variant having the amino acid modification when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound, the structural change rate of the amino acid site where the modified amino acid is located is large; (2) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to the low molecular weight compound with the structure when not bound to the low molecular weight compound, the presence of the modified amino acid does not inhibit the structural change of the antigen-binding domain variant; A method for manufacturing a library that includes [this library]. [C9] The low molecular weight compound is selected from at least one selected from adenosine, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and S-(5'-Adenosyl)-L-homocysteine ​​(SAH), according to any one of [C6] to [C8]. A library manufactured by the manufacturing method described in any one of the following: [C10] [C6] through [C8].

[0017] This disclosure also includes the embodiments described below as illustrative examples. [D1] A method for screening antigen-binding molecules that contain an antigen-binding domain whose binding activity to the antigen changes in an MTA-dependent manner. [D2] The method according to [D1], comprising comparing the antigen-binding activity of the antigen-binding domain in the presence of a first concentration of MTA with the antigen-binding activity in the presence of a different concentration (second concentration) of MTA. [D3] The method according to [D1] or [D2], comprising the step of selecting an antigen-binding molecule containing an antigen-binding domain that has different binding activity to the antigen in the presence of a first concentration of MTA and a binding activity to the antigen in the presence of a second concentration of MTA. [D4] The method according to any one of [D2] to [D3], comprising the step of selecting an antigen-binding molecule that includes an antigen-binding domain whose binding activity to the antigen in the presence of the first concentration of MTA is higher than the binding activity to the antigen in the presence of the second concentration of MTA. [D5] The method according to any one of [D2] to [D3], comprising the step of selecting an antigen-binding molecule that includes an antigen-binding domain whose binding activity to the antigen in the presence of the first concentration of MTA is lower than its binding activity to the antigen in the presence of the second concentration of MTA. [D6] The following steps (a) to (c): (a) A step of contacting an antigen-binding molecule containing an antigen-binding domain with an antigen in the presence of a first concentration of MTA, (b) A step of 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) A step of isolating an antigen-binding molecule containing the antigen-binding domain dissociated in step (b), The method described in [D1], including the method described in [D1]. [D7] The following steps (a) to (d); (a) A step of contacting an antigen-binding molecule containing an antigen-binding domain with an antigen in the presence of a first concentration of MTA, (b) A step to confirm that the antigen-binding molecule containing the antigen-binding domain has bound to the antigen in step (a), (c) A step of placing an antigen-binding molecule containing the antigen-binding domain bound to the antigen in the presence of a second concentration of MTA, and (d) A step of isolating an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity is weaker than the criterion confirmed in step (c) that it was bound to the antigen in step (b), The method described in [D1], including the method described in [D1]. [D8] The method according to [D6] or [D7], which confirms that the antigen-binding domain can bind to the MTA before performing step (a). [D9] The following steps (a) to (d): (a) A step of bringing an antigen-binding molecule containing an antigen-binding domain into contact with an antigen in the presence of a first concentration of MTA, (b) A step of confirming that the antigen-binding molecule containing the antigen-binding domain in step (a) does not bind to the antigen, (c) A step of binding an antigen-binding molecule containing an antigen-binding domain that does not bind to the antigen to the antigen in the presence of a second concentration of MTA, and (d) A step of isolating an antigen-binding molecule containing the antigen-binding domain bound to the antigen in step (c), The method described in [D1], including the method described in [D1]. [D10] The following steps (a) to (c): (a) A step of contacting an antigen with a library on which antigen-binding molecules containing antigen-binding domains are displayed in the presence of a first concentration of MTA. (b) A step of 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) A step of isolating an antigen-binding molecule containing the antigen-binding domain dissociated in step (b), The method described in [D1], including the method described in [D1]. [D11] The following steps (a) to (d); (a) A step of contacting an antigen with a library on which antigen-binding molecules containing antigen-binding domains are displayed in the presence of a first concentration of MTA. (b) A step of selecting an antigen-binding molecule that includes an antigen-binding domain bound to an antigen in step (a), (c) A step of placing an antigen-binding molecule containing the antigen-binding domain selected in step (b) in the presence of a second concentration of MTA, and (d) A step of isolating an antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity is weaker than the criterion selected in step (b) above, The method described in [D1], including the method described in [D1]. [D12] Before step (a) above, the following steps (1) to (2): (1) A step of contacting a library on which antigen-binding molecules containing antigen-binding domains are displayed with MTA, and (2) A step of selecting an antigen-binding molecule containing an antigen-binding domain bound to the MTA in step (1), The method according to [D10] or [D11], wherein the library that is contacted with the antigen in the presence of a first concentration of MTA in step (a) is a library on which antigen-binding molecules containing antigen-binding domains selected in steps (1) and (2) are displayed. [D13] The following steps (a) to (d): (a) A step of contacting an antigen with a library on which antigen-binding molecules containing antigen-binding domains are displayed in the presence of a first concentration of MTA, (b) A step of selecting an antigen-binding molecule that includes an antigen-binding domain that does not bind to the antigen in step (a), (c) A step of binding an antigen-binding molecule containing the antigen-binding domain selected in step (b) to an antigen in the presence of a second concentration of MTA, and (d) A step of isolating an antigen-binding molecule containing the antigen-binding domain bound to the antigen in step (c), The method described in [D1], including the method described in [D1]. [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 binding activity of the antigen-binding domain to an antigen in the presence of MTA with the binding activity to an antigen in the absence of MTA. [D16] The method according to [D1] or [D15], comprising the step of selecting an antigen-binding domain that has different binding activity to the antigen in the presence of MTA and in the absence of MTA. [D17] The method according to [D1] or [D15], comprising the step of selecting an antigen-binding domain whose binding activity to the antigen in the presence of MTA is higher than that of the antigen in the absence of MTA. [D18] The following steps (a) to (c): (a) A step of contacting an antigen-binding molecule containing an antigen-binding domain with an antigen in the presence of MTA, (b) A step of placing the antigen-binding molecule containing the antigen-binding domain bound in step (a) in the absence of MTA, and (c) A step of isolating an antigen-binding molecule containing the antigen-binding domain dissociated in step (b), The method described in [D1], including the method described in [D1]. [D19] The following steps (a) to (d); (a) A step of contacting an antigen-binding molecule containing an antigen-binding domain with an antigen in the presence of MTA, (b) A step to confirm that the antigen-binding molecule containing the antigen-binding domain has bound to the antigen in step (a), (c) A step of placing the antigen-binding molecule containing the antigen-binding domain bound to the antigen in the absence of MTA, and (d) A step of isolating an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity is weaker than the criterion confirmed in step (c) that it was bound to the antigen in step (b), The method described in [D1], including the method described in [D1]. [D20] The method according to [D18] or [D19], wherein, before performing step (a), the antigen-binding molecule containing the antigen-binding domain is capable of binding to the MTA. [D21] The following steps (a) to (d): (a) A step of bringing an antigen-binding molecule containing an antigen-binding domain into contact with an antigen in the presence of MTA, (b) A step of confirming that the antigen-binding molecule containing the antigen-binding domain in step (a) does not bind to the antigen, (c) A step of binding an antigen-binding molecule containing an antigen-binding domain that does not bind to the antigen to the antigen in the absence of MTA, and (d) A step of isolating an antigen-binding molecule containing the antigen-binding domain bound to the antigen in step (c), The method described in [D1], including the method described in [D1]. [D22] The following steps (a) to (c): (a) A step of contacting an antigen with a library on which antigen-binding molecules containing antigen-binding domains are displayed, in the presence of MTA. (b) A step of placing the antigen-binding molecule containing the antigen-binding domain bound in step (a) in the absence of MTA, and (c) A step of isolating an antigen-binding molecule containing the antigen-binding domain dissociated in step (b), The method described in [D1], including the method described in [D1]. [D23] The following steps (a) to (d); (a) A step of contacting an antigen with a library on which antigen-binding molecules containing antigen-binding domains are displayed in the presence of MTA, (b) A step of selecting an antigen-binding molecule that includes an antigen-binding domain bound to an antigen in step (a), (c) A step of placing an antigen-binding molecule containing the antigen-binding domain selected in step (b) in the absence of MTA, and (d) A step of isolating an antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity is weaker than the criterion selected in step (b) above, The method described in [D1], including the method described in [D1]. [D24] Before step (a) above, the following steps (1) to (2): (1) A step of contacting a library on which antigen-binding molecules containing antigen-binding domains are displayed with MTA, and (2) A step of selecting an antigen-binding molecule containing an antigen-binding domain bound to the MTA in step (1), The method according to [D22] or [D23], wherein the library that is contacted with the antigen in the presence of the MTA in step (a) is a library on which antigen-binding molecules containing the antigen-binding domain selected in steps (1) and (2) are displayed. [D25] The following steps (a) to (d): (a) A step of contacting an antigen with a library on which antigen-binding molecules containing an antigen-binding domain are displayed in the presence of MTA, (b) A step of selecting an antigen-binding molecule that includes an antigen-binding domain that does not bind to the antigen in step (a), (c) A step of binding an antigen-binding molecule containing the antigen-binding domain selected in step (b) to an antigen in the absence of MTA, and (d) A step of isolating an antigen-binding molecule containing the antigen-binding domain bound to the antigen in step (c), The method described in [D1], including the method described in [D1]. [D26] The method according to any one of [D10] to [D14] or [D22] to [D25], wherein the library displaying the antigen-binding molecule containing the antigen-binding domain is a naive human antibody display library, or a synthetic human antibody display library, or the library described in any one of [B1] to [B14], or the library described in [C5]. [D27] The method according to any one of [D1] to [D26], wherein the antigen-binding domain is an antibody variable region or a monodomain antibody.

[0018] This disclosure also includes the embodiments described below as illustrative examples. [F1] A method for producing an antigen-binding molecule containing an antigen-binding domain whose binding activity to the antigen changes in an MTA-dependent manner. [F2] The method according to [F1], comprising comparing the binding activity of the antigen-binding domain to an antigen in the presence of a first concentration of MTA with the binding activity to an antigen in the presence of a different concentration (second concentration) of MTA. [F3] The method according to [F1] or [F2], comprising the step of selecting an antigen-binding domain that has different binding activity to an antigen in the presence of a first concentration of MTA and a second concentration of MTA. [F4] The method according to any one of [F2] to [F3], comprising the step of selecting an antigen-binding domain whose binding activity to the antigen in the presence of the first concentration of MTA is higher than the binding activity to the antigen in the presence of the second concentration of MTA. The method according to any one of [F2] to [F3], comprising the step of selecting an antigen-binding domain having a binding activity to an antigen in the presence of the first concentration of MTA lower than the binding activity to the antigen in the presence of the second concentration of MTA. The method according to any one of [F3] to [F5], further comprising the step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the selected antigen-binding domain is operably linked is introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the culture solution of the cell. [F7] The following steps (a) to (d): (a) A step of contacting an antigen-binding domain with an antigen in the presence of a first concentration of MTA. (b) A step of placing the antigen-binding domain bound in step (a) in the presence of a second concentration of MTA. (c) A step of isolating the antigen-binding domain dissociated in step (b), and (d) A step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (c) is operably linked is introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the culture solution of the cell. The method according to [F1], comprising the above steps. [F8] The following steps (a) to (e); (a) A step of contacting an antigen-binding domain with an antigen in the presence of a first concentration of MTA. (b) A step of confirming that the antigen-binding domain has bound to the antigen in step (a). (c) A step of placing the antigen-binding domain bound to the antigen in the presence of a second concentration of MTA. (d) A step of isolating an antigen-binding domain whose antigen-binding activity is weaker than the criterion for confirming binding to the antigen in step (b) in step (c), and (e) A step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) is operably linked is introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the culture solution of the cell. The method according to [F1], comprising the above steps. [F9] The method according to [F7] or [F8], wherein before performing the step (a), it is confirmed that the antigen-binding domain is capable of binding to MTA. [F10] The following steps (a) to (e): (a) A step of contacting an antigen-binding domain with an antigen in the presence of a first concentration of MTA, (b) A step of confirming that the antigen-binding domain does not bind to the antigen in the step (a), (c) A step of binding an antigen-binding domain that does not bind to the antigen to the antigen in the presence of a second concentration of MTA, (d) A step of isolating the antigen-binding domain bound to the antigen in the step (c), and (e) A step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in the step (d) is operably linked is introduced, and recovering an antigen-binding molecule containing the antigen-binding domain from the cell culture solution, The method according to [F1], comprising the above steps. [F11] The following steps (a) to (d): (a) A step of contacting a library in which an antigen-binding domain is displayed with an antigen in the presence of a first concentration of MTA, (b) A step of placing the antigen-binding domain bound in the step (a) in the presence of a second concentration of MTA, (c) A step of isolating the antigen-binding domain dissociated in the step (b), and (d) A step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in the step (c) is operably linked is introduced, and recovering an antigen-binding molecule containing the antigen-binding domain from the cell culture solution, The method according to [F1], comprising the above steps. [F12] The following steps (a) to (e); (a) A step of contacting a library in which an antigen-binding domain is displayed with an antigen in the presence of a first concentration of MTA, (b) A step of selecting the antigen-binding domain bound to the antigen in the step (a), (c) A step of placing the antigen-binding domain selected in step (b) in the presence of a second concentration of MTA. (d) A step of isolating antigen-binding domains in step (c) whose antigen-binding activity is weaker than the criteria selected in step (b), and (e) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [F13] Before step (a) above, the following steps (1) to (2): (1) A step of bringing a library on which antigen-binding domains are displayed into contact with MTA, and (2) A step of selecting the antigen-binding domain that has been bound to the MTA in step (1), The method according to [F11] or [F12], wherein the library that is contacted with the antigen in the presence of a first concentration of MTA in step (a) is a library on which the antigen-binding domains selected in steps (1) and (2) are displayed. [F14] The following steps (a) to (e): (a) A step of contacting an antigen with a library on which antigen-binding domains are displayed in the presence of a first concentration of MTA, (b) A step of selecting an antigen-binding domain that does not bind to the antigen in step (a), (c) A step of binding the antigen-binding domain selected in step (b) to an antigen in the presence of a second concentration of MTA. (d) A step of isolating the antigen-binding domain bound to the antigen in step (c), and (e) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [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], comprising comparing the binding activity of the antigen-binding domain to an antigen in the presence of MTA with the binding activity to an antigen in the absence of MTA. [F17] The method according to [F1] or [F16], comprising the step of selecting an antigen-binding domain that has different binding activity to the antigen in the presence of MTA and in the absence of MTA. [F18] The method according to [F1] or [F16], comprising the step of selecting an antigen-binding domain that has high binding activity to the antigen in the presence of MTA compared to the binding activity to the antigen in the absence of MTA. [F19] The method according to any one of [F16] to [F18], further comprising the step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the selected antigen-binding domain has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. [F20] The following steps (a) to (d): (a) A step of contacting the antigen-binding domain with the antigen in the presence of MTA, (b) A step of placing the antigen-binding domain bound in step (a) in the absence of MTA, (c) A step of isolating the antigen-binding domain dissociated in step (b), and (d) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (c) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [F21] The following steps (a) to (e); (a) A step of contacting the antigen-binding domain with the antigen in the presence of MTA, (b) A step to confirm that the antigen-binding domain has bound to the antigen in step (a), (c) A step of placing the antigen-binding domain bound to the antigen in the absence of MTA, (d) A step of isolating antigen-binding domains whose antigen-binding activity is weaker than the criterion confirmed in step (c) that they bound to the antigen in step (b), and (e) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [F22] The method according to [F20] or [F21], wherein the antigen-binding domain is capable of binding to the MTA before performing step (a). [F23] The following steps (a) to (e): (a) A step of bringing the antigen-binding domain into contact with the antigen in the presence of MTA, (b) A step to confirm that the antigen-binding domain does not bind to the antigen in step (a), (c) A step of binding an antigen-binding domain that does not bind to the antigen to the antigen in the absence of MTA, (d) A step of isolating the antigen-binding domain bound to the antigen in step (c), and (e) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [F24] The following steps (a) to (d): (a) A step of contacting a library on which antigen-binding domains are displayed with an antigen in the presence of MTA, (b) A step of placing the antigen-binding domain bound in step (a) in the absence of MTA, (c) A step of isolating the antigen-binding domain dissociated in step (b), and (d) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (c) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method according to [F1], comprising [F25] The following steps (a) to (e); (a) A step of contacting a library displaying an antigen-binding domain with an antigen in the presence of MTA, (b) A step of selecting an antigen-binding domain that binds to the antigen in the step (a), (c) A step of placing the antigen-binding domain selected in the step (b) in the absence of MTA, (d) A step of isolating an antigen-binding domain whose antigen-binding activity is weaker than the criterion selected in the step (b), and (e) A step of culturing a cell into which a vector in which a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in the step (d) is operably linked is introduced, and recovering an antigen-binding molecule containing the antigen-binding domain from the cell culture solution, The method according to [F1], comprising [F26] Before the step (a), the following steps (1) to (2): (1) A step of contacting a library displaying an antigen-binding domain with MTA, and (2) A step of selecting an antigen-binding domain that binds to MTA in the step (1), The method according to [F24] or [F25], comprising, wherein the library contacted with the antigen in the presence of MTA in the step (a) is a library displaying the antigen-binding domain selected in the steps (1) and (2). [F27] The following steps (a) to (e): (a) A step of contacting a library displaying an antigen-binding domain with an antigen in the presence of MTA, (b) A step of selecting an antigen-binding domain that does not bind to the antigen in the step (a), (c) A step of binding the antigen-binding domain selected in the step (b) to the antigen in the absence of MTA, (d) A step of isolating an antigen-binding domain that binds to the antigen in the step (c), and (e) A step of culturing cells into which a vector operably linked to a polynucleotide encoding an antigen-binding molecule containing the antigen-binding domain isolated in step (d) has been introduced, and recovering the antigen-binding molecule containing the antigen-binding domain from the cell culture medium. The method described in [F1], including the method described in [F1]. [F28] The method according to one of [F11] to [F15] or [F24] to [F27], wherein the library on which the antigen-binding domain is displayed is a naive human antibody display library, or a synthetic human antibody display library, or the library described in any one of [B1] to [B14], or the 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 monodomain antibody.

[0019] This disclosure also includes the embodiments described below as illustrative examples. [E1] An antigen-binding molecule that specifically binds to MTA. [E2] An antigen-binding molecule that binds to MTA, wherein the antigen-binding molecule is substantially not bound to adenosine, as described in [E1]. [E3] An antigen-binding molecule that binds to MTA, wherein the antigen-binding molecule is substantially incompatible with (5'-Adenosyl)-L-homocysteine ​​(SAH), AMP, ADP, and / or ATP, as described in [E1] or [E2]. A method for measuring MTA concentration using an antigen-binding molecule described in any one of the following: [E4] [E1] to [E3]. [E5] The measurement method described in [E4], wherein the MTA concentration is the MTA concentration in the tissue. [E6] A measurement method described in either [E4] or [E5] that measures MTA concentration using an antigen-antibody reaction. [E7] A measurement method described in any one of [E4] to [E5], using immunohistochemistry. [E8] A measurement method described in any one of [E4] to [E5], using the imaging method. [E9] A measurement method described in any one of [E4] to [E5], using in vivo imaging. [E10] A method for diagnosing a disease using an antigen-binding molecule described in any one of [E1] through [E3]. [E11] The diagnosis is the method described in [E10] for determining whether or not a disease is present, or for predicting the effectiveness of treatment for the disease. [E12] The disease is cancer, and the method according to any one of [E10] to [E11]. [E13] The method according to [E12], wherein the cancer is a cancer in which MTA is accumulated in the cancer tissue. [E14] The method according to either [E12] or [E13], wherein the cancer is a cancer tissue in which the gene encoding MTAP is deficient or has reduced expression, or has a mutation or splicing variant that reduces enzyme activity. A disease diagnostic kit containing an antigen-binding molecule described in any one of the following categories: [E15] [E1] through [E3]. [E16] The diagnosis is the kit described in [E15] for determining whether or not a disease is present, or for predicting the effectiveness of treatment for the disease. [E17] The disease is cancer, a kit as described in any one of [E15] to [E16]. [E18] The cancer is a cancer in which MTA is accumulated in the cancer tissue, as described in [E17]. [Effects of the Invention]

[0020] The antigen-binding molecule comprising an antigen-binding domain whose binding activity to an antigen changes in an MTA-dependent manner, and the pharmaceutical composition comprising the same, do not act systemically in normal tissues or blood, but act reversibly in cancer, thereby exerting therapeutic effects while avoiding side effects and enabling the treatment of cancer. Furthermore, by using the library of antigen-binding molecules contained herein, which includes multiple antigen-binding domains with different sequences and whose antigen-binding activity changes in an MTA-dependent manner, it is possible to efficiently obtain various antigen-binding molecules with MTA-dependent antigen-binding activity that are useful for treating cancer tissue-specific diseases as described above, in a short amount of time. [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the intracellular MTA concentration for 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-free cell line. [Figure 2] This figure shows the MTA concentration in the culture medium from which each cell line was cultured. The vertical axis represents the MTA concentration in the culture medium, and the horizontal axis represents the culture time, cell line name, and MTAP deficiency status. MTAP- indicates an MTAP-deficient cell line, and MTAP+ indicates an MTAP-free cell line. [Figure 3] This figure shows the MTA concentration in the culture medium when each cell line was cultured in a medium pre-supplied with MTA. The vertical axis represents the MTA concentration in the culture medium, and the horizontal axis represents the culture time. Each spot represents the measured data. The graph on the left shows the MTA concentration in the culture medium when HT-1376 cells (non-MTAP-deficient cells) were cultured, and the graph on the right shows the MTA concentration in the culture medium when SK-MES-1 cells (non-MTAP-deficient cells) were cultured. [Figure 4] This figure shows the MTA concentration in tumors of tumor-bearing mice. The vertical axis represents the MTA concentration in the tumor, and the horizontal axis represents the name of the cell line transplanted into the mouse. Each spot indicates the measured data. [Figure 5]This figure shows the relationship between the amount of MTAP DNA in human clinical samples and the tissue MTA concentration. The graph on the left shows the results of measurements taken from bladder cancer clinical samples, and the graph on the right shows the results of measurements taken from esophageal cancer clinical samples. Each spot in the graph represents a clinical sample, and the light-colored spots indicate samples where the tissue MTA concentration was below the detection limit. The vertical axis represents the tissue MTA concentration, and the horizontal axis represents ΔCt, which is the MTAP gene Ct value minus the ΨX4 gene Ct value. [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 normal liver tissue. The vertical axis represents the extracellular MTA concentration in the tissue, and the horizontal axis represents the name of the cell line transplanted into the mouse. Each spot represents the measured data, and empty spots represent data below the lower limit of MTA quantification. [Figure 7] This figure shows the binding amount of C03H-BH076N17 / C03L-KT0 to hIL-6R under different concentrations of MTA or adenosine. The vertical axis shows the amount of hIL-6R bound per unit of solid-phase antibody, and the horizontal axis shows the concentration of MTA or adenosine. [Figure 8] This figure shows the binding mechanism between SMB0002hFab and adenosine. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and adenosine is shown as a ball-and-stick model. The amino acid residues that form interactions with adenosine are shown as stick models. The dashed lines and their numerical values ​​indicate the distance between hydrogen bonds, CH-π interactions, or π-π interactions between each amino acid residue and adenosine. The unit is Å. [Figure 9] This figure shows the variable regions extracted and superimposed from two molecules, SMB0002hFab_1 and SMB0002hFab_2, which are contained within the asymmetric unit of the crystal structure of SMB0002hFab. In the figure, SMB0002hFab_1 is shown in gray and SMB0002hFab_2 is shown in black. [Figure 10]This figure shows SMB0002hFab_1, one of the two molecules in the asymmetric unit of the crystal structure of SMB0002hFab, and the adenosine and variable region extracted from the SMB0002hFab-adenosine complex (SMB0002hFab-adenosine complex), superimposed on each other. In the figure, SMB0002hFab_1 is shown in gray, and the SMB0002hFab-adenosine complex is shown in black. [Figure 11] This figure shows SMB0002hFab_2, one of the two molecules in the asymmetric unit of the crystal structure of SMB0002hFab, and the adenosine and variable region extracted from the SMB0002hFab-adenosine complex (SMB0002hFab-adenosine complex) superimposed. In the figure, SMB0002hFab_2 is shown in gray, and the SMB0002hFab-adenosine complex is shown in black. [Figure 12] This figure shows the binding amount of clones obtained after panning of heavy chain variable region phage display libraries to MTA. The vertical axis shows the absorbance obtained by phage ELISA when MTA is not immobilized, and the horizontal axis shows the absorbance obtained when MTA is immobilized. [Figure 13] This figure shows the binding amount of clones obtained after panning of a light chain variable region phage display library to MTA. The vertical axis shows the absorbance obtained by phage ELISA when MTA is not immobilized, and the horizontal axis shows the absorbance when MTA is 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 in the presence of MTA or adenosine via SPR. The vertical axis represents the binding amount (from -100 to 200 RU), and the horizontal axis represents the reaction time (from -100 to 1200 seconds, with 0 seconds being the antigen reaction initiation time). [Figure 15] This figure shows the variable region and MTA extracted from the crystal structure of the MTA0303Fab and MTA complex. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. [Figure 16]This figure shows the binding mechanism between MTA0303Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA is shown as a ball-and-stick model. The amino acid residues that form interactions with MTA are shown as stick models. The dashed lines and their numerical values ​​indicate the distances between hydrogen bonds, CH-π interactions, π-π interactions, and sulfur-π interactions between each amino acid residue and MTA. The unit is Å. [Figure 17] This figure shows the binding mechanism between MTA0303Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. The amino acid residues that form interactions with MTA are shown as stick models. The dashed lines and their numerical values ​​indicate the hydrogen bond distance between the amino acid residues and MTA. The unit is given in Å. [Figure 18] This figure shows the variable region and MTA extracted from the crystal structure of the MTA0330Fab and MTA complex. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA as a ball-and-stick model. [Figure 19] This figure shows the binding mechanism between MTA0330Fab and MTA. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and MTA is shown as a ball-and-stick model. Amino acid residues that form interactions with MTA are shown as stick models. The dashed lines and their numerical values ​​indicate the distance between hydrogen bonds, CH-π interactions, or π-π interactions between each amino acid residue and MTA. The unit is Å. [Figure 20] This is a diagram of the crystal structure of the MTA0303Fab and MTA complex. In the diagram, the heavy chain of the antibody is shown in black, the light chain in light gray, and MTA as 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 MTA0330Fab and MTA complex. In the diagram, the heavy chain of the antibody is shown in black, the light chain in light gray, and MTA as 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] This figure shows a superposition of the 1H-15N trosis spectra of MTA0303Fab in both the MTA-bound and unbound states. The spectrum of the bound state is shown in black, and the spectrum of the unbound state is shown in gray. [Figure 23] This figure shows a superposition of the 1H-13C SOFAST-HMQC spectra of MTA0303Fab in both the MTA-bound and unbound states. The bound state spectrum is shown in black, and the unbound state spectrum in gray. [Figure 24] This figure shows a superposition of the 1H-15N trosis spectra of MTA0330Fab in both the MTA-bound and unbound states. The spectrum of the bound state is shown in black, and the spectrum of the unbound state is shown in gray. [Figure 25] This figure shows a superposition of the 1H-13C SOFAST-HMQC spectra of MTA0330Fab in both the MTA-bound and unbound states. The spectrum of the bound state is shown in black, and the spectrum of the unbound state is shown in gray. [Figure 26] This figure shows that in a normal environment where small molecules are absent, small molecule switch antibodies do not bind to the antigen, but in target tissues where small molecules are present at high concentrations, they do bind to the antigen. [Figure 27] This diagram illustrates how small molecules act as switches by being sandwiched between a complex of small antibodies and an antigen. Without small molecules, the interaction between the antibody and antigen is insufficient, and the antibody cannot bind to the antigen. However, with small molecules present, the antibody can bind to the antigen by being sandwiched between the antibody and the antigen. [Figure 28] This figure shows the T cell activation ability of a bispecific antibody possessing an antigen-binding domain that binds to IL-6R in an MTA-dependent manner and an antigen-binding domain that binds to CD3, in the presence or absence of MTA or ADO, as tested using NFAT-RE-luc2-Jurkat cells. The X axis represents the antibody concentration (μg / mL), and the Y axis represents the relative luminescence (RLU). [Figure 29]This figure shows the amount of anti-IL-6R antibody that binds to hIL-6R in an MTA-dependent manner, measured using BiacoreT200, under different MTA concentrations. The vertical axis represents the amount of hIL-6R bound per unit of solid-phase antibody, and the horizontal axis represents the MTA concentration. [Figure 30] This sensorgram shows the time course of the binding amount of anti-IL-6R antibody to the antigen (hIL-6R) measured using the Octet RED384 system, which binds to IL-6R in an MTA-dependent manner. The upper and lower graphs show the measurement results with 100 μM and 10 μM MTA, respectively. [Modes for carrying out the invention]

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

[0023] Amino acid modification For modifying amino acids in the amino acid sequence of antigen-binding molecules, 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 employed. In addition, several known methods can be used to modify amino acids by substituting them with amino acids other than natural ones (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 tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is suitably used.

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

[0025] In this specification, expressions that include a number representing a specific position followed by a one-letter or three-letter code of the original and modified amino acids may be used as appropriate to represent amino acid modifications. For example, the modification N100bL or Asn100bLeu, used when making amino acid substitutions in the antibody variable region, represents the substitution of Asn at position 100b, represented by Kabat numbering, to Leu. That is, the number represents the position of the amino acid as represented by Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the original amino acid, and the one-letter or three-letter code of the amino acid listed after it represents the substituted amino acid. Similarly, the modification P238D or Pro238Asp, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238, represented by EU numbering, to Asp. In other words, the numbers represent the position of the amino acid as expressed in EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution.

[0026] antigen In this specification, the structure of an "antigen" is not limited to a specific structure, as long as it includes an epitope to which an antigen-binding domain binds. In one embodiment, an antigen is a peptide, polypeptide, or protein with four or more amino acids. Examples of antigens include molecules such as: 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, Adresin, 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-Stimulating 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, β-NGF, BOK, Bombecin, 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, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)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-activating 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-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, Glucagon, Glut4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth Hormone Releasing Factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like proliferation Factor 1, Integrin Alpha 2, Integrin Alpha 3, Integrin Alpha 4, Integrin Alpha 4 / Beta 1, Integrin Alpha 4 / Beta 7, Integrin Alpha 5 (Alpha V), Integrin Alpha 5 / Beta 1, Integrin Alpha 5 / Beta 3, Integrin Alpha 6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, polynuclear respiratory 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, Thymus 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 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-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-likereceptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP High-pressure washer CA125, high-pressure washers and wheelbarrows TW EAK, TXB2, Ung, uPAR, uPAR-1, VC AM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGF R, VEGFR-3(flt-4), VEGI, VIM, VLA, VLA-1, VLA-4, and VNR Liquid classes WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WN T7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9A、WNT9B、WNT10A、WNT10B、WNT11、WNT16、X CL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1. DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, low-density LDL, PCSK9, prekallikrein RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B tau VAP1 IL-31 IL-31R Nav1.1 Nav 1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1 Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9 factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, antioxidant factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, and factor IX、factor IXa、factor X、factor Xa、factor XI、factor XIa、factor XII、factorExamples include receptors for hormones and growth factors, as well as 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 other antigens. Antigens include those expressed on cancer cells, immune cells, stromal cells, etc., in cancer tissue. It is preferable.

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

[0028] Examples of antigens as described above include membrane-bound molecules expressed on cell membranes and soluble molecules secreted extracellularly from cells. When an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes in an MTA-dependent manner binds to a soluble molecule secreted from a cell, it is preferable that the antigen-binding molecule has neutralizing activity, as described later.

[0029] There are no limitations on the solutions in which the soluble molecule may exist; it can be present in any biological fluid, i.e., any fluid that fills the spaces between blood vessels or tissues and cells within a living organism. In one non-limiting embodiment, the soluble molecule to which the antigen-binding molecule of this disclosure binds may be present in extracellular fluid. Extracellular fluid in vertebrates refers to the general term for components of bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid, or synovial fluid, as well as cellular permeable fluids such as alveolar fluid (bronchial alveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor) (fluids in various glandular lumens resulting from the active transport and secretory activity of cells, and fluids in the gastrointestinal tract and other body cavities).

[0030] When an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes in an MTA-dependent manner, as described herein, binds to a membrane-type molecule expressed on a cell membrane, suitable examples of such antigen-binding molecules include those that have cytotoxic activity or the ability to bind or bind cytotoxic substances, as described below. In addition, an antigen-binding molecule that has neutralizing activity instead of, or in addition to, the property of having cytotoxic activity or the ability to bind or bind cytotoxic substances is also a suitable, non-limiting embodiment.

[0031] antigen-binding domain In this specification, the "antigen-binding domain" may be any domain structure as long as it binds to the target antigen. Examples of such domains include, for instance, the variable regions of the heavy and light chains of antibodies; a module called the A domain, consisting of about 35 amino acids, found in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229); Adnectin, which contains the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925); Affibody, which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A (International Publication WO1995 / 001937); and DARPins (Designed Ankyrin Repeat), which are regions exposed on the molecular surface of ankyrin repeats (ARs), which have a structure in which a turn containing 33 amino acid residues and two antiparallel helical and loop subunits are repeatedly stacked. Preferred examples include Anticalin, which consists of four loop regions supporting one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands in lipocalin molecules such as proteins (International Publication WO2002 / 020565), neutrophil gelatinase-associated lipocalin (NGAL), etc. (International Publication WO2003 / 029462), and recessed regions of parallel sheet structures within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules are repeatedly stacked, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854). Preferred examples of antigen-binding domains in this disclosure include antigen-binding domains that include 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", or "F(ab')2".

[0032] antigen binding molecule In this disclosure, the term "antigen-binding molecule" is used in its broadest sense, and specifically, it includes various molecular types as long as they contain an antigen-binding domain. An antigen-binding molecule may consist only of an antigen-binding domain, or it may contain 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 bound, 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 an existing stable α / β barrel protein structure or other three-dimensional structure is used as a scaffold (base), and only a portion of that structure is library-formed for the construction of an antigen-binding domain, may also be included in the antigen-binding molecules of this disclosure.

[0033] antibody In this specification, an antibody means an immunoglobulin that is naturally occurring or produced by partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma or serum that exist naturally, or from the culture supernatant of antibody-producing hybridoma cells, or they can be partially or completely synthesized using methods such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and subclasses of those isotypes. Nine classes (isotypes) of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Antibodies in this disclosure may include IgG1, IgG2, IgG3, and IgG4 from among these isotypes. Multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242 as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, but any of these may be used in this disclosure. In particular, the sequence of human IgG1 may be either DEL or EEM, with the amino acid sequence at positions 356-358 represented by EU numbering. Furthermore, while multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, any of these may be used in this disclosure.

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

[0035] Variable region The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH 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 particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, 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 the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs 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) Antigen contact 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), including 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 in accordance with Kabat et al.

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

[0038] Fc area The Fc region contains an amino acid sequence derived from the constant region of the antibody heavy chain. The Fc region is the portion of the antibody heavy chain constant region that includes the hinge, CH2, and CH3 domains, starting from the N-terminus of the hinge region of the papain cleavage site at approximately the 216th amino acid in EU numbering. The Fc region can be obtained from human IgG1, but is not limited to a specific subclass of IgG. A preferred example of such an Fc region is one that has binding activity to FcRn in the acidic pH range, as described later. Another preferred example of such an Fc region is one that has binding activity to the Fcγ receptor, as described later. An example of such an Fc region is the Fc region represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).

[0039] Low molecular weight antibody The antibodies used in this disclosure are not limited to full-length antibody molecules, but may also be low-molecular-weight antibodies or modified versions thereof. Low-molecular-weight antibodies include antibody fragments in which a portion of a full-length antibody (e.g., whole antibody such as whole IgG) is missing, and are not particularly limited as long as they have binding activity to the antigen. The low-molecular-weight antibodies of this disclosure are not particularly limited as long as they are a portion of a full-length antibody, but preferably include a heavy chain variable region (VH) and / or a light chain variable region (VL). The amino acid sequence of VH or VL may be substituted, deleted, added and / or inserted. Furthermore, a portion of VH and / or VL may be deleted as long as it has binding activity to the antigen. The variable regions may also be chimeric or humanized. Specific examples of antibody fragments include, for example, Fab, Fab', F(ab')2, Fv, etc. Furthermore, specific examples of low-molecular-weight antibodies include, for example, Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), Diabody, and sc(Fv)2 (single chain (Fv)2). Multimers of these antibodies (e.g., dimers, trimers, tetramers, polymers) are also included in the low-molecular-weight antibodies of this disclosure.

[0040] Antibody fragments can be produced by treating antibodies with enzymes such as papain and pepsin, or by constructing genes encoding these antibody fragments, introducing them into expression vectors, and then expressing them in suitable host cells (e.g., 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, See 132-137).

[0041] A diabody refers to a bivalent, low-molecular-weight antibody constructed by gene fusion (Holliger et al. (Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993), European Publication EP404097, and PCT Publication WO1993 / 011161, etc.). A diabody is a dimer composed of two polypeptide chains, and typically, each polypeptide chain is linked by a linker that is too short, for example, about 5 residues, to allow the VL and VH to bind to each other within the same chain. Because the linker between the VL and VH encoded on the same polypeptide chain is short, they cannot form a single-chain variable region fragment and instead form a dimer, resulting in a diabody having two antigen-binding sites.

[0042] The scFv is obtained by linking the H chain variable region and the L chain variable region of an antibody. In this scFv, the H chain variable region and the L chain variable region are linked via a linker, preferably a peptide linker (Huston et al. (Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883)). The H chain variable region and the L chain variable region in the scFv may be derived from any antibody described herein. There are no particular restrictions on the peptide linker used to link the variable regions, but for example, any single-chain peptide consisting of about 3 to 25 residues, or the peptide linkers described later, can be used. The PCR method described above can be used as a method for linking the variable regions. The DNA sequence encoding the H chain or H chain variable region of the antibody, and the DNA sequence encoding the L chain or L chain variable region, either all or a portion of the DNA sequence encoding a desired amino acid sequence, are used as templates, and sequences corresponding to the sequences at both ends thereof are used. The DNA encoding scFv can be amplified by PCR using a pair of primers. Then, by performing a PCR reaction with a pair of primers, one containing DNA encoding the peptide linker portion and the other having sequences designed so that both ends are linked to the H chain and L chain respectively, DNA with the desired sequence can be obtained. Furthermore, once the DNA encoding scFv is produced, an expression vector containing it and recombinant cells transformed with the expression vector can be obtained according to conventional methods. The resulting recombinant cells can then be cultured to express the DNA encoding the scFv, thereby obtaining the scFv itself.

[0043] sc(Fv)2 is a low-molecular-weight antibody formed by linking two VH molecules and two VL molecules with a linker or the like to create a single chain (Hudson et al. (J. Immunol. Methods (1999) 231, 177-189)). sc(Fv)2 can be produced, for example, by linking scFv molecules with a linker.

[0044] Furthermore, antibodies are preferred in which two VHs and two VLs are arranged in the order VH, VL, VH, VL ([VH]linker[VL]linker[VH]linker[VL]) with the N-terminus of the single-chain polypeptide as the starting point. The order of the two VHs and two VLs is not limited to the above configuration and they may be arranged in any order. For example, the following configuration can also be given. -[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] As the linker for binding the variable region of the antibody, a linker similar to the linker described in the section on antigen-binding molecules above may be used. For example, particularly preferred embodiments of sc(Fv)2 in this 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 binding four antibody variable regions, typically three linkers are required, but the same linker may be used for all of them, or different linkers may be used.

[0046] To obtain such low-molecular-weight antibodies, antibodies can be treated with enzymes, such as papain or pepsin, to generate antibody fragments, or DNA encoding these antibody fragments or low-molecular-weight antibodies can be constructed, introduced into an expression vector, and then expressed in a suitable host cell (e.g., 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, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0047] Furthermore, one non-limiting aspect of the antibodies in this disclosure is a chimeric antigen receptor, which is formed by incorporating a fusion of an antibody or fragment thereof that recognizes an antigen in place of a T cell receptor and the signaling domain of a T cell into a T cell, as well as T cells into which the chimeric antigen receptor is incorporated. However, the disclosure is not limited to these examples.

[0048] Single-domain antibody One preferred example of the antigen-binding domain of the present invention is a single-domain antibody (sdAb).

[0049] In this specification, the term "monodomain antibody" is used without regard to its structure, as long as the domain alone can exhibit antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL domains, monodomain antibodies are known to exhibit antigen-binding activity solely through their own domain structure, without pairing with other domains. Monodomain antibodies usually have a relatively low molecular weight and exist in monomeric form.

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

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

[0052] Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. 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 created 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) is one example.

[0053] Method for producing antibodies having desired binding activity against an antigen. Methods for producing antibodies that have desired binding activity against antigens that are molecules different from small molecule compounds, and that are independent of small molecule compounds including MTA, are known to those skilled in the art. The following is an example of a method for producing antibodies that bind to IL-6R (anti-IL-6R antibodies). Antibodies that bind to antigens other than IL-6R can also be produced as appropriate in accordance with the following examples.

[0054] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known means. Mammalian-derived monoclonal antibodies 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 using genetic engineering techniques. The monoclonal antibodies of the present invention include "humanized antibodies" and "chimeric antibodies".

[0055] Monoclonal antibody-producing hybridomas can be produced, for example, by using known techniques as follows: Mammals are immunized according to a standard immunization method using the IL-6R protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells by a standard cell fusion method. Next, hybridomas that produce anti-IL-6R antibodies can be selected by screening monoclonal antibody-producing cells using a standard screening method.

[0056] Specifically, the production of monoclonal antibodies is carried out as follows: First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody acquisition, can be obtained by expressing the IL-6R gene whose nucleotide sequence is disclosed in SEQ ID NO: 2. That is, a suitable host cell is transformed by inserting the 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 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. Alternatively, 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 against 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. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. Furthermore, it can be obtained by degrading the IL-6R protein using a proteolytic enzyme, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited to any special form. 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. Preferably, the number of amino acids constituting the peptide to be used as a sensitizing antigen is at least 5, 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] Furthermore, fusion proteins obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as sensitization antigens. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitization antigens. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in-frame, and then inserting the fusion gene into an expression vector as described above. The method for producing fusion proteins is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). Methods for obtaining IL-6R used as a sensitization antigen and immunization methods using it are specifically described in international publications WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0059] While the mammals immunized with the sensitizing antigen are not limited to specific animals, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred.

[0060] The animals described above are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to mammals by injection intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen, diluted to an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified. After emulsification, the sensitizing antigen is administered to mammals several times every 4 to 21 days. A suitable carrier may 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] Furthermore, hybridomas that produce the desired antibody can also be produced 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, and the sensitized antigen is expressed in the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages. -Membrane proteins such as IL-6R can maintain their structure and provide immune stimulation. - There is no need to purify immune antigens.

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

[0063] After the mammal is immunized in this manner and an increase in antibody titers binding to IL-6R in the serum is confirmed, immune cells are collected from the mammal and used for cell fusion. Splenocytes, in particular, may be used as preferred immune cells.

[0064] Mammalian myeloma cells are used as the cells fused with the aforementioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers 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) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells lacking 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 using the normal cell's salvage pathway and thus proliferate even in HAT-selective medium.

[0065] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0066] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), 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] Cell fusion between the immune cells and myeloma cells is basically performed according to known methods, such as the method of Köhler and Myrstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0068] More specifically, the cell fusion can be carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and additional adjuvants such as dimethyl sulfoxide may be added as desired to further enhance fusion efficiency.

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

[0070] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v). The desired fused cells (hybridomas) are formed by the gradual mixing of the mixture. Subsequently, the appropriate culture medium mentioned above is added sequentially, and the process of centrifugation and removal of the supernatant is repeated, thereby removing cell fusion agents and other substances unfavorable to hybridoma growth.

[0071] The hybridomas obtained in this manner can be selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture medium can be continued for a sufficient time (usually several days to several weeks) to kill cells other than the desired hybridoma (non-fusion cells). Subsequently, screening and single cloning of hybridomas that produce the desired antibody is performed using a standard limiting dilution method.

[0072] The hybridomas obtained in this way can be selected by using a selective culture medium corresponding to the selection markers present in the myeloma used for cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that successfully fuse with normal cells can be selectively proliferated in the HAT culture medium. Culturing with the HAT culture medium is continued for a sufficient amount of time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, generally, the desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas that produce the desired antibody can be performed using the usual limiting dilution method.

[0073] Screening and single cloning of desired antibodies can be suitably carried out by known antigen-antibody reaction-based screening methods. 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 allows for the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

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

[0075] Alternatively, the binding activity of antibodies against 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 culture supernatant of hybridomas is brought into contact 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 bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability, selected through these screenings, can be cloned by methods such as limiting dilution.

[0076] The hybridomas that produce monoclonal antibodies in this manner can be subcultured in a normal culture medium. Furthermore, these hybridomas can be stored for extended periods in liquid nitrogen.

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

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

[0079] For example, cDNA encoding the variable region (V region) of the anti-IL-6R antibody can be obtained from hybridoma cells that produce anti-IL-6R antibodies. To do this, total RNA is usually extracted from the hybridoma first. Methods such as the following 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), etc. Alternatively, kits for directly extracting total mRNA from cells are commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). mRNA can be obtained from hybridomas using such kits. From the obtained mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Furthermore, for cDNA synthesis and amplification, 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) may be used as appropriate. Furthermore, during the process of synthesizing cDNA, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA.

[0081] The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus prepared, introduced into E. coli or other organisms, and after colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector contains the target cDNA sequence is confirmed by known methods, such as dideoxynucleotide chain intermination.

[0082] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a convenient approach. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, yielding a 5'-RACE cDNA library. Commercially available kits, such as the SMART RACE cDNA amplification kit, can be used as appropriate for synthesizing the 5'-RACE cDNA library.

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

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

[0085] Using the amplified PCR product, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to IL-6R can be used as an indicator to screen for the desired antibody. For example, when the goal is to obtain an antibody against IL-6R, it is even more preferable that the antibody binds to IL-6R specifically. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) A step of contacting IL-6R expressing cells with an antibody containing a V region encoded by cDNA obtained from a hybridoma, (2) A step to detect the binding of IL-6R-expressing cells to an antibody, and (3) A step of selecting an antibody that binds to IL-6R expressing cells.

[0086] Methods for detecting the binding of antibodies to IL-6R-expressing cells are known. Specifically, the binding of antibodies to IL-6R-expressing cells can be detected using methods such as FACS, as mentioned earlier. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of antibodies.

[0087] As a screening method for antibodies using binding activity as an indicator, the panning method using phage vectors is also suitably employed. When antibody genes are obtained from a polyclonal antibody-expressing cell population as a library of heavy chain and light chain subclasses, the screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy chain and light chain can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding scFv with the desired binding activity. By repeating this operation as needed, scFv with the desired binding activity can be enriched.

[0088] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that appear infrequently in the base sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, insertion of a restriction enzyme that provides an adhesive end is preferable. By inserting the cDNA encoding the V region of the anti-IL-6R antibody, digested as described above, into a suitable expression vector, an antibody expression vector can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the constant region and the variable region originate from different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allologous chimeric antibodies are also included in the chimeric antibodies of this invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region. A chimeric antibody expression vector is constructed by in-frame fusion of the two, which have been digested with the same combination of restriction enzymes.

[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 regulatory region. This expression regulatory region includes, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, recombinant cells expressing DNA encoding the anti-IL-6R antibody can be obtained by transforming a suitable host cell with this expression vector.

[0090] For antibody gene expression, the DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is incorporated into separate expression vectors. The vectors containing both the H and L chains can simultaneously transform (co-transfect) the same host cells, thereby expressing antibody molecules with both H and L chains. Alternatively, the host cells can be transformed by incorporating the DNA encoding both the H and L chains 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 suitable hosts. These expression systems can all be applied to isolate the antigen-binding domain of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells may be used as appropriate. Specifically, 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 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)) (2) Amphibian cells: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0092] Alternatively, as for plant cells, antibody gene expression systems using cells from the Nicotiana genus, such as Nicotiana tabacum, are known. Callus-cultured cells can be used as appropriate for plant cell transformation.

[0093] Furthermore, the following types of fungal cells can be used: - Yeast: Saccharomyces species such as Saccharomyces serevisiae, and Pichia species such as methanol-utilizing yeast Pichia pastoris. - Filamentous fungi: Aspergillus species such as Aspergillus niger.

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

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

[0096] When the antigen-binding molecules described herein are administered to humans, the antigen-binding domain in 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. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be manufactured using known methods.

[0097] As a method for producing antibodies with desired binding activity against specific small molecule compounds, antibodies with desired binding activity against small molecule compounds can be obtained using a method similar to that for producing antibodies that bind to ordinary protein antigens. One example of a method for producing sensitizing antigens used to obtain antibodies against small molecule compounds is the method of linking Mariculture Keyhole Limpet Hemocyanin (KLH) with the small molecule compound. As an example of a non-limited sensitizing antigen produced to obtain antibodies against MTA, 6'-MTA-Keyhole Limpet Hemocyanin (6'-MTA-KLH) is given. 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, it is expected that linking it with MTA will enhance the production of antibodies against MTA. The small molecule compounds linked to KLH are not limited to MTA; sensitizing antigens can be created using a similar method for various synthetically available small molecule compounds. Non-limited examples include AMP, ADP, ATP, adenosine, or SAH. The design of small molecule immunogens is also disclosed in International Publication WO2013 / 180200. Furthermore, the antigen to be linked to the low-molecular-weight compound is not limited to KLH; for example, a compound linked to biotin may be used as the sensitizing antigen, and other compounds that can be linked to low-molecular-weight compounds may also be used besides KLH and biotin. This disclosure also includes the embodiments described below as illustrative examples. [1] Biotinylated MTA. [2] Biotin-2'-MTA. [3] 6'-MTA-biotin. [4] Use of biotinylated MTAs as described in [1] to [3] for screening antigen-binding molecules that bind to MTA. [5] Use of biotinylated MTA as described in [1] to [3] as an immunogen to obtain antigen-binding molecules that bind to MTA. [6] A method for screening antigen-binding molecules that bind to MTA using biotinylated MTA as described in [1] to [3].

[0098] Multiple specific antigen-binding molecules or multiple paratopic antigen-binding molecules An antigen-binding molecule containing at least two antigen-binding domains, wherein at least one antigen-binding domain binds to a first epitope in the 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 in terms of the specificity of its reaction. When an antigen-binding molecule binds to two different epitopes through two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a bispecific antigen-binding molecule. Furthermore, when an antigen-binding molecule binds to three different epitopes through three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a triplicate antigen-binding molecule.

[0099] The paratopes in the antigen-binding domain that bind to the first epitope in the antigen molecule and the paratopes in the antigen-binding domain that bind to the second epitope, which has a different structure from the first epitope, are structurally different from each other. Therefore, an antigen-binding molecule containing at least two antigen-binding domains, in which at least one antigen-binding domain binds to the first epitope in the antigen molecule and at least one other antigen-binding domain binds to the second epitope in the antigen molecule, is called a multiple paratopic antigen-binding molecule from the standpoint of its structural specificity. When an antigen-binding molecule binds to two different epitopes through two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a double paratopic antigen-binding molecule. Furthermore, when an antigen-binding molecule binds to three different epitopes through three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a triple paratopic antigen-binding molecule.

[0100] Bispecific antibodies and methods for producing them Polyvalent, multispecific or multiparatopic antigen-binding molecules containing one or more antigen-binding domains and methods for preparing them are 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 publications WO1996 / 034103 or WO1999 / 023221. By using the multispecific or multiparatopic antigen-binding molecules and methods for preparing them described herein, it is possible to produce the antigen-binding molecules of this disclosure.

[0101] As one embodiment of the multispecific or multiparatopic antigen-binding molecules and methods for their preparation described above, bispecific antibodies and methods for their production are exemplified below. A bispecific antibody is an antibody that contains two variable regions that specifically bind to different epitopes. IgG-type bispecific antibodies can be secreted by a hybrid hybridoma (quadroma) produced by fusing two hybridomas that produce IgG antibodies (Milstein et al. (Nature (1983) 305, 537-540)).

[0102] When producing bispecific antibodies using the recombinant method described in the antibody section above, a method can be employed in which genes encoding heavy chains containing the two target variable regions are introduced into cells and co-expressed. However, even considering only the combinations of heavy chains in such co-expression methods, a mixture exists in a 2:1:1 ratio of molecules, consisting of (i) a pair of heavy chains containing a variable region that binds to the first epitope and a pair of heavy chains containing a variable region that binds to the second epitope, (ii) a pair of heavy chains containing only a variable region that binds to the first epitope, and (iii) a pair of heavy chains containing only a variable region that binds to the second epitope. It is difficult to purify the antigen-binding molecule containing the target 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 constituting the heavy chains with appropriate amino acid substitutions. Specifically, this method involves substituting an amino acid side chain in the CH3 domain of one heavy chain with a larger side chain (knob) and a smaller side chain in the CH3 domain of the other heavy chain with a smaller side chain (hole), thereby allowing the knob to be positioned within the hole, promoting the formation of heterogeneous heavy chains and inhibiting the formation of homogeneous heavy chains (International Publication WO1996027011, Ridgway et al. (Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat. Biotech. (1998) 16, 677-681)).

[0104] Furthermore, there are known techniques for producing bispecific antibodies by utilizing methods for controlling the association of polypeptides, or heterogeneous polymers composed of polypeptides, for the association of heavy chains. Specifically, by modifying the amino acid residues that form interfaces within the heavy chain, the association of heavy chains with identical sequences is inhibited, and a method is employed to control the formation of two heavy chains with different sequences, which can be used to produce bispecific antibodies (International Publication WO2006 / 106905). Such methods can also be employed when producing bispecific antibodies.

[0105] cancer In this specification, the term "cancer" is used in general to refer to malignant neoplasms, which may be metastatic or non-metastatic. For example, non-limiting examples of carcinomas that originate from epithelial tissues such as the digestive tract or skin include brain tumors, skin cancer, cervical cancer, esophageal cancer, lung cancer, gastric 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 that originate from non-epithelial tissues (stroma) such as muscle include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Furthermore, non-limiting examples of hematopoietic blood cancers include malignant lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; leukemias, including acute myelocytic leukemia and chronic myelocytic leukemia; and multiple myeloma. The term “neoplasm,” as used herein, means any newly arising pathological tissue tumor. In this disclosure, a neoplasm is a tumor that results in the formation of a tumor, which is partly characterized by angiogenesis. Neoplasms can be benign, such as hemangiomas, gliomas, and teratomas, or malignant, such as carcinomas, sarcomas, gliocytomas, astrocytomas, neuroblastomas, and retinoblastomas.

[0106] The term "cancer tissue" means tissue containing at least one cancer cell. Therefore, 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. In this specification, "tumor" means a foci of tumor tissue. The term "tumor" is generally used to mean either a benign or malignant neoplasm.

[0107] In this disclosure, "cancer tissue in which MTA accumulates" means cancer tissue in which MTA is detected in a larger amount compared to normal tissue. Examples of normal tissue to be used for comparison include adjacent normal tissue to the cancer tissue or tissue from a healthy person. Furthermore, cancer tissue lacking methylthioadenosine phosphorylase (MTAP), which metabolizes MTA, or cancer tissue with reduced MTAP function, is also a form of cancer tissue in which MTA accumulates. Specifically, cancer tissue in which the gene encoding MTAP is deficient or has reduced expression, cancer tissue expressing mutations or splicing variants that reduce MTAP activity, or cancer tissue with reduced MTAP enzymatic activity are all forms of cancer tissue in which MTA accumulates. Reduced expression or function of MTAP can be determined by comparison with normal tissue, and non-limiting examples of normal tissue to be used for comparison include adjacent normal tissue to the cancer tissue or tissue from healthy individuals.

[0108] Cancer-associated fibroblast (CAF) In this disclosure, "cancer-associated fibroblast (CAF)" refers to a heterogeneous population of cells with diverse origins, such as endothelial cells, that are present around cancer tissue. Non-limiting characteristics of CAF include the creation of a microenvironment favorable to cancer progression through the promotion of cancer cell proliferation, angiogenesis, vascular invasion by cancer cells, and regulation of immune responses. Furthermore, as one non-limiting aspect of CAF, examples 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 this disclosure, "tumor-associated macrophage (TAM)" refers to macrophages present in and around cancer tissue. For example, macrophage populations that form the tumor microenvironment along with fibroblasts and vascular endothelial cells are examples. Non-limiting characteristics of TAM include the induction of neovascularization through the production of various angiogenic factors, such as suppressing antitumor immunity by promoting the production of anti-inflammatory factors and the infiltration of regulatory T cells. Furthermore, as one non-limiting aspect of TAM, cells expressing markers selected from CD163, CD204, IL-10, TGF-β, and Prastaglandin E2 are examples.

[0110] Effector Cells In this disclosure, "effector cell" refers to a T cell (CD4 + (Helper lymphocytes) T cells and / or CD8 +It can be used in the broadest sense to include white blood cells 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, but a suitable example of an effector cell is CD8 + Examples include (cytotoxic) T cells, NK cells, or macrophages. Any membrane-bound molecule expressed on the cell membrane of effector cells can be used as an antigen to which at least one antigen-binding domain of the antigen-binding molecule of this disclosure binds. Preferred membrane-bound molecules include, but are not limited to, polypeptides constituting the TCR, CD3, CD2, CD28, CD44, CD16, CD32, CD64, or NKG2D or NK cell-activating ligands.

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

[0112] MTA related products As used herein, the term "MTA analogue" refers to low-molecular-weight compounds other than MTA that share a structure partially with MTA. Examples of MTA analogues include low-molecular-weight compounds that have adenosine as a common backbone in their molecules, and low-molecular-weight compounds that have a side chain containing a sulfur atom or oxygen atom at the 5th carbon of adenosine. Furthermore, although not limited to these, metabolites of the polyamine biosynthesis 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)) are preferably cited as MTA analogues. [ka] SAM [ka] SAH Furthermore, adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) are examples of non-limiting embodiments of MTA analogs. [ka] Adenosine [ka] Adenosine monophosphate (AMP) [ka] Adenosine diphosphate (ADP) [ka] Adenosine triphosphate (ATP)

[0113] low molecular compound As used in this disclosure, the term "low molecular weight compound" refers to naturally occurring or non-naturally occurring chemical substances other than "biomolecules" present in living organisms. Examples of low molecular weight compounds, though not limited to these, include 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. Examples of non-limiting embodiments of low molecular weight compounds include cancer tissue-specific compounds, inflammatory 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 significantly present in cancer tissue compared to non-cancer tissue.

[0115] For example, in some embodiments, cancer tissue-specific compounds may be compounds defined by qualitative cancer tissue specificity, such as being present in cancer tissue but not in non-cancer tissue, or not being present in cancer tissue but being present in non-cancer tissue. In other embodiments, cancer tissue-specific compounds may be compounds defined by quantitative cancer tissue specificity, such as being present in cancer tissue at different concentrations (e.g., high or low concentrations) compared to non-cancer tissue. For example, cancer tissue-specific compounds may be present differentially at any concentration. However, generally, cancer tissue-specific compounds are 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 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 10 3 double, at least 10 4 double, at least 10 5 double, at least 10 6It is possible for the compound to be present at concentrations increasing by a factor of two or more, up to infinity (i.e., absent in non-cancerous tissue), or generally at concentrations decreasing to 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., absent). Cancer tissue-specific compounds are preferably present differentially at statistically significant concentrations (i.e., p-value less than 0.05 and / or q-value less than 0.10, as determined by either Welch's t-test or Wilcoxon's rank-sum test). 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.

[0116] Cancer tissue-specific metabolites The term "metabolism" refers to the chemical changes that occur within the tissues of living organisms, and includes "anabolism" and "catabolism." Anabolism refers to the biosynthesis or accumulation of molecules, while catabolism refers to the breakdown of molecules. "Metabolites" are intermediates or products resulting from metabolic processes. "Primary metabolites" refer to metabolites directly involved in the growth or reproduction of cells or organisms, while "secondary metabolites" refer to products such as antibiotics and dyes that result from the biosynthesis of substances that are not directly involved in the growth or reproduction of cells or organisms and do not directly participate in life phenomena common to cells or organisms. Metabolites can be metabolites of "biomolecules" or "low molecular weight" molecules. "Biomolecules" are polymers consisting of one or more repeating units. Biomacromolecules are generally molecules with a molecular weight of approximately 5000 or more that are found in biological systems and form structures such as cells that make up living organisms and the intercellular matrix and intertissue matrix attached to them. These include polysaccharides (such as carbohydrates), peptides (this term is used to include polypeptides and proteins), and polynucleotides, as well as their analogues, such as amino acid analogs or compounds composed of or containing non-amino acid groups.

[0117] One non-limiting embodiment of cancer tissue-specific metabolites described herein is preferably cancer cell-specific low molecular weight metabolites (Eva Gottfried, Katrin Peter and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132). Furthermore, metabolites highly produced by immune cells infiltrating cancer tissue and metabolites highly produced by 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, inhibitory dendritic cells, inhibitory T cells, exhausted T cells, myeloma-derived suppressor cells (MDSCs), etc. The metabolites in the present invention also include compounds released from inside cells to outside cells when cells present in cancer tissue (cancer cells, immune cells, stromal cells) undergo cell death by apoptosis, necrosis, etc.

[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, etc.)) or analyses 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 methods 1741-1753), metabolic analysis, particularly metabolic profiling, is used as appropriate. Specifically, metabolic profiling, using methods such as 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)), and ELISA, individually or in combination, may be used as appropriate to identify metabolites in the test sample.

[0119] These studies have revealed intratumoral heterogeneity, comprised of altered concentration gradients of metabolites (e.g., glucose or oxygen) and growth factors that enable cancer cells to grow under low oxygen pressure conditions (Dang and Semenza (Trends Biochem. Sci. (1999) 24, 68-72)). These studies also utilize cell line models to understand changes in energy utilization pathways at different degrees of tumor malignancy (Vizan et al. (Cancer Res. (2005) 65, 5512-5515)). As a non-limiting aspect of the technical components of the metabolic platform, examples include sample extraction, separation, detection, spectroscopic analysis, data normalization, class-specific metabolite depiction, pathway mapping, confirmation, and functional characterization of candidate metabolites, as described by 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 "inflammation tissue-specific compound" refers to a compound that is significantly present in inflammatory tissue compared to non-inflammatory tissue. In this specification, "inflammation tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveoli) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis • Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that are being rejected in organ transplants • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure • Visceral fat in metabolic syndrome • Skin tissue in atopic dermatitis and other skin inflammations • Spinal nerves in herniated discs and chronic lower back pain

[0121] Inflammatory tissue-specific metabolites Inflammation tissue-specific metabolites are metabolites produced in high concentrations by immune cells infiltrating inflammatory tissue, and metabolites produced in high concentrations specifically by normal cells that are damaged in inflammatory tissue. Examples of infiltrating immune cells include effector T cells, mature dendritic cells, neutrophils, granule cells (mast cells), and basophils. Furthermore, the metabolites in this invention also include compounds released from inside cells to outside cells when cells (immune cells, normal cells) present in inflammatory tissue undergo cell death through apoptosis, necrosis, etc.

[0122] As used herein, the term "non-natural compound" refers to a chemical substance of non-natural origin and its metabolites. One aspect of the invention is a non-natural chemical substance and its metabolites that have the property of accumulating in target tissue after being administered into the body from outside the body. Examples of non-natural compounds include (1) capecitabine (Xeloda) and its metabolite 5-FU (fluorouracil), and (2) TH-302 and bromoisophosphamide mustard (Br-IPM). 5-FU is a metabolite of capecitabine (Xeloda) and is known to be metabolized by cytidine deaminase and thymidine phosphorylase, which are cancer tissue-specific metabolic enzymes (Desmoulin F. et al. Drug Metab Dispos. 2002). Furthermore, TH-302 is known to be converted to Br-IPM by reduction under hypoxic conditions, such as around cancer tissue (Duan JX, et al. J Med Chem. 2008). For example, when capecitabine (xeloda) is administered, it is metabolized to 5-FU by cancer-specific metabolic enzymes, etc., so the concentration of 5-FU at the site of the cancer increases (Desmoulin F. et al. Drug Metab Dispos. 2002). Therefore, it is thought that antibodies that use 5-FU as a switch can selectively bind to the target antigen only at the site of the cancer. In addition, it is also thought that molecules produced under cancer-specific hypoxic or acidic environments can be used as switches, other than metabolic enzymes. For example, since TH-302 (Duan JX, et al. J Med Chem. 2008) is metabolized to Br-IPM under hypoxic conditions, it is thought that antibodies that use Br-IPM as a switch can selectively bind to the target antigen only at the site of the cancer. For example, known methods of administering non-natural compounds to a living organism include, but are not limited to, oral administration, eye drop administration, transdermal administration, nasal administration, intravenous administration, and transpulmonary administration.

[0123] Examples of non-limiting embodiments of the small molecule compounds in this disclosure include, for example, MTA, SAM, SAH, adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).

[0124] Examples of non-limiting embodiments of low-molecular-weight compounds in this disclosure include the following compounds:

[0125] (1) Primary metabolites of glycolysis or the Krebs cycle, such as lactic acid, succinic acid, and citric acid. As an unspecified embodiment of the low-molecular-weight compounds or cancer tissue-specific compounds used in the present invention, particularly cancer cell-specific metabolites, primary metabolites produced as a result of glucose metabolism, such as lactic acid, succinic acid, and citrate, which are present in higher concentrations in cancer tissue than in surrounding non-cancerous tissue, are preferred. Glycolytic phenotypes characterized by the upregulation of glycolytic enzymes (Embden-Myerhof pathway) such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH) have been conventionally known as the Warburg effect and are characteristic of solid tumors.

[0126] In other words, it is thought that the high expression of the M2 iso-type pyruvate kinase, which is necessary for erobic glycolysis under anaerobic conditions, rather than the M1 iso-type, in tumor cells is advantageous 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 the equilibrium reaction by lactate dehydrogenase (LDH) under anaerobic conditions. This feedback inhibition promotes mitochondrial respiration (Krebs cycle) and suppresses cell proliferation, so it is said that the upregulation of LDH, hexokinase, and glucose transporter (GLUT) plays an important role in tumor cell proliferation (Fantin et al. (Cancer Cell (2006) 9, 425-434)). Glucose is metabolized in glycolysis, and its final metabolite, lactate, is co-transported with protons to the area surrounding the tumor, resulting in a change in the pH of the surrounding tissue to acidic conditions. It is known that lactate, the final product of glycolysis, and succinate and citrate, which are produced by the promotion of mitochondrial respiration, accumulate in cancer tissue (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). As an unspecified embodiment of the low-molecular-weight compound, cancer tissue-specific compound, and especially cancer cell-specific metabolite used in the present invention, primary metabolites produced by this glycolytic metabolism, such as lactate, succinate, and citrate, are preferably mentioned. Furthermore, it is known that succinate, which is present in high concentrations inside cells, leaks out of cells due to cell death (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is thought that the concentration of succinate increases in cancer tissue where cell death occurs frequently.

[0127] (2) Amino acids such as alanine, glutamic acid, and aspartic acid In addition to the glucose metabolism described above, it is known that amino acid metabolism is also altered in tumor cells, which require a continuous supply of essential and non-essential amino acids necessary for the biosynthesis of biomacromolecules under anaerobic conditions. Glutamine is the most widely distributed amino acid in the body, acting as a nitrogen carrier with two nitrogen atoms in its side chain. Tumor cells with increased rates of glutamine uptake into cells are thought to function as glutamine traps. This increased uptake of glutamine and its conversion to glutamate and lactate is called "glutaminolysis" and is thought 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 a decrease in plasma glutamine levels while glutamate concentrations increase (Droge et al. (Immunobiology (1987) 174, 473-479). And lung cancer tissue 13 Metabolic studies of 14C 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 14C-labeled citric acid. Suitable examples of low-molecular-weight compounds and cancer tissue-specific compounds used in the present invention include alanine, glutamic acid, and aspartic acid, which accumulate in high concentrations in cancer tissue through glutamic degradation and other processes.

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

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

[0130] [ka] Anthranilic acid

[0131] [ka] 3-Hydroxykynurenine

[0132] Kynurenine is converted to kynurenic acid (represented by the following formula) by kynurenine aminotransferase. In the present invention, as an unspecified embodiment of the low molecular weight compound, cancer tissue-specific compound, and in particular cancer cell-specific metabolite, kynurenine and its metabolites, such as amino acid metabolites including anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, are preferably mentioned.

[0133] [ka] kynurenic acid

[0134] (4) Metabolites of arachidonic acid such as prostaglandin E2 Prostaglandin E2 (PGE2) (represented by the formula below) is a metabolite of arachidonic acid called a plastonoid, which contains 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 suppresses their apoptosis (Sheng et al. (Cancer Res. (1998) 58, 362-366)). It is known that cyclooxygenase expression is altered in many cancer cells. That is, while COX-1 is constitutively expressed in almost all tissues, COX-2 has been found to be mainly induced in tumors by certain inflammatory cytokines and oncogenes (Warner and Mitchell (ibid.)). COX-2 overexpression has also been reported to be associated with a 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). Small molecule compounds such as prostaglandins and leukotrienes, metabolites of arachidonic acid, are known to act as stimulants that control autocrine and / or paracrine proliferation of cancer (Nat. Rev. Cancer (2012) 12 (11)). 782-792). As an unspecified embodiment of the low molecular weight compounds, cancer tissue-specific compounds, and especially cancer cell-specific metabolites and immune cell-specific metabolites infiltrating cancer tissue used in the present invention, arachidonic acid metabolites such as prostaglandin E2 are preferably mentioned. In addition to prostaglandin E2, thromboxane A2 (TXA2) is produced in increased amounts in cancer tissues such as colorectal cancer (J. Lab. Clin. Med. (1993) 122, 518-523), and is also preferably mentioned as an unspecified embodiment of the arachidonic acid metabolites 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.). As a non-limited embodiment of the low-molecular-weight compounds, inflammatory tissue-specific compounds, and especially inflammatory cell-specific metabolites and immune cell-specific metabolites that infiltrate inflammatory tissue used in the present invention, arachidonic acid metabolites such as prostaglandin E2 are preferably mentioned.

[0137] (5) Nucleosides having 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 cell types release adenine nucleotides in the forms of ATP, ADP, and AMP. Adenosine is metabolized by extracellular enzymes on the cell surface, such as extracellular-5'-nucleotidase (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 significant increase in extracellular adenosine concentration has been reported in hypoxic tissues found in solid tumors (Blay and Hoskin (Cancer Res. (1997) 57, 2602-2605)). CD73 is expressed on the surface of tumor and immune cells (Kobie et al. (J. Immunol. (2006) 177, 6780-6786), and in breast cancer (Canbolat et al. (Breast Cancer Res. Treat.)). Increased activity has been observed 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 suggested that the accumulation of adenosine in cancer tissue 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, suppressor T cells and other cells infiltrating cancer tissue also express ATP-degrading enzymes 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 tissue via adenosine receptors such as the A2A receptor (Curr. Med. Chem. (2011), 18, 5217-23). ​​In the present invention, as an unspecified embodiment of the low-molecular-weight compound and cancer tissue-specific compound, suitable examples include ATP, ADP, AMP, or adenosine, which accumulate in high concentrations in cancer tissue through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is broken down into inosine by adenosine deaminase, leading to a high concentration of inosine accumulation.

[0138] Furthermore, it is known that ATP concentrations are high in alveoli where inflammation occurs due to bronchial asthma (Nat. Med. (2007) 13, 913-919). It is also known that ATP concentrations are high in alveoli where inflammation occurs due to COPD (Am. J. Respir. Crit. Care Med. (2010) 181, 928-934). In addition, high adenosine concentrations have been observed in the synovial fluid of rheumatoid arthritis patients (Journal of Pharmaceutical and Biomedical Analysis (2004) 36 877-882). Moreover, it is known that ATP concentrations are high in tissues where rejection reactions occur due to GVHD (Nat. Med. (2010) 16, 1434-1438). Furthermore, it is known that adenosine concentrations are elevated in fibrotic tissues of 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). In addition, elevated ATP concentrations have been observed in fibrotic tissues of patients with pulmonary fibrosis (Am. J. Respir. Crit. Care Med. (2010) 182, 774-783). As an unspecified embodiment of the low-molecular-weight compound and inflammatory tissue-specific compound used in the present invention, suitable examples include ATP, ADP, AMP, or adenosine, which accumulate in high concentrations in inflammatory tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is broken down into inosine by adenosine deaminase, leading to a high concentration of inosine accumulation.

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

[0140] Furthermore, recent studies have shown that uric acid released from cells undergoing necrosis promotes inflammatory responses (J. Clin. Invest. (2010) 120 (6), 1939-1949). As an unspecified embodiment of the low-molecular-weight compounds and inflammatory tissue-specific compounds used in the present invention, uric acid, which accumulates in high concentrations in inflammatory tissues through the metabolism of purine nucleotides such as ATP, is also a suitable example.

[0141] (7) 1-Methylnicotinamide It is known that the enzyme nicotinamide N-methyltransferase is highly expressed in several human cancer tissues. When this enzyme produces the stable metabolite 1-methylnicotinamide from nicotinamide, it consumes the methyl group of S-adenosylmethionine (SAM), which acts as a methyl donor. It has been proposed that high expression of nicotinamide N-methyltransferase contributes to tumorigenesis through a mechanism that impairs the DNA methylation ability associated with a decrease in SAM concentration in cancer cells (Ulanovskaya et al. (Nat. Chem. Biol. (2013) 9 (5) 300-306)). 1-methylnicotinamide, a stable metabolite of this enzyme, is known to be secreted extracellularly by cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). As an unspecified embodiment of the low-molecular-weight compounds and cancer tissue-specific compounds used in the present invention, 1-methylnicotinamide, which accumulates in high concentrations in cancer tissue through the metabolism of such nicotinamides, is also a suitable example. The small molecule compounds in this disclosure may interact with antigen-binding molecules. Amino acid residues in the antigen-binding molecule that may interact with the small molecule compounds may be located in the antigen-binding domain or in other locations. While the antigen-binding domain is given as an example of a site in the antigen-binding molecule that interacts with the small molecule compounds, the disclosure is not limited to this.

[0142] Antigen-binding domain that specifically binds to the antigen In this specification, the term "antigen-binding domain that specifically binds to an antigen" is used when the antigen-binding domain is specific to a particular epitope among several epitopes contained in a given antigen. Furthermore, if the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope. Here, substantially non-binding is determined according to the method described in the section on binding activity above, and means that the binding activity of the specific binding molecule to molecules other than the aforementioned target molecule is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the aforementioned target molecule. In the case of an antigen-binding domain whose binding activity to the antigen changes in a manner dependent on MTA or a low-molecular-weight compound other than MTA, the binding of the antigen-binding domain to the antigen is measured under conditions in which the binding activity of the antigen-binding domain to the antigen is high (for example, under a specific concentration of MTA or in the absence of MTA, under a specific concentration of a low-molecular-weight compound other than MTA or in the absence of a low-molecular-weight compound other than MTA).

[0143] Antigen-binding activity and method for confirming antigen-binding activity The term “binding activity” refers to the strength of the combined non-covalent interactions between one or more binding sites on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Here, “binding activity” is not strictly limited to the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, if the members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). If the members of a binding pair are capable of both monovalent and polyvalent binding, binding activity is the sum of these binding forces. The binding activity of molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or “analyte binding per unit amount of ligand.” Binding activity can be measured by conventional methods known in the art, including those described herein. Conditions other than the concentration of the target tissue-specific compound can be appropriately determined by those skilled in the art. Specific and illustrative examples of methods 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, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 This is the dissociation constant (KD) of M).

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

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

[0147] In one embodiment, antibody binding activity can be measured using a bio-layer interferometry (BLI) method, such as the Octet RED96e system or the Octet RED 384 system (Pall ForteBio). By using the system according to the supplier's instructions, qualitative binding characteristic analysis and kinetic analysis of antigen-antibody reactions can be performed. As one non-limiting embodiment of the specific measurement method, the change in the amount of binding between antibody and antigen can be measured by immobilizing the antibody on a Protein A (ProA) biosensor (Pall ForteBio) and then interacting it with the antigen as an analyte. For example, when measuring the amount of antibody bound to an antigen in the presence of MTA, the binding reaction between the antibody and analyte can be measured in a buffer containing 3000 nM analyte diluted with 20 mM ACES, 150 mM NaCl, and 0.05% (w / v) Tween 20 at pH 7.4, to which MTA has been added at final concentrations of 0, 10, and 100 μM, as the binding phase. The dissociation reaction between the antibody and analyte can then be measured by using the same buffer as the binding phase but without 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 how the binding of the antibody and antigen in the presence of MTA reversibly dissociates in the absence of MTA.

[0148] For antigen-binding activity, the kd (dissociation rate constant) can be used if the antigen is a soluble molecule, and the apparent kd (apparent dissociation rate constant) can be used if the antigen is a membrane molecule. Both the kd (dissociation rate constant) and the 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 binding activity of a test antigen-binding molecule containing an antigen-binding domain whose binding activity to the antigen changes in a low-molecular-weight compound-dependent manner, the interaction between the test antigen-binding molecule and the antigen can be performed under specific concentrations or in the absence 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 binding activity to the antigen in the presence of that low-molecular-weight compound, the binding activity can be evaluated in the presence of that low-molecular-weight compound. Similarly, if a molecule does not exhibit binding activity to the antigen in the presence of a specific low-molecular-weight compound, but exhibits binding activity to the antigen in the absence of that low-molecular-weight compound, the binding activity can be evaluated in the absence of that low-molecular-weight compound. If the antigen-binding activity under conditions where a specific low-molecular-weight compound is present at a low concentration is higher than the antigen-binding activity under conditions where that low-molecular-weight compound is present at a high concentration, the binding activity can be evaluated under conditions where that low-molecular-weight compound is present at a high concentration. Similarly, if the antigen-binding activity under conditions where a specific low-molecular-weight compound is present at a high concentration is lower than the antigen-binding activity under conditions where that low-molecular-weight compound is present at a low concentration, the binding activity can be evaluated under conditions where that low-molecular-weight compound is present at a low concentration. Conditions that may affect the binding activity between an antigen and an antigen-binding molecule are not limited to the presence / absence of the low-molecular-weight compound or its concentration, but are exemplified by, but not limited to, ion concentration, ion composition, or temperature. Furthermore, it is certainly possible to evaluate binding activity under conditions in which multiple different factors are combined.

[0150] Epitope An epitope, meaning an antigenic determinant present in an antigen, refers to a site on an antigen to which the antigen-binding domain of an antigen-binding molecule disclosed herein binds. The site on an antigen to which an antigen-binding molecule binds in this disclosure may be defined by evaluating whether or not the antigen-binding molecule binds.

[0151] An epitope can be defined by its structure. It can also be defined by the binding activity of an antigen-binding molecule that recognizes it. If the antigen is a peptide or polypeptide, the epitope can be identified by the amino acid residues that make up the epitope. Furthermore, if the epitope is a glycan, it can be identified by its specific glycan structure.

[0152] A linear epitope is an epitope that contains an epitope whose primary amino acid sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, for example, about eight to about ten, or six to twenty amino acids, in a specific sequence.

[0153] A structural epitope, in contrast to a linear epitope, is an epitope in which the primary sequence of amino acids containing the epitope is not a single defining component of the recognized epitope (for example, an epitope whose primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope). A structural epitope may contain a larger number of amino acids than a linear epitope. In relation to the recognition of structural epitopes, antibodies recognize the three-dimensional structure of the peptide or protein. For example, if a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the structural epitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0154] The structure of the antigen-binding domain that binds to an epitope is called a paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. This binding force between the epitope and paratope is called affinity. The sum of the binding forces when multiple antigens and multiple antigen-binding molecules bind is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding forces (affinity) work synergistically, resulting in avidity that is higher than affinity.

[0155] The following is an example of how to confirm the binding of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to an epitope. However, methods for confirming the binding of a test antigen-binding molecule containing an antigen-binding domain for antigens other than IL-6R to an epitope can also be carried out as appropriate in accordance with the following examples.

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

[0157] Furthermore, the recognition of a structural epitope by a test antigen-binding molecule containing an antigen-binding domain for IL-6R can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. When the test antigen-binding molecule containing an antigen-binding domain for IL-6R comes into contact with IL-6R-expressing cells, it binds strongly to the cells, while the antigen-binding molecule does not substantially bind to the linear peptide consisting of the amino acid sequence constituting the extracellular domain of immobilized IL-6R. Here, substantially non-binding means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.

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

[0159] In the ELISA format, the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to IL-6R-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, the test polypeptide aggregate is added to an ELISA plate immobilized with IL-6R-expressing cells, 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, the binding activity of the test antigen-binding molecule to IL-6R-expressing cells can be compared by creating a dilution series of the test antigen-binding molecule and determining the antibody binding titer against IL-6R-expressing cells.

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

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

[0162] The same epitope-binding antibody The sharing of an epitope between a test antigen-binding molecule containing an antigen-binding domain for IL-6R and another antigen-binding molecule can be confirmed by competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

[0163] Specifically, in a cross-blocking assay, IL-6R protein coated on wells of a microtiter plate is pre-incubated in the presence or absence of candidate competing antigen-binding molecules, after which the test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to the IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecules that compete 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 the well coated with IL-6R protein.

[0164] The amount of antigen-binding molecules bound to the wells via the IL-6R protein can be easily measured by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using an avidin peroxidase conjugate and an appropriate substrate. Cross-blocking assays utilizing enzymatic labeling such as peroxidase are specifically called competitive ELISA assays. Antigen-binding molecules can be labeled with other detectable or measurable labeling substances. Specifically, radiolabeling and fluorescent labeling are well known.

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

[0166] If the structure of the epitope to which the test antigen-binding molecule, which contains an antigen-binding domain for IL-6R, binds has been identified, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by comparing the binding activity of both antigen-binding molecules to peptides into which amino acid mutations have been introduced in the peptide constituting the epitope.

[0167] One method for measuring such binding activity is to compare the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced in the aforementioned ELISA format. Alternatively, the binding activity to the mutated peptide bound to a column can be measured by quantitatively determining the antigen-binding molecule eluted into the eluate after the test antigen-binding molecule and the control antigen-binding molecule have been passed through the column. Methods for adsorbing the mutated peptide onto a column as a fusion peptide with, for example, GST, are well known.

[0168] Furthermore, if the identified epitope is a stereoepitope, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, in which these cells are suspended in a suitable buffer such as PBS. Next, FITC-labeled antibodies that can recognize the test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, which has been washed with a buffer as appropriate. The fluorescence intensity and cell count of the cells stained with the labeled antibody are measured using FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are adjusted to the desired concentration by appropriately diluting them with a suitable buffer. For example, concentrations between 10 μg / ml and 10 ng / ml are used. 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, it is possible to measure the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, which is represented by the amount of labeled antibody bound to them.

[0169] In this method, for example, "substantially ineffective binding to mutant IL-6R-expressing cells" can be determined by the following method. First, the test antigen-binding molecule and the control antigen-binding molecule bound to cells expressing mutant IL-6R are stained with a labeled antibody. Next, the fluorescence intensity of the cells is detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. By calculating this comparison value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of polypeptide aggregates based on Equation 1 below, the percentage increase in fluorescence intensity due to the binding of the antigen-binding molecule can be determined.

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

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

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

[0173] When evaluating whether test antigen-binding molecules containing antigen-binding domains whose binding activity to the antigen changes in a low-molecular-weight compound-dependent manner compete or bind to the same epitope, it is possible to perform the interaction between the test antigen-binding molecule and the antigen under specific concentrations or in the absence of the low-molecular-weight compound, and it is preferable to keep the concentration conditions of the low-molecular-weight compound the same among the test antigen-binding molecules.

[0174] Antigen-binding domain whose binding activity to the antigen changes in a low-molecular-weight compound-dependent manner. In this specification, "antigen-binding domain whose binding activity to an antigen changes in a low-molecular-weight compound-dependent manner" means an antigen-binding domain whose binding activity to an antigen that is a different molecule from the low-molecular-weight compound changes in the presence of different concentrations of the low-molecular-weight compound.

[0175] Antigen-binding domain whose binding activity to the antigen changes in an MTA-dependent manner. The antigen-binding domains in this disclosure whose binding activity to an antigen changes in an MTA-dependent manner are antigen-binding domains that exhibit different binding activity to antigens that are molecules different from MTA under conditions of different MTA concentrations. The antigens to which these antigen-binding domains bind may be membrane molecules or soluble molecules. Furthermore, the antigens to which these antigen-binding domains bind are antigens expressed in diseased tissue, more preferably antigens expressed in cancer tissue, and even more preferably antigens expressed in cancer tissue where MTA has accumulated. The antigens expressed in cancer tissue may be antigens expressed on cancer cells, or antigens expressed on cancer stromal cells or immune tissues within the cancer tissue.

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

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

[0178] As long as the antigen-binding activity of the antigen-binding domain in the presence of MTA is weaker than the antigen-binding activity in the absence of MTA, and the antigen-binding activity in the absence of MTA is stronger than the antigen-binding activity in the presence of MTA, the ratio of antigen-binding activity in the absence of MTA to antigen-binding activity in the presence of MTA is not particularly limited. However, preferably, the value of KD(in the presence of MTA) / KD(in the absence of MTA), which is the ratio of the dissociation constant (KD) of the antigen in the presence of MTA to the KD in the absence of MTA, is 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD(in the presence of MTA) / KD(in the absence of MTA) is not particularly limited and may be any value such as 400, 1000, 10000, etc., as long as it can be produced by the art. If no antigen-binding activity is observed in the presence of MTA, this upper limit becomes an infinite value. If the antigen-binding domain's binding activity to the antigen in the presence of MTA is weaker than its binding activity to the antigen in the absence of MTA, the antigen-binding domain may include an antigen-binding domain that does not substantially bind to the antigen in the presence of MTA.

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

[0180] The conditions for the presence of MTA can be set to an appropriate MTA concentration. As a non-limiting example, the condition for the presence of 100 μM of MTA can be considered as the conditions for the presence of MTA. Furthermore, as a non-limiting aspect of the MTA concentration described as "in the presence of MTA" in this disclosure, the concentrations exemplified below as thresholds for distinguishing between low and high concentrations of MTA may be applied.

[0181] An example of an antigen-binding domain whose antigen-binding activity changes in an MTA-dependent manner is an antigen-binding domain in which antigen-binding activity is stronger in the presence of high concentrations of MTA than in the presence of low concentrations of MTA, or an antigen-binding domain in which antigen-binding activity is weaker in the presence of high concentrations of MTA than in the presence of low concentrations of MTA.

[0182] As long as the antigen-binding activity of the antigen-binding domain in the presence of a low concentration of MTA is weaker than the antigen-binding activity in the presence of a high concentration of MTA, the ratio of the antigen-binding activity in the presence of a high concentration of MTA to the antigen-binding activity in the presence of a low concentration of MTA is not particularly limited. Preferably, the value of KD(low concentration of MTA) / KD(high concentration of MTA), which is the ratio of the dissociation constant (KD) of the antigen in the presence of a low concentration of MTA to the KD in the presence of a high concentration of MTA, is 2 or more. More preferably, the value of KD(low concentration of MTA) / KD(high concentration of MTA) is 10 or more. Even more preferably, the value of KD(low concentration of MTA) / KD(high concentration of MTA) is 40 or more. The upper limit of the KD(low concentration of MTA) / KD(high concentration of MTA) value is not particularly limited and can be any value such as 400, 1000, 10000, etc., as long as it can be produced by the art. If no binding activity to the antigen is observed in the presence of a low concentration of MTA, this upper limit becomes an infinite value. Antigen-binding activity in the presence of high concentrations of MTA and antigen-binding activity in the presence of low concentrations of MTA are stronger antigen-binding domains that include antigen-binding domains that do not substantially bind to the antigen in the presence of low concentrations of MTA.

[0183] As long as the antigen-binding activity of the antigen-binding domain in the presence of low concentrations of MTA is stronger than the antigen-binding activity in the presence of high concentrations of MTA, the ratio of the antigen-binding activity in the presence of high concentrations of MTA to the antigen-binding activity in the presence of high concentrations of MTA is not particularly limited. However, preferably, the value of KD(high concentration of MTA) / KD(low concentration of MTA), which is the ratio of the dissociation constant (KD) of the antigen in the presence of high concentrations of MTA to the KD in the presence of low concentrations of MTA, is 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD(high concentration of MTA) / KD(low concentration of MTA) is not particularly limited and may be any value such as 400, 1000, 10000, etc., as long as it can be produced by the art. If no binding activity to the antigen is observed in the presence of high concentrations of MTA, this upper limit becomes infinite. Antigen-binding activity in the presence of high concentrations of MTA is weaker than antigen-binding activity in the presence of low concentrations of MTA. Antigen-binding domains include antigen-binding domains that do not substantially bind to the antigen in the presence of high concentrations of MTA.

[0184] Furthermore, as another indicator showing the ratio of the binding activity of the antigen-binding domain (or antigen-binding molecule containing said domain) in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA in the presence of MTA, the value of kd(low MTA concentration) / kd(high MTA concentration), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of kd(low MTA concentration) / kd(high MTA concentration) is not particularly limited and may be any value such as 50, 100, 200, etc., as long as it can be produced according to the common technical knowledge of those 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. Examples of such non-limiting methods include, but are not limited to, techniques using flow cytometry, ELISA, cavilery electrophoresis, or liquid chromatography. Furthermore, even when the KD value cannot be directly calculated, it is possible to evaluate the relative binding activity of the test molecule by comparing it to a reference molecule for which the KD value has been calculated using Biacore, determining whether the test molecule has stronger or weaker binding activity than the reference molecule.

[0186] In one non-limiting embodiment of thresholds for distinguishing between low and high concentrations of MTA, the low concentration condition can be appropriately set from values ​​of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, or 0 M as the threshold. 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 twice, at least five times, at least ten times, at least 50 times, at least 100 times, and at least 10 times each threshold. 3 double, at least 10 4 double, at least 10 5 double, at least 10 6 It can be set appropriately from double the value.

[0187] One non-limiting embodiment of the antigen-binding domain in this disclosure whose binding activity to an antigen changes in an MTA-dependent manner is an antigen-binding domain whose binding activity to an antigen is substantially unaffected by one or more small molecule compounds selected from adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP. Here, adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP have adenosine as a common skeleton in their molecules and are compounds similar to MTA. It is generally considered difficult to obtain an antigen-binding molecule in which the binding activity of the antigen-binding domain in this disclosure is substantially unaffected by the presence of these similar molecules and whose binding activity changes in an MTA-dependent manner. However, in this disclosure, we constructed a library for obtaining antibodies that bind to antibodies in an MTA-dependent manner, as exemplified in the various examples described later, and by screening this library, we succeeded in obtaining an antigen-binding domain with excellent MTA-dependent specificity that binds to the antigen in an MTA-specific and dependent manner and does not show binding to the antigen in a dependent manner to small molecule compounds that exhibit a structure similar to MTA. In this specification, "antigen binding activity is substantially unaffected by the small molecule compound" means that the antigen binding activity of the antigen-binding domain in the presence of the small molecule compound and the antigen-binding activity of the antigen-binding domain in the absence of the small molecule compound are at least 0.5 to 2 times each other.

[0188] In one non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is subject to MTA-dependent changes in antigen-binding activity, is substantially unaffected by adenosine. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is MTA-dependently altered in its antigen-binding activity, 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, which is MTA-dependently altered in its binding activity to the antigen, is substantially unaffected by the SAM. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is MTA-dependently altered in its antigen-binding activity, is substantially unaffected by any of adenosine, AMP, ADP, or ATP. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is MTA-dependently altered in its antigen-binding activity, is substantially unaffected by either 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, which is MTA-dependently altered in its antigen-binding activity, is substantially unaffected by adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), or SAM. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is MTA-dependently altered in its antigen-binding activity, is substantially unaffected by any of adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, or ATP.

[0189] One non-limiting embodiment of the antigen-binding domain of this disclosure whose binding activity to an antigen changes in an MTA-dependent manner is an antigen-binding domain whose binding activity to an antigen changes in a manner dependent on one or more small molecules selected from adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, and ATP. In a non-limiting embodiment, the antigen-binding activity of an antigen-binding domain whose binding activity to an antigen changes in an MTA-dependent manner may also change in an adenosine-dependent manner. An antigen-binding domain whose binding activity changes in a manner dependent on both MTA and adenosine molecules is not bound by any particular theory, but it can be understood that the binding activity to an antigen changes due to interaction with a common structure in both MTA and adenosine molecules. In that case, even if the concentration of MTA decreases in the solvent in which the binding activity is being evaluated, if the concentration of adenosine is present at a level that exceeds the degree of decrease in binding activity due to the decrease in MTA concentration, the binding activity may be maintained or strongly detected. Also, even if the concentration of MTA increases in the solvent in which the binding activity is being evaluated, if the concentration of adenosine decreases by more than the degree of increase in binding activity due to the increase in MTA concentration, the binding activity may be maintained or weakly detected. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is altered in an MTA-dependent manner, is also altered 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, which is altered in an MTA-dependent manner, is also altered in a SAM-dependent manner. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is altered in an MTA-dependent manner, is also altered in a way that is dependent on adenosine, AMP, ADP, or ATP. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is altered in an MTA-dependent manner, is also altered in a manner dependent on either 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, which is MTA-dependently altered in its antigen-binding activity, is also altered in a manner dependent on adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), or SAM. In another, non-limiting embodiment, the antigen-binding activity of the antigen-binding domain of the present disclosure, which is MTA-dependently altered in its antigen-binding activity, is also altered in a manner dependent on adenosine, S-(5'-Adenosyl)-L-homocysteine ​​(SAH), SAM, AMP, ADP, or ATP.

[0190] An example of an antigen-binding domain in which the binding activity to an antigen changes in an MTA-dependent manner, as described herein, is an antigen-binding domain containing an antibody variable region and / or a monodomain antibody.

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

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

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

[0194] The antigen-binding domain in this disclosure may have amino acid residues that interact with the MTA. These amino acid residues may be located at the interface that directly interacts with the antigen within the antigen-binding domain, or at other locations. The "interface that directly interacts with the antigen" refers to the region where the antigen and the antigen-binding molecule are in close proximity to each other in the structure of the complex of the antigen and the antigen-binding domain, as analyzed by methods such as crystal structure analysis. A distance of 4 to 6 Å can be given as an example of the distance at which the antigen and the antigen-binding molecule are in close proximity to each other, but in the structure of the complex of the antigen and the antigen-binding molecule, "the antigen and the antigen-binding molecule are in close proximity to each other" refers to the region where they are relatively close within the complex, and is not limited to the distance given above. The amino acid residues that interact with the MTA may be residues that interact with the MTA when the antigen-binding molecule is bound to the antigen, or residues that interact with the MTA when the antigen is absent. Furthermore, the amino acid residues that interact with the MTA may be residues that interact with the MTA both when the antigen-binding molecule is bound to the antigen and when the antigen is absent. Furthermore, the amino acid residues that interact with the MTA may be just one residue or multiple residues in the antigen-binding domain.

[0195] In embodiments where the antigen-binding domain includes an antibody heavy chain variable region and an antibody light chain variable region, the amino acids that interact with the MTA may be located in the CDR or FR within the antibody variable region. In embodiments where the antigen-binding domain includes a single-domain antibody, the amino acids that interact with the MTA may be located in the CDR or FR within the single-domain antibody.

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

[0197] As one non-limiting aspect of this disclosure, amino acid residues of the antigen-binding molecule that interact with MTA can be identified by crystal structure analysis of the complex of MTA and two antigen-binding molecules. Here, "interacting with MTA" means a state in which atoms of the side chain or main chain of the amino acid forming the antigen-binding molecule and atoms of the low-molecular-weight compound form an antigen-binding molecule-MTA interaction at a distance that can affect the MTA-binding activity, or a state in which, in embodiments in which the antigen-binding domain in the antigen-binding molecule includes an antibody heavy chain variable region and an antibody light chain variable region, a certain amino acid residue contributes to MTA binding, including indirect effects such as stabilizing the conformation of the three-dimensional structure such as the CDR loop when MTA binds, and a state that satisfies both of these conditions. In this specification, the "state in which intermolecular interactions are formed" can be determined, for example, from the crystal structure analysis of a complex of MTA and an antigen-binding molecule, based on the interatomic distance between the non-hydrogen atoms constituting the side chain or main chain of the amino acid forming the antigen-binding molecule and the non-hydrogen atoms constituting the MTA. For example, the above interatomic distance is preferably within 3.0 Å, 3.2 Å, 3.4 Å, 3.6 Å, 3.8 Å, 4.0 Å, 4.2 Å, 4.4 Å, 4.6 Å, 4.8 Å, or 5.0 Å, but is not limited thereto. More preferably, the above interatomic distance is within 3.6 Å, 3.8 Å, 4.0 Å, or 4.2 Å. More precisely, the possibility of direct interaction can be determined based on information regarding interatomic distances in the three-dimensional structure, the types of intermolecular interactions formed, and the types of atoms. More accurately, it can be determined from the effect of introducing mutations in amino acid residues, such as modifications to Ala or Gly, on the activity of small molecule compounds, but it is not limited to this. Regarding the "indirectly influencing state" as used herein, for example, it is possible to estimate whether it indirectly affects MTA binding by analyzing in detail the conformation of each amino acid residue and the intermolecular interactions with surrounding residues from the three-dimensional structure of the complex of MTA and the antigen-binding molecule. More accurately, it can be determined from the effect that the introduction of amino acid residue mutations, such as modifications to Ala or Gly, has on MTA activity.

[0198] As one non-limiting aspect of this disclosure, amino acid residues of the antigen-binding molecule that interact with MTA can be identified by crystal structure analysis of a complex of MTA, antigen, and antigen-binding molecule. Here, "interacting with MTA" means a state in which atoms of the side chain or main chain of the amino acid forming the antigen-binding molecule and atoms of the low-molecular-weight compound form an antigen-binding molecule-MTA interaction at a distance that can affect MTA-binding activity in the presence of the antigen, or a state in which, in embodiments in which the antigen-binding domain in the antigen-binding molecule includes an antibody heavy chain variable region and an antibody light chain variable region, a certain amino acid residue contributes to MTA binding, including indirect effects such as stabilizing the three-dimensional structure of a CDR loop or the like to the conformation when MTA binds in the presence of the antigen, and a state that satisfies both of these conditions. In this specification, the "state in which intermolecular interactions are formed" can be determined, for example, from the crystal structure analysis of a complex of MTA, antigen, and antigen-binding molecule, based on the interatomic distance between the non-hydrogen atoms constituting the side chain or main chain of the amino acid that forms the antigen-binding molecule in the presence of the antigen and the non-hydrogen atoms constituting the MTA. For example, the above interatomic distance is preferably within 3.0 Å, 3.2 Å, 3.4 Å, 3.6 Å, 3.8 Å, 4.0 Å, 4.2 Å, 4.4 Å, 4.6 Å, 4.8 Å, or 5.0 Å, but is not limited thereto. More preferably, the above interatomic distance is within 3.6 Å, 3.8 Å, 4.0 Å, or 4.2 Å. More precisely, the possibility of direct interaction can be determined based on information regarding interatomic distances in the three-dimensional structure, the types of intermolecular interactions formed, and the types of atoms. More accurately, it can be determined from the effect of introducing mutations in amino acid residues, such as modifications to Ala or Gly, on the activity of small molecule compounds, but it is not limited to this. Regarding the "indirectly influencing state" as used herein, for example, it is possible to estimate whether it indirectly affects MTA binding in the presence of an antigen by analyzing in detail the conformation of each amino acid residue and the intermolecular interactions with surrounding residues from the three-dimensional structure of the complex of MTA, antigen, and antigen-binding molecule. More precisely, it can be determined from the effect of introducing mutations into amino acid residues, such as modifications to Ala or Gly, on MTA activity.

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

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

[0201] Furthermore, as an example of an antigen-binding domain that interacts with the MTA, the antigen-binding domain is an antibody variable region, and the amino acid residue that interacts with the MTA is an amino acid residue located at at least one amino acid site selected from the group of amino acid sites at positions 33, 50, 52, 54, 56, 57, 58, 99, 100, 100a, 91, 95c, and 96 of the heavy chain within the amino acid sequence of the antibody variable region, as identified by Kabat numbering. Furthermore, the antigen-binding domain is an antibody variable region, and an example of such an antigen-binding domain may be one that contains at least one amino acid selected from the heavy chain A33, I50, G52, D54, S56, T57, W58, G99, Y100, T100a, the light chain S91, Y95c, and N96 (Kabat numbering).

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

[0203] The antigen-binding molecules of this disclosure may include at least a portion of an Fc region that mediates binding to an Fcγ receptor and / or binding to an FcRn. For example, in one non-limiting embodiment, the antigen-binding molecule may 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 does not naturally link in nature. For example, a fusion protein may include 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 an FcRn). The amino acid sequences may be present in separate proteins that are carried together in the fusion protein, or they may normally be present in the same protein but are incorporated into a new rearrangement in the fusion polypeptide. Fusion proteins may be produced, for example, by chemical synthesis or by recombination techniques that create polynucleotides in which the peptide regions are encoded in the desired relationship and express them.

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

[0205] For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·Ser Ser·Gly·Gly Gly·Gly·Gly·Ser(Sequence ID: 19) Ser·Gly·Gly·Gly (Sequence ID: 20) Gly·Gly·Gly·Gly·Ser(Sequence ID: 21) Ser·Gly·Gly·Gly·Gly(Sequence code:22) Gly·Gly·Gly·Gly·Gly·Ser(Sequence code: 23) Ser·Gly·Gly·Gly·Gly·Gly(Sequence code:24) Gly·Gly·Gly·Gly·Gly·Gly·Ser(Sequence code: 25) Ser·Gly·Gly·Gly·Gly·Gly·Gly(array number:26) (Gly·Gly·Gly·Gly·Ser(Sequence ID:21))n (Ser·Gly·Gly·Gly·Gly(Sequence No. 22))n [where n is an integer greater than or equal to 1] is a preferred example. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0206] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), etc., and these crosslinking agents are commercially available.

[0207] When multiple linkers are used to connect each domain, all linkers may be of the same type, or different types may be used. In addition to the linkers exemplified above, linkers with peptide tags such as His tags, HA tags, myc tags, and FLAG tags may also be used as appropriate. Furthermore, properties that bind to each other through hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or combinations thereof may also be suitably utilized. For example, the affinity between CH1 and CL of an antibody may be utilized, or the Fc region originating from the aforementioned bispecific antibody may be used in the association of hetero-Fc regions. Moreover, disulfide bonds formed between domains may also be suitably utilized.

[0208] To link each domain by peptide bonds, the 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 may be used individually or in combination as appropriate for the preparation of the antigen-binding molecules of this disclosure. In this disclosure, the terms “linked,” “fused,” “linked,” and “fused” are interchangeable. These terms refer to linking two or more elements or components, such as polypeptides, to form a single structure by any means, including the chemical bonding or recombination methods described above. In-frame fusion refers to the linking of two or more reading frame units to form a longer, continuous reading frame that maintains the correct reading frame of the polypeptides, when the two or more elements or components are polypeptides. When two Fab molecules are used as antigen-binding domains, an antibody that is an antigen-binding molecule of this disclosure, in which the antigen-binding domains and a constant region containing the Fc region are linked in-frame by peptide bonds without the use of a linker, may be used as a preferred antigen-binding molecule of this disclosure.

[0209] In another aspect, this 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 concentration of MTA increases. In one aspect, the antigen-binding activity of the antigen-binding molecule is higher in target tissues than in non-target tissues. In several aspects, the antigen-binding molecule is an antibody. While not bound by any particular theory, the changes in plasma dynamics described above can be interpreted as follows: As the antigen-binding activity of the antigen-binding molecule increases in a concentration-dependent manner of MTA, the antigen-binding ability of the antigen-binding molecule in tissues other than the target tissue decreases. As a result, the antigen-dependent elimination (clearance) of the antigen-binding molecule in tissues other than the target tissue decreases. Reduced antigen-dependent elimination (clearance) in most tissues in the body (tissues other than the target tissue) collectively leads to high plasma retention of the antigen-binding molecule. Whether an antigen-binding molecule in the present invention has high plasma retention can be determined by a relative comparison with a control antigen-binding molecule. In some embodiments, an antigen-binding molecule whose antigen-binding activity increases as the concentration of MTA increases has 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 dependent on the concentration of MTA. In a particular embodiment, an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of the compound means an antigen-binding molecule in which the difference in antigen-binding activity in the presence and absence of MTA is, for example, less than 2 times, less than 1.8 times, less than 1.5 times, less than 1.3 times, less than 1.2 times, or less than 1.1 times. From a comparative standpoint, it is desirable that the antigen-binding molecules of this disclosure and the control antigen-binding molecules have substantially equal antigen-binding activity in the presence of a sufficient amount of MTA.

[0210] Here, the magnitude of antigen-dependent disappearance of antigen-binding molecules detected in vivo is thought to change depending on the quantitative balance between antigens and antigen-binding molecules present in the plasma. Generally, the more antigens and the fewer antigen-binding molecules present in the plasma, the easier it is to detect antigen-dependent disappearance of antigen-binding molecules. Conversely, the less antigens and the more antigen-binding molecules present in the plasma, the harder it is to detect antigen-dependent disappearance of antigen-binding molecules. The antigen-binding molecules of this 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 for sufficient antigen-dependent disappearance to be detected. If the amount of antigen in the plasma is low, the amount of antigen may be increased by some artificial means before evaluating plasma retention.

[0211] In another aspect, the present invention provides an antigen-binding molecule having low plasma antigen accumulation capacity. In a further aspect, the antigen-binding activity of the antigen-binding molecule increases as the concentration of MTA increases. In certain embodiments, 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. While not bound by any particular theory, the changes in plasma dynamics described above can be interpreted as follows: As the antigen-binding activity of the antigen-binding molecule increases in a concentration-dependent manner of MTA, the antigen-binding ability of the antigen-binding molecule in tissues other than the target tissue decreases. As a result, the ability of the antigen-binding molecule to form antigen-antibody complexes in tissues other than the target tissue decreases. Generally, it is known that when an antigen-binding molecule such as an antibody binds to an antigen, the clearance of the antigen decreases and the antigen concentration in plasma increases (antigen accumulation). A decrease in the ability to form antigen-antibody complexes in most tissues in the body (tissues other than target tissues) leads, overall, to low antigen accumulation (in other words, low antigen accumulation ability of antigen-binding molecules). In the present invention, it can be determined whether an antigen-binding molecule has low plasma antigen accumulation ability by relative comparison with a control antigen-binding molecule. In some embodiments, an antigen-binding molecule whose antigen-binding activity increases as the concentration of MTA increases has a lower plasma antigen accumulation ability compared to a control antigen-binding molecule. In one embodiment, the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of MTA. In a particular embodiment, an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of MTA means an antigen-binding molecule in which the difference in antigen-binding activity in the presence and absence of the compound is, for example, less than 2 times, less than 1.8 times, less than 1.5 times, less than 1.3 times, less than 1.2 times, or less than 1.1 times. From a comparative standpoint, it is desirable that the antigen-binding molecule of the present invention and the control antigen-binding molecule have substantially equal antigen-binding activity in the presence of a sufficient amount of the compound.

[0212] Here, the amount of antigen-antibody complexes formed in vivo is thought to depend on the amount of antigen and antibody present in the plasma. Generally, the more antigen and antibody levels in the plasma increase, the more antigen-antibody complexes are formed, and conversely, the less antigen and antibody levels in the plasma decrease, the less antigen-antibody complexes are formed. The antigen-binding molecule of the present invention does not need to exhibit low plasma antigen accumulation ability under all conditions; it is sufficient to exhibit low plasma antigen accumulation ability under appropriate conditions that allow for sufficient antigen-antibody complex formation. If the amount of antigen in the plasma is low, the amount of antigen should be increased by some artificial means before evaluating the plasma antigen accumulation ability. That's good too.

[0213] Fcγ receptor (FcγR) An Fcγ receptor (also written as FcγR) is a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and essentially means any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes FcγRI(CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes H131 and R131, i.e., FcγRIIa (H) and FcγRIIa (R)); FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc, which are isoforms FcγRII(CD32); and FcγRIIIa (including allotypes V158 and F158, i.e., FcγRIIIa (V) and FcγRIIIa FcγRIII(CD16), including (F)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR species or FcγR isoforms or allotypes. FcγR may be of any biological origin, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR species include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16) and FcγRIII-2(FcγRIV,CD16-2), as well as any undiscovered mouse FcγR species 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 and amino acid sequences of human FcγRI are assigned to SEQ ID NOs: 9 (NM_000566.3) and 10 (NP_000557.1), respectively. The polynucleotide and amino acid sequences of human FcγRIIa (allotype H131) are assigned to 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 and amino acid sequences of FcγRIIb are assigned to SEQ ID NOs: 9 (NM_000566.3) and 10 (NP_000557.1), respectively. The acid sequences are described in SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIa are described in SEQ ID NOs: 15 (BC033678.1) and 16 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are described in SEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (the numbers in parentheses indicate database registration numbers such as RefSeq). Whether or not the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be confirmed by the FACS and ELISA formats described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

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

[0215] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains an ITIM that transmits inhibitory signals. In B cells, crosslinking between FcγRIIb and the B cell receptor (BCR) suppresses the activation signal from the BCR, resulting in suppression of antibody production by the BCR. In macrophages, crosslinking between FcγRIII and FcγRIIb suppresses phagocytic activity and the production of inflammatory cytokines. Fcγ receptors that have the ability to transmit i...

Claims

1. A library mainly consisting of nucleic acids encoding antigen-binding molecules containing multiple antigen-binding domains with different sequences from each other, and / or antigen-binding molecules containing multiple antigen-binding domains with different sequences from each other, The aforementioned antigen-binding domain is an antigen-binding domain that interacts with 5'-methylthioadenosine (MTA). The antigen to which the aforementioned antigen-binding domain binds is a molecule other than MTA. The antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), The antibody variable region is an antibody variable region modified that has amino acids different from the amino acids located at one or more amino acid sites in the unmodified antibody variable region having binding activity to MTA. The aforementioned unmodified antibody variable region is a library that substantially does not bind to adenosine and / or S-(5'-Adenosyl)-L-homocysteine ​​(SAH).

2. The library according to claim 1, wherein the unmodified antibody variable region is any of the following: a) Antibody variable region including the heavy chain variable region shown in SEQ ID NO: 46 and the light chain variable region shown in SEQ ID NO: 47; b) Antibody variable region including the heavy chain variable region indicated by SEQ ID NO: 50 and the light chain variable region indicated by SEQ ID NO: 51; c) Antibody variable region including the heavy chain variable region shown in SEQ ID NO: 48 and the light chain variable region shown in SEQ ID NO: 49; d) An antibody variable region including the heavy chain variable region shown in SEQ ID NO: 52 and the light chain variable region shown in SEQ ID NO:

53.

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

4. The following steps (a) and (b): (a) A step of identifying an amino acid site in an unmodified antigen-binding domain having binding activity to MTA that satisfies at least one of the following (i) to (vi): (i) Amino acid sites exposed on the surface of the unmodified antigen-binding domain; (ii) Amino acid sites located in regions with a large rate of structural change when comparing the structure of the unmodified antigen-binding domain when bound to MTA with the structure when not bound to MTA; (iii) Amino acid sites not involved in binding with MTA; (iv) Amino acid sites that do not significantly reduce binding to MTA; (v) an amino acid site with diverse amino acid occurrence frequency in the animal species to which the unmodified antigen-binding domain belongs; or (vi) Amino acid sites that are not important for the formation of the canonical structure; (b) A step of designing a library comprising nucleic acids encoding the antigen-binding domain unmodified and nucleic acids encoding multiple variants of the antigen-binding domain unmodified, each having an amino acid modification at one or more amino acid sites identified in step (a), and having different sequences from each other, the antigen-binding domain unmodified, The amino acid modification described above satisfies at least one of the following steps (1) to (3): (1) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to MTA with the structure when not bound to MTA, the rate of structural change at the amino acid site where the amino acid modification is located is large; (2) When comparing the structure of the antigen-binding domain variant having the amino acid modification when bound to MTA with the structure when not bound to MTA, the presence of the amino acid modification does not inhibit the structural change of the antigen-binding domain variant; (3) The antigen-binding domain modified product having the amino acid modification does not have significantly reduced binding activity to MTA compared to the unmodified antigen-binding domain product. Includes, The aforementioned unmodified antigen-binding domain does not substantially bind to adenosine and / or S-(5'-Adenosyl)-L-homocysteine ​​(SAH), A method for producing a library, wherein the unmodified antigen-binding domain and the modified antigen-binding domain are, respectively, an unmodified antibody variable region containing an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and a modified antibody variable region containing an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

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

6. The method according to claim 4 or 5, wherein the unmodified antigen-binding domain is any of the following: a) Antibody variable region including the heavy chain variable region shown in SEQ ID NO: 46 and the light chain variable region shown in SEQ ID NO: 47; b) Antibody variable region including the heavy chain variable region indicated by SEQ ID NO: 50 and the light chain variable region indicated by SEQ ID NO: 51; c) Antibody variable region including the heavy chain variable region shown in SEQ ID NO: 48 and the light chain variable region shown in SEQ ID NO: 49; d) An antibody variable region including the heavy chain variable region shown in SEQ ID NO: 52 and the light chain variable region shown in SEQ ID NO: 53.

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