Antigen-binding molecules whose antigen-binding ability varies depending on the concentration of the compound and libraries thereof

Abstract

The subject of the present invention is to provide: target tissue specific antigen binding molecules, antigen binding activity according to the concentration of non-natural compounds and the antigen binding molecules that change, comprising a library of multiple antigen binding molecules that are different from each other, a pharmaceutical composition comprising the antigen binding molecules, a method for screening the antigen binding molecules, and a method for manufacturing the same, etc. The inventors have created antigen binding domains or antigen binding molecules comprising antigen binding domains whose antigen binding activity depends on the concentration of low molecular weight compounds, and then created multiple antigen binding domains or antigen binding domains that are different from each other, and found that by using the library, the above-mentioned problems can be solved. By using the antigen binding molecules of the present application invention, various diseases caused by target tissues can be treated with target tissue specificity.

Description

[0001] The present application is a divisional application of international application number PCT / JP2014 / 082060, international application date December 4, 2014, Chinese application number 201480074824.1, and invention name “Antigen-binding molecules whose antigen-binding ability varies according to the concentration of the compound and their library”. Technical Field

[0002] The present invention relates to a library of antigen-binding domains or antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity varies according to the concentration of a low-molecular-weight compound. The present invention also relates to antigen-binding domains or antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity varies according to the concentration of a non-natural compound, methods for producing and screening such antigen-binding molecules, and pharmaceutical compositions comprising such antigen-binding molecules. Background Art

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. IgG-type antibody drugs are already widely marketed, and many more are currently under development (Non-Patent Documents 1 and 2).

[0004] As cancer therapeutics using antibody drugs, rituximab for CD20 antigen, cetuximab for EGFR antigen, Herceptin for HER2 antigen, etc. have been approved so far (non-patent document 3). These antibody molecules bind to antigens expressed in cancer cells and exert toxicity against cancer cells through ADCC, etc. It is known that this cytotoxicity based on ADCC, etc. depends on the number of antigens expressed on the target cells of the therapeutic antibody (non-patent document 4). Therefore, from the perspective of the effect of the therapeutic antibody, it is preferred that the expression level of the target antigen is high. However, even if the expression level of the antigen is high, when the antigen is expressed in normal tissues, toxicity such as ADCC will also occur for normal cells, thus causing a problem of large side effects. Therefore, the target antigen of the therapeutic antibody as a cancer therapeutic drug is preferably specifically expressed on cancer cells. For example, antibody molecules against EpCAM antigen, which is known as a cancer antigen, are considered promising as cancer therapeutics, but it is known that EpCAM antigen is also expressed in the pancreas. In fact, it is reported that in clinical trials, by administering anti-EpCAM antibodies, pancreatitis side effects were observed due to cytotoxicity to the pancreas (non-patent document 5).

[0005] Following the success of antibody drugs that exert cytotoxicity based on ADCC activity, reports have been made of second-generation modified antibody molecules that exert potent cytotoxicity through, for example, removal of fucose from the N-type sugar chain of the Fc region of native human IgG1 (Non-Patent Document 6), and enhanced ADCC activity achieved by enhancing binding to FcγRIIIa through amino acid substitution in the Fc region of native human IgG1 (Non-Patent Document 7). As antibody drugs that exert cytotoxic activity against cancer cells through mechanisms other than the aforementioned NK cell-mediated ADCC activity, there have also been reports of modified antibody molecules that exert potent cytotoxicity through, for example, the formation of an Antibody Drug Conjugate (ADC) by conjugating a drug with potent cytotoxicity to an antibody (Non-Patent Document 8), and low-molecular-weight antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to cancer cells (Non-Patent Document 9).

[0006] Even if the antibody molecule that plays a strong cytotoxic role in this way can also play a cytotoxic role on cancer cells that do not express much antigen, on the other hand, it can also play a cytotoxic role on normal tissues that do not express much antigen. In fact, compared with the natural human IgG1 for EGFR antigen, i.e., cetuximab, the bispecific antibody for CD3 and EGFR, i.e., EGFR-BiTE, plays a strong cytotoxic role on cancer cells by supplementing T cells to cancer cells, thereby playing an anti-tumor effect. On the other hand, it has also been confirmed that since EGFR is also expressed in normal tissues, serious side effects occur when EGFR-BiTE is given to cynomolgus monkeys (non-patent literature 10). In addition, the ADC formed by combining the antibody for CD44v6 expressed in large quantities in cancer cells with mertansine, i.e., bivatuzumab mertansine, has been observed to have severe skin toxicity and liver toxicity (non-patent literature 11) in clinical practice because CD44v6 is also expressed in normal tissues.

[0007] Thus, when using antibodies that can exert potent cytotoxicity against cancer cells that express only a small amount of antigen, the target antigen must be expressed with high cancer-specificity. However, as shown by the fact that HER2, the target antigen of Herceptin, and EGFR, the target antigen of cetuximab, are also expressed in normal tissues, it is believed that the number of highly cancer-specifically expressed cancer antigens is limited. Therefore, while these antibodies can enhance cytotoxicity against cancer cells, side effects caused by cytotoxicity to normal tissues also become a problem.

[0008] Furthermore, ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, has recently been shown to prolong overall survival in patients with metastatic melanoma (Non-Patent Document 12). However, since ipilimumab systemically inhibits CTLA4, while tumor immunity is enhanced, severe side effects such as autoimmune disease caused by systemic immune activation have become a problem (Non-Patent Document 13).

[0009] On the other hand, antibody drugs for diseases other than cancer are known to exert therapeutic effects in inflammatory and autoimmune diseases by inhibiting inflammatory cytokines (Non-Patent Document 14). It is also known that, for example, Remicade and Humira, which target TNF, and Actemra, which targets IL-6R, have high therapeutic effects on rheumatoid arthritis, but on the other hand, side effects of infectious diseases are observed due to systemic neutralization of these cytokines (Non-Patent Document 15).

[0010] As a technology applicable to second-generation antibody drugs, various technologies have been developed, and it has been reported that effector function, antigen binding ability, pharmacokinetics, stability are improved, or that immunogenicity risk is reduced (non-patent literature 16), but there is no report for solving the side effects as described above, that the technology capable of target tissue-specific antibody drug effects is capable of. For example, for lesions such as cancerous tissue or inflammatory tissue, it has been reported that there are pH-dependent antibodies (patent literature 1 and 2) that utilize the pH in these target tissues to be acidic conditions. However, the reduction (i.e., the rise in hydrogen ion concentration) of the pH in cancerous tissue or inflammatory tissue compared to normal tissue is very small, and it is difficult to make antibodies that act on the slight rise in the concentration of hydrogen ions with extremely small molecular weight. At the same time, there are also situations in which pH is acidic conditions in normal tissues such as osteoclast bone resorption areas or tissues other than target lesions. It is considered that the conditions of pH are utilized as environmental factors specific to the lesion site and that there are also many problems. On the other hand, it has been reported that there is a method for preparing antibodies that begin to play antigen binding activity by being cut off by a protease expressed in the lesion site such as cancerous tissue or inflammatory tissue (patent literature 3). However, the cleavage of antibodies using proteases is irreversible, so the antibodies that have been cleaved at the lesion site return to normal tissues along with the bloodstream and can also bind to antigens in normal tissues, which is considered a problem. In addition, it is believed that there are also problems with the cancer specificity of such proteases. Therefore, in order to exert the drug effect while avoiding side effects, it is not known that there is a technology that acts reversibly in the cancer or inflammation site that is the lesion site without acting systemically in normal tissues or blood. In addition, it is not known that there is a method for controlling the activity and pharmacological action of antibodies by non-invasive administration of exogenous compounds.

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: International Publication No. WO2003 / 105757

[0014] Patent Document 2: International Publication No. WO2012 / 033953

[0015] Patent Document 3: International Publication No. WO2010 / 081173

[0016] Non-patent literature

[0017] Non-patent literature 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

[0018] Non-patent document 2: The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59(3), 389-396

[0019] Non-patent document 3: Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10(5), 317-327

[0020] Non-patent document 4: Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, Shepard HM, Cancer Immunol. Immunotherapy (1993) 37, 255-263

[0021] De Bono JS, Tolcher AW, Forero A, Vanhove GF, Takimoto C, Bauer RJ, Hammond LA, Patnaik A, White ML, Shen S, Khazaeli MB, RowinskyEK, LoBuglio AF,Clin.Cancer Res.(2004)10(22),7555-7565

[0022] Volume 6: Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies.Satoh M,Iida S,Shitara K.,Expert Opin.Biol.Ther.(2006)6(11),1161-1173

[0023] Drug Discov 7: Optimizing engagement of the immune system by anti-tumor bodies: an engineer's perspective.Desjarlais JR, Lazar GA, Zhukovsky EA, ChuSY., Drug Discov. Today(2007)12(21-22),898-910

[0024] Volume 8:Antibody-drug conjugates:targeted drug delivery forcancer.Alley SC,Okeley NM,Center PD.,Curr.Opin.Chem.Biol.(2010)14(4),529-537

[0025] No. 9:BiTE:Teaching antibodies to engage T-cells for cancertherapy.Baeuerle PA,Kufer P,Bargou R.,Curr.Opin.Mol.Ther.(2009)11(1),22-30

[0026] LutterbueseR,Raum T,Kischel R,Hoffmann P,Mangold S,Rattel B,Friedrich M,Thomas O,Lorenczewski G,Rau.10:T cell-engaging BiTE antibodies specific for EGFRpotently eliminate KRAS-and BRAF-mutated colorectal cancer cells D,Schaller E,Herrmann I,Wolf A,Urbig T,Baeuerle PA,KuferP.,Proc.Natl.Acad.Sci.USA(2010)107(28),12605-12610

[0027] Note 11: Phase I trial of the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma S.,OralOncol.(2008)44(9),823-829

[0028] Expert Opin.Biol.Ther.,(2012)Apr 14(doi:10.1517 / 14712598.2012.675325) Trinh VA,HwuWJ.,Expert Opin.Biol.Ther.

[0029] Issue 13:A NOVEL IMMUNOMODULATING THERAPY OF IPILIMUMAB CAUSINGAUTOIMMUNE HYPOPHYSITIS:A CASE REPORT AND REVIEW.Juszczak A,Gupta A,Karavitaki N,Middleton MR,Grossman A.,Eur.J.Endocrinol.(2012)Apr 10(doi:10.1530 / AXIS-12-0167)

[0030] Non-patent literature 14: The Japanese experience with biologic therapies for rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev. Rheumatol. (2010) 6(11), 644-652

[0031] Non-patent literature 15: Current evidence for the management of rheumatoidarthritis with biological disease-modifying antirheumatic drugs: a systematic literature review informing the EULAR recommendations for the management of RA. Nam JL, Winthrop KL, van Vollenhoven RF, Pavelka K, Valesini G, Hensor EM, Worthy G, Landewe R, Smolen JS, Emery P, Buch MH.,Ann.Rheum.Dis.(2010)69(6),976-986

[0032] Non-patent literature 16: Antibody engineering for the development of therapeutic antibodies. Kim SJ, Park Y, Hong HJ., Mol. Cells. (2005) 20(1), 17-29. Summary of the Invention

[0033] Technical problem to be solved by the invention

[0034] In view of the above background, if antibodies can be obtained whose binding to target antigens is controlled by the concentration of low molecules specifically present or produced in the target tissue (hereinafter, sometimes referred to as "small molecule switch antibodies" in this application specification), such antibodies will be extremely useful because they can act reversibly on lesions such as tumor sites and inflammatory sites while avoiding side effects. In addition, if antibodies can be obtained whose binding to antigens is controlled by the concentration of non-natural compounds, such antibodies will be extremely useful because they can be controlled by activating the activity of the antibody at the lesion site, by administering pharmacological exogenous compounds, or by administering exogenous compounds that can be administered non-invasively.

[0035] However, there have been no reports of obtaining such antibodies by conventional methods of immunizing non-human animals with antigens or by methods utilizing antibody libraries derived from humans and non-human animals.

[0036] Therefore, it is strongly desired to provide antibodies whose binding to any target antigen is controlled by the concentration of low-molecular or non-natural compounds specifically present or produced in the target tissue (low-molecular switch antibodies), and further to provide methods for efficiently obtaining such antibodies in a short period of time.

[0037] Methods used to solve technical problems

[0038] The present inventors have conducted intensive research to achieve the above-mentioned objectives and, as a result, have created antigen-binding molecules comprising antigen-binding domains whose antigen-binding activity varies according to the concentration of a target tissue-specific compound. Furthermore, the present inventors have found that the antigen-binding molecules or pharmaceutical compositions comprising the antigen-binding molecules are useful for treating diseases arising from target tissues, and have also found that the antigen-binding molecules are useful for treating diseases arising from target tissues comprising administering the antigen-binding molecules, and that the antigen-binding molecules are useful in the manufacture of medicines for treating diseases arising from target tissues.

[0039] Furthermore, the present inventors have successfully created a library comprising a plurality of antigen-binding molecules having sequences different from each other, wherein the antigen-binding domains of the antigen-binding molecules contain amino acid residues involved in binding to low molecules, and wherein the low molecules can cause the antigen-binding activity of the antigen-binding molecules to vary depending on environmental factors in the body or on the administration of non-natural compounds. The present inventors have also successfully created a method for screening and producing the antigen-binding molecules using the library, thereby completing the present invention.

[0040] The present invention is based on the above findings and specifically includes the following exemplary embodiments.

[0041] [Scheme 1]

[0042] A library mainly comprising:

[0043] (i) multiple antigen-binding domains or antigen-binding molecules comprising antigen-binding domains that differ in sequence from one another, or

[0044] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0045] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound.

[0046] [Scheme 2]

[0047] The library of scheme [1] is produced by a method comprising the following steps (a) and (b):

[0048] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0049] (i) one or more amino acid sites that are not involved in binding to the low molecular weight compound,

[0050] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs,

[0051] (iii) one or more amino acid positions that are not important for the formation of the canonical structure; and

[0052] Step (b) designing a library comprising: nucleic acids encoding unaltered versions of the antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain, each having an alteration at one or more amino acid positions identified in step (a) and having sequences different from each other.

[0053] [Scheme 3]

[0054] The library described in Scheme [2] is produced by a method comprising the following steps (a) to (d):

[0055] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0056] (i) one or more amino acid sites not involved in binding to the low molecular weight compound,

[0057] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs,

[0058] (iii) one or more amino acid positions that are not important for the formation of the canonical structure;

[0059] Step (b) preparing multiple variants of the antigen-binding domain or antigen-binding molecule comprising the antigen-binding domain, each having a sequence different from another, with alterations at one or more amino acid positions identified in step (a);

[0060] Step (c) identifying one or more amino acid changes that do not substantially change the low molecular compound binding activity of each variant; and

[0061] Step (d) produces a library comprising nucleic acids encoding unaltered variants and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain identified in step (c) that have altered sequences at one or more amino acids.

[0062] [Scheme 4]

[0063] The library described in Scheme [1] is produced by a method comprising the following steps 1) and 2):

[0064] Step 1) contacting a library comprising a plurality of antigen-binding molecules having low-molecular-weight compound-binding activity with a low-molecular-weight compound; and

[0065] Step 2) Nucleic acids encoding multiple variants of antigen-binding molecules having low-molecular-weight compound-binding activity are concentrated from the library.

[0066] [Scheme 5]

[0067] The library of embodiment [4], wherein the antigen-binding molecules are antigen-binding molecules comprising the heavy chain variable region and light chain variable region of an antibody, and are produced by a method comprising any one of the following steps 1) to 3):

[0068] Step 1) Designing a library by concentrating nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound binding activity from the library described in Scheme [4], wherein the library described in Scheme [4] comprises nucleic acids encoding one or more variants in which amino acids in the heavy chain variable region are altered;

[0069] Step 2) Designing a library by concentrating nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound binding activity from the library described in scheme [4], wherein the library described in scheme [4] comprises nucleic acids encoding one or more variants in which amino acids in the light chain variable region are altered;

[0070] In step 3), nucleic acids encoding antigen-binding molecules enriched from the variable region libraries in steps 1) and 2) are combined to design a library.

[0071] [Scheme 6]

[0072] The library according to any one of schemes [1] to [5] is characterized in that the antigen-binding molecule is a fusion polypeptide of an antigen-binding domain and at least a portion of a viral coat protein.

[0073] [Scheme 7]

[0074] The library according to any one of schemes [1] to [5], wherein the antigen-binding molecules are antigen-binding molecules comprising heavy chains and light chains of antibodies, and the library further comprises a step of designing a synthetic library of the heavy chains and / or light chains.

[0075] [Scheme 8]

[0076] The library described in Scheme [7] is characterized in that the heavy chain and / or light chain of the antibody contains a framework sequence derived from the germline.

[0077] [Scheme 9]

[0078] The library according to any one of schemes [1] to [8], wherein the low molecular weight compound is a target tissue-specific compound or a non-natural compound.

[0079] [Scheme 10]

[0080] The library according to any one of schemes [1] to [9], wherein the target tissue is cancer tissue or inflammatory tissue.

[0081] [Scheme 11]

[0082] The library described in Scheme

[10] , wherein the cancer tissue-specific compound is at least one compound selected from the group consisting of nucleosides having a purine ring structure, amino acids and their metabolites, lipids and their metabolites, primary metabolites of sugar metabolism, and nicotinamide and its metabolites.

[0083] [Scheme 12]

[0084] The library according to any one of schemes [1] to

[11] , wherein the low molecular weight compound is kynurenine, adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

[0085] [Scheme 13]

[0086] The library according to any one of claims 1 to 12, wherein the amino acid site not involved in binding to the low-molecular compound is a site other than any one or more of the following amino acids:

[0087] H chain: 97, 100c, 101, 94, 95, 100d, 100e, 33, 50, 52, 56, 57, 58, 99, 100, 100a, 54, 55 (Kabat numbering);

[0088] L chain: 49, 55, 95c, 96, 95a, 95b (Kabat numbering).

[0089] [Scheme 14]

[0090] A method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a low-molecular-weight compound, comprising the following steps (a) to (g):

[0091] Step (a) contacting the library described in any one of Schemes [1] to

[13] with an antigen in the absence of a low molecular weight compound;

[0092] Step (b) selecting the antigen-binding domain that does not bind to the antigen in step (a);

[0093] Step (c) contacting the antigen-binding domain selected in step (b) with an antigen in the presence of a low molecular weight compound;

[0094] Step (d) selecting the antigen-binding domain that binds to the antigen in step (c);

[0095] Step (e) ligating the polynucleotide encoding the antigen-binding domain selected in step (d) to a polynucleotide encoding a polypeptide comprising an Fc region;

[0096] Step (f) culturing the cells into which the vector has been introduced, wherein the vector is operably linked to the polynucleotide obtained in step (e); and

[0097] Step (g) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (f).

[0098] [Scheme 15]

[0099] A method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a low molecular weight compound, comprising the following steps (a) to (e):

[0100] Step (a) contacting the library described in any one of schemes [1] to

[13] with an antigen in the presence of a low molecular weight compound;

[0101] Step (b) using a low-molecular-weight compound at a lower concentration than that in step (a) to dissociate and recover the antigen-binding domain;

[0102] Step (c) ligating the polynucleotide encoding the antigen-binding domain recovered in step (b) to a polynucleotide encoding a polypeptide comprising an Fc region;

[0103] Step (d) culturing the cells into which the vector has been introduced, wherein the vector is operably linked to the polynucleotide obtained in step (c); and

[0104] Step (e) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (d).

[0105] [Scheme 16]

[0106] The method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a low molecular weight compound as described in claim

[14] or

[15] , further comprising the following steps (a) to (b):

[0107] Step (a) contacting the library of any one of schemes [1] to

[13] with a low molecular weight compound; and

[0108] Step (b) selects the antigen-binding domain recovered in step (a).

[0109] [Scheme 17]

[0110] The method for producing an antigen-binding molecule according to any one of schemes

[14] to

[16] , wherein the low molecular weight compound is kynurenine, adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

[0111] [Scheme 18]

[0112] An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a non-natural compound.

[0113] [Scheme 19]

[0114] A pharmaceutical composition comprising the antigen-binding molecule of Scheme

[18] .

[0115] It is obvious to those skilled in the art that combinations of one or more of the above-described aspects are also encompassed by the present invention, as long as there is no technical contradiction based on their common technical knowledge.

[0116] Effects of the Invention

[0117] The antigen-binding domains or antigen-binding molecules comprising the antigen-binding domains of the present invention whose antigen-binding activity varies depending on the concentration of a low-molecular-weight compound, and pharmaceutical compositions comprising the same, do not act systemically in normal tissues or blood but rather act reversibly in target tissues, such as lesions or sites of cancer or inflammation. This allows them to exert their pharmacological efficacy while avoiding side effects and to treat diseases arising from these target tissues.

[0118] Furthermore, by using the library of the present invention comprising a plurality of antigen-binding domains whose sequences differ and whose antigen-binding activity varies depending on the concentration of a low-molecular-weight compound, or antigen-binding molecules comprising such domains, it is possible to obtain various antigen-binding molecules useful for the treatment of tissue-specific diseases as described above in a short period of time.

[0119] In one embodiment of the library of the present invention, it is characterized by identifying amino acid sites in the antigen-binding molecule domain that are not involved in binding to low-molecular compounds, and designing a library containing nucleic acids encoding antigen-binding domains with different sequences in such a way that the amino acid at that site is one to several amino acids. This provides a library of antigen-binding molecules whose antigen-binding ability changes in the presence of compounds, which can be obtained more efficiently than using methods based on immunization of non-human mammals or antibody libraries derived from humans or non-human animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] [ Figure 1 ] is a diagram showing that a small molecule switch antibody does not bind to an antigen in a normal environment where low molecules do not exist, but binds to an antigen in a target tissue where low molecules exist at a high concentration.

[0121] [ Figure 2 ] is a diagram showing that low molecules function as switches by being sandwiched between a low-molecular-weight antibody and an antigen. Without the low molecule, the interaction between the antibody and antigen is insufficient, preventing the antibody from binding to the antigen. However, with the low molecule present, it becomes sandwiched between the antibody and antigen, enabling the antibody to bind to the antigen.

[0122] [ Figure 3 ] is a diagram showing the structure of 2'-adenosine-PEG-peptide, an adenosine analog used for immunization of rabbits.

[0123] [ Figure 4 ] is a diagram showing the structure of 5'-adenosine-PEG-peptide, an adenosine analog used for immunization of rabbits.

[0124] [ Figure 5] is a diagram showing the structure of 2'-adenosine-PEG-biotin obtained by replacing the peptide portion of the adenosine analog used for immunization of rabbits with biotin.

[0125] [ Figure 6 ] is a diagram showing the structure of 5'-adenosine-PEG-biotin obtained by replacing the peptide portion of the adenosine analog used for immunization of rabbits with biotin.

[0126] [ Figure 7 ] is a graph showing the comparative results of the binding activity of various antibodies obtained from rabbit B cell clones toward 2'-adenosine-PEG-biotin. The vertical axis represents the value (N_Bound_100) obtained by dividing the amount of binding when each antibody interacts with 2'-adenosine-PEG-biotin by the amount captured by each antibody (RU), while the horizontal axis represents the value (N_Stability_100) obtained by dividing the value after 60 seconds of dissociation of 2'-adenosine-PEG-biotin from each antibody following interaction with 2'-adenosine-PEG-biotin by the amount captured by each antibody (RU).

[0127] [ Figure 8A ] is a sensorgram showing the binding (interaction) of clone SMB0002 with adenosine by surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction of 100 (Duplicate), 50, 25, 12.5, 6.25, and 3.13 nM adenosine with SMB0002.

[0128] [ Figure 8B ] is a sensorgram showing the binding (interaction) of clone SMB0002 with ATP by surface plasmon resonance analysis. The sensorgrams show the interaction of 5000, 1250, 313, and 78.1 nM ATP with SMB0002, from the top.

[0129] [ Figure 9 ] is a graph showing the results of a competition ELISA showing that clone SMB0002 binds to adenosine and ATP.

[0130] [ Figure 10A ] is a sensorgram showing the binding (interaction) of clone SMB0002 with AMP by surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction between AMP and SMB0002 at 500, 250 (duplicate), 125, 62.5, 31.3, 15.6, and 7.81 μM, respectively.

[0131] [ Figure 10B] is a sensorgram showing the binding (interaction) of clone SMB0002 with ADP from surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction between SMB0002 and ADP at concentrations of 2000, 1000 (duplicate), 500, 250, 125, 62.5, and 31.3 μM, respectively.

[0132] [ Figure 11A ] is a diagram showing the binding mode of the SMB0002 antibody to the adenine ring of adenosine. In the figure, the H chain of the antibody is represented by a thick line, the L chain is represented by a thin line, and adenosine is represented by a ball-and-stick model. The amino acid residues within are represented by stick models. The dotted line indicates the residues between the antibody and the adenine ring. Hydrogen bonds within a distance of .

[0133] [ Figure 11B ] is a diagram showing the binding pattern of the SMB0002 antibody to the ribose moiety of adenosine. In the figure, the H chain of the antibody is represented by a thick line, the L chain is represented by a thin line, and adenosine is represented by a ball-and-stick model. The amino acid residues within are shown in stick models. The dotted line indicates the residues between the antibody and the ribose moiety. The dotted area indicates the region where the phosphate group is expected to exist when AMP is bound.

[0134] [ Figure 12 ] is a sensorgram showing the binding (interaction) of humanized SMB0002 with adenosine from surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction of 200, 100, 50 (duplicate), 25, 12.5, 6.25, and 3.125 nM adenosine with humanized SMB0002.

[0135] [ Figure 13 ] is a sensorgram showing the binding (interaction) between humanized SMB0002 and AMP from surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction between humanized SMB0002 and AMP at concentrations of 500, 250, 125 (duplicate), 62.5, 31.3, 15.6, and 7.8 μM, respectively.

[0136] [ Figure 14 ] is a sensorgram showing the binding (interaction) of humanized SMB0002 with ADP from surface plasmon resonance analysis. The sensorgrams, from the top, show the interaction between humanized SMB0002 and ADP at 1000 (duplicate), 500, 250, 125, and 62.5 μM, respectively.

[0137] [ Figure 15] is a sensorgram showing the binding (interaction) of humanized SMB0002 with ATP from surface plasmon resonance analysis. The sensorgrams, from the top, show the interaction between 1000 (duplicate), 500, 250, 125, and 62.5 μM ATP and humanized SMB0002.

[0138] [ Figure 16 ] is a graph showing the results of ELISA for binding of clone 6RNMSC1-2_F02 to human IL-6R. The vertical axis represents the absorbance value used to evaluate the binding activity of the antibody to human IL-6R based on the presence or absence of each low-molecular-weight substance.

[0139] [ Figure 17 ] is a graph showing the results of ELISA for binding of clone 6RNMSC1-3_G02 to human IL-6R. The vertical axis represents the absorbance value used to evaluate the binding activity of the antibody to human IL-6R based on the presence or absence of each low-molecular-weight substance.

[0140] [ Figure 18 ] is a graph showing the results of ELISA for antibody binding to human IL-6R. The vertical axis represents the absorbance value used to evaluate the binding activity of the antibody to human IL-6R based on the presence or absence of each amino acid or amino acid metabolite.

[0141] [ Figure 19 ] is a sensorgram showing the interaction between 6RNMSC1-2_F02 and 1 μmol / L IL-6R in the presence of 100 μmol / L kynurenine, 10 mmol / L ATP, and the absence of kynurenine and ATP. The solid line represents the interaction in the presence of kynurenine, the dotted line represents the interaction in the presence of ATP, and the dashed line represents the interaction in the absence of kynurenine.

[0142] [ Figure 20 ] In the presence of 100 μmol / L kynurenine, 6RNMSC1-2_F02 was allowed to interact with IL-6R immobilized on sensor chip CM5, and then the dissociation of 6RNMSC1-2_F02 from IL-6R was observed under buffer conditions containing 100 μmol / L kynurenine or a buffer condition without kynurenine. The vertical axis of the figure represents the value obtained by normalizing the binding amount of 6RNMSC1-2_F02 in the presence of 100 μmol / L kynurenine to 100, and the horizontal axis represents the elapsed time (seconds) after the start of the interaction. The solid line represents the dissociation of 6RNMSC1-2_F02 from IL-6R in the presence of kynurenine, and the dotted line represents the dissociation of 6RNMSC1-2_F02 from IL-6R in the absence of kynurenine.

[0143] [ Figure 21] is a graph evaluating the response to IL-6R immobilized on sensor chip CM5, using 5 μg / L 6RNMSC1-2_F02 as the analyte for 180 seconds. The vertical axis represents the change in response (RU) before and after interaction with 6RNMSC1-2_F02, and the horizontal axis represents the kynurenine concentration (μmol / L) in the solution.

[0144] [ Figure 22 Figure 1 is a sensorgram showing the binding (interaction) between clone 6RNMSC1-2_F02 and kynurenine using surface plasmon resonance analysis. From the top, the sensorgrams show the interaction between 6RNMSC1-2_F02 and kynurenine at concentrations of 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 mM, respectively. Kinetic parameters are: ka = 709 (1 / s), kd = 0.17 (1 / s), and KD = 0.239 (mmol / L).

[0145] [ Figure 23 ] is a sensorgram showing the binding (interaction) between clone 6RNMSC1-2_F02 and 3-hydroxy-DL-kynurenine from surface plasmon resonance analysis. The sensorgrams show, from the top, the interaction between 6RNMSC1-2_F02 and 3-hydroxy-DL-kynurenine at concentrations of 0.625, 0.313, 0.156, 0.078, and 0.039 mM, respectively.

[0146] [ Figure 24 ] is a sensorgram showing the binding (interaction) between clone 6RNMSC1-2_F02 and compound RO0635389-000-001 by surface plasmon resonance analysis. The sensorgrams show the interaction between 6RNMSC1-2_F02 and compound RO0635389-000-001 at 0.625, 0.313, and 0.156 mM, respectively, from the top.

[0147] [ Figure 25 ] is a sensorgram showing the binding (interaction) between clone 6RNMSC1-2_F02 and compound RO0635390-000-001 by surface plasmon resonance analysis. The sensorgrams show the interaction between compound RO0635390-000-001 and 6RNMSC1-2_F02 at 0.625, 0.313, and 0.156 mM, respectively, from the top.

[0148] [ Figure 26 ] is an Octet sensorgram showing changes in the binding (interaction) of clone 6RNMSC1-2_F02 to IL6R depending on the presence (solid line) and absence (dashed line) of kynurenine. The vertical axis represents the response to IL6R.

[0149] [ Figure 27 ] is an Octet sensorgram showing changes in the binding (interaction) of clone 6RNMSC1-2_F02 to IL6R depending on the presence (solid line) and absence (dashed line) of 3-hydroxy-DL-kynurenine. The vertical axis represents the response to IL6R.

[0150] [ Figure 28 ] is an Octet sensorgram showing changes in the binding (interaction) of clone 6RNMSC1-2_F02 to IL6R in the presence (solid line) and absence (dashed line) of compound RO0635389-000-001. The vertical axis represents the response to IL6R.

[0151] [ Figure 29 ] is an Octet sensorgram showing changes in the binding (interaction) of clone 6RNMSC1-2_F02 to IL6R in the presence (solid line) and absence (dashed line) of compound RO0635390-000-001. The vertical axis represents the response to IL6R.

[0152] [ Figure 30 ] is a diagram showing the binding pattern of the 6RNMSC1-2_F02 Fab fragment to kynurenine. In the figure, the H chain of the antibody is represented by a thick line, the L chain is represented by a thin line, and kynurenine is represented by a ball-and-stick model. The amino acid residues within are represented by stick models. The dotted line indicates the residues between the antibody and kynurenine. Hydrogen bonds or electrostatic interactions within a distance of 1.

[0153] [ Figure 31 Figure 1 is a sensorgram showing the binding (interaction) of the H49Y variant of clone 6RNMSC1-2_F02 with kynurenine from surface plasmon resonance analysis. The sensorgrams, from the top, show the interaction between kynurenine and 6RNMSC1-2_F02H49Y at concentrations of 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 mM, respectively. Kinetic parameters are: ka = 2543 (1 / s), kd = 0.24 (1 / s), and KD = 0.095 (mmol / L).

[0154] [ Figure 32 ] is a sensorgram showing the binding (interaction) between clone F02h011 / F02l003 and kynurenine, in which a mutation was introduced into the framework sequence of 6RNMSC1-2_F02 to restore the germline sequence. The sensorgrams, from the top, show the interaction between F02h011 / F02l003 and kynurenine at concentrations of 1000, 500, 250, 125, and 62.5 μM, respectively.

[0155] [ Figure 33 ] is a sensorgram showing the binding (interaction) between F02h011 / F021098, a clone modified to enhance kynurenine binding to F02h011 / F021003, and kynurenine. The sensorgrams, from the top, show the interaction between F02h011 / F021098 and kynurenine at concentrations of 500, 250, 125, 62.5, 31.3, and 15.6 μM, respectively.

[0156] [ Figure 34 ] is a diagram showing the binding pattern of the 6RNMSC1-2_F02 Fab fragment to kynurenine. In the figure, the heavy chain of the antibody is represented by a black thick line, the light chain is represented by a gray thin line, and kynurenine is represented by a ball-and-stick model. The amino acid residues within are represented by stick models.

[0157] [ Figure 35 Figure 1 is a diagram showing the binding pattern of the 6RNMSC1-2_F02 Fab fragment to kynurenine. In the figure, the heavy chain of this antibody is represented by a thick black line, the light chain by a thin gray line, and kynurenine is represented by a ball-and-stick model. Light chain Ser56 (Kabat numbering) is represented by a stick model. The dotted line and the number above the dotted line represent the shortest distance between the non-hydrogen atoms of light chain Ser56 and kynurenine.

[0158] [ Figure 36 Figure 1 is a diagram showing the binding pattern of the 6RNMSC1-2_F02 Fab fragment to kynurenine. The heavy chain of this antibody is represented by a thick black line, the light chain by a thin gray line, and kynurenine is represented by a ball-and-stick model. Gly50 on the heavy chain and Asp28 on the light chain (Kabat numbering) are represented by stick models.

[0159] [ Figure 37 ] is a graph showing the binding amount (Binding response (RU)) when 1 μM of each clone was allowed to interact with IL-6R immobilized on the sensor chip CM5 for 120 seconds in the presence or absence of 1 mM of each low molecular weight agent.

[0160] [ Figure 38 ] is a graph showing the binding response (RU) when 10 μg / mL of each clone was allowed to interact with IL-6R immobilized on an Octet sensor for 120 seconds in the presence or absence of 1 mM of each low molecular weight agent.

[0161] [ Figure 39] is a graph showing the ELISA results for clones 6RFHm12-4_040, 6RFHm12-4_078, 6RFHm14-4_087, 6RFHm14-4_093, 6RFHm17-4_006, and 6RFHm17-4_010 obtained from the kynurenine library Ver.A against hIL-6R under various conditions. 6RNMSC1-2_F02 was used as a positive control. The vertical axis represents the absorbance value used to evaluate the hIL-6R binding activity of the antibodies. Details of each condition are shown in Table 38.

[0162] [ Figure 40 ] is a graph showing the ELISA results for hIgA-Fc under various conditions for clones hIAFHm12-4_018, hIAFHm12-4_061, hIAFHm14-4_001, hIAFHm14-4_041, hIAFHm17-4_026, and hIAFHm17-4_072 obtained from the kynurenine library Ver.A. The vertical axis represents the absorbance value used to evaluate the hIgA-Fc binding activity of the antibodies. The details of each condition are shown in Table 41.

[0163] [ Figure 41 ] is a graph showing the ELISA results for clones I6FHm12-4_068, I6FHm12-4_094, I6FHm14-4_007, I6FHm14-4_030, I6FHm17-4_016, and I6FHm17-4_036 obtained from the kynurenine library Ver.A against hIL-6 under various conditions. The vertical axis represents the absorbance value used to evaluate the hIL-6 binding activity of the antibodies. The details of each condition are shown in Table 44.

[0164] [ Figure 42 ] is a graph evaluating the ability of ATP to inhibit the binding of ATNLSA1-4_D12 to biotin-labeled antigen (5'-adenosine-PEG-biotin, a mixture of ATP-PEG-biotin).

[0165] [ Figure 43 ] is a diagram showing the concept of rationally designed antibody library that can obtain low-molecular-weight switch antibodies for any antigen by sandwiching a low-molecular-weight switch between the antibody and the antigen and libraryizing the antibody variable region portion that contacts the antigen.

[0166] [ Figure 44] is a graph showing the results of ELISA against human IL-6 by clone I6RLSA1-6_011, obtained from a rationally designed antibody library using antibodies that bind to ATP / adenosine as templates, in the presence or absence of 10 mM ATP and adenosine. The vertical axis represents the absorbance value used to evaluate the antibody's human IL-6 binding activity. A clone obtained from the rationally designed antibody library that exhibited human IL-6 binding activity regardless of the presence or absence of low-molecular-weight antibodies was used as a positive control. M13KO7 helper phage was used as a negative control.

[0167] [ Figure 45 ] is a graph showing the results of ELISA for the human IL-6 receptor in the presence and absence of 10 mM ATP and adenosine using clones 6RRLSA1-6_037 and 6RRLSA1-6_045, obtained from a rationally designed antibody library using antibodies that bind to ATP / adenosine as templates. The vertical axis represents the absorbance value used to evaluate the human IL-6 receptor binding activity of the antibodies. M13KO7 helper phage was used as a negative control (denoted as nega in the figure).

[0168] [ Figure 46 ] is a graph showing the results of ELISA for HSA in the presence or absence of 10 mM ATP and adenosine using clone HSADSA1-6_020, obtained from a rationally designed antibody library using antibodies that bind to ATP / adenosine as templates. The vertical axis represents the absorbance value used to evaluate the antibody's HSA binding activity. A clone obtained from the rationally designed antibody library that exhibited human HSA binding activity regardless of the presence or absence of low-molecular-weight antibodies was used as a positive control. M13KO7 helper phage was used as a negative control. DETAILED DESCRIPTION

[0169] The following definitions and detailed description are provided to facilitate understanding of the invention described in this specification.

[0170] amino acids

[0171] In the present specification, amino acids are represented by single-letter codes or three-letter codes, or both, for example, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V.

[0172] Amino acid changes

[0173] In order to change the amino acids in the amino acid sequence of the antigen-binding molecule, known methods such as site-specific mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR can be suitably used. In addition, as a method for amino acid mutation replacement with amino acids other than natural amino acids, various known methods can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) in which a tRNA formed by binding a non-natural amino acid to a complementary amber suppressor tRNA containing the UAG codon (amber codon), which is one of the stop codons, can also be preferably used.

[0174] In this specification, the term "and / or" used to indicate the site of amino acid modification includes all appropriate combinations of "and" and "or". Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes variants with the following amino acid modifications:

[0175] (a) 33 positions, (b) 55 positions, (c) 96 positions, (d) 33 and 55 positions, (e) 33 and 96 positions, (f) 55 and 96 positions, (g) 33, 55, and 96 positions.

[0176] In addition, in this specification, as a representation of an amino acid change, it is also possible to appropriately use a representation in which the single-letter code or three-letter code of the amino acid before and after the change is recorded before and after the number representing a specific position. For example, the change N100bL or Asn100bLeu used when adding an amino acid replacement contained in an antibody variable region represents that the Asn at position 100b, as represented by Kabat numbering, is replaced with Leu. That is, the number represents the position of the amino acid represented by Kabat numbering, the single-letter code or three-letter code of the amino acid before it represents the amino acid before the replacement, and the single-letter code or three-letter code of the amino acid after it represents the amino acid after the replacement. Similarly, the change P238D or Pro238Asp used when adding an amino acid replacement to the Fc region contained in an antibody constant region represents that the Pro at position 238, as represented by EU numbering, is replaced with Asp. That is, the numbers indicate the positions of amino acids expressed by EU numbering, the single-letter code or three-letter code of the amino acid preceding them indicates the amino acid before substitution, and the single-letter code or three-letter code of the amino acid following them indicates the amino acid after substitution.

[0177] antigen

[0178] In this specification, the structure of "antigen" is not limited to a specific structure as long as it contains the epitope bound by the antigen binding domain. In another sense, the antigen can be an inorganic substance or an organic substance. As antigens, the following molecules can be exemplified: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, 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-2BMP-2a, BMP-3 osteogenic factor (O steogenin), BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C 5a, 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, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (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, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2 , CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, C XCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement inhibitory factor (decay-promoting factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, Eotaxin 1, EpCAM,Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Activation Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrinogen, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, cleavage factor, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF -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-α1, GFR-α2, GFR-α3, 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 IIIBgp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human myocardial globulin, 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)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 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, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MI P, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adhesion factor, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4, or neurotrophin-6, Neurturin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF,PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaloids Phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific (pan-specific TGF-β), TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, 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 (BAFFR), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L(RELT),TNFRSF1A (TNF RICD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbRTNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (FasApo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23(DcTRAILR1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHTHVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a adhesin (Conectin), DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125,Tumor-associated antigen expression Lewis-Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT 5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prokaryotic cell death protein kinase (KKL), RON, TMEM16F, SOD1, chromogranin White A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, Thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones and growth factors. Antigens are preferably those expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues or inflammatory tissues.

[0179] Receptors are also described in the examples of the above antigens. However, when these receptors are present in soluble forms in biological fluids, they can also be used as antigens bound by antigen-binding molecules of the present invention comprising antigen-binding domains whose antigen-binding activity varies depending on the concentration of low molecular weight compounds (e.g., target tissue-specific compounds). As a non-limiting example of such soluble receptors, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence shown in SEQ ID NO: 1, described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), can be exemplified.

[0180] Examples of the above antigens also include membrane-bound molecules expressed on cell membranes and soluble molecules secreted from cells. When an antigen-binding molecule of the present invention comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a target tissue-specific compound binds to a soluble molecule secreted from cells, the antigen-binding molecule preferably has neutralizing activity as described below.

[0181] There is no limitation on the solution present for soluble molecules, and the soluble molecules can be present in biological fluids, i.e., all liquids that fill the vascular or tissue cell spaces in the organism. In a non-limiting embodiment, the soluble molecules combined by the antigen binding molecules of the present invention can be present in extracellular fluid. With respect to vertebrates, extracellular fluid refers to the composition in the bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid or joint fluid, alveolar fluid (bronchoalveolar washing fluid), ascites, pleural effusion, pericardial fluid, cystic fluid or aqueous humor (aqueous humor) and other cell permeable fluids (liquids in various glandular cavities produced by the result of the active transport and secretion activity of cells and other body cavity fluids such as the lumen of the digestive tract).

[0182] When the antigen binding molecules of the present invention comprising antigen binding domains whose antigen binding activity varies according to the concentration of low molecular weight compounds (e.g., target tissue-specific compounds) are combined with membrane-type molecules expressed in cell membranes, preferred examples of the antigen binding molecules include antigen binding molecules with cytotoxicity as described later, or antigen binding molecules with cell-damaging substances or antigen binding molecules with the ability to combine cell-damaging substances. In addition, alternatives having cytotoxicity, or combining cell-damaging substances or having the ability to combine cell-damaging substances, or in addition to this property, antigen binding molecules with neutralizing activity can also be preferably cited as a non-limiting scheme.

[0183] Epitope

[0184] An epitope, representing an antigenic determinant present in an antigen, refers to the site on the antigen to which the antigen-binding domain in the antigen-binding molecules disclosed in this specification binds. Therefore, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by its binding activity against an antigen in an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can also be defined by the amino acid residues that constitute the epitope. Furthermore, when the epitope is a sugar chain, the epitope can also be defined by the specific sugar chain structure.

[0185] A linear epitope is an epitope that comprises a recognized primary sequence of amino acids. A linear epitope typically contains at least 3 amino acids in the native sequence, and most commonly contains at least 5, such as about 8 to about 10, 6 to 20 amino acids.

[0186] In contrast to linear epitopes, three-dimensional structural epitopes are epitopes in which the primary sequence of amino acids containing the epitope is not a single specified component of the recognized epitope (for example, the primary sequence of amino acids does not need to be the epitope recognized by the antibody that specifies the epitope). Three-dimensional structural epitopes can contain an increased number of amino acids relative to linear epitopes. Regarding the recognition of three-dimensional structural epitopes, antibodies recognize the three-dimensional structure of peptides or proteins. For example, when protein molecules fold to form a three-dimensional structure, certain amino acids and / or polypeptide main chains that form the three-dimensional structural epitope become side by side, allowing antibodies to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-specific rotation labeling, and electron paramagnetic resonance spectroscopy, but are not limited thereto. For example, reference is made to Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0187] The structure of the antigen-binding domain that binds to an epitope is called a paratope. The epitope and the paratope are stably bound by hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between the epitope and the paratope. The binding force between the epitope and the paratope is called affinity. The sum of the binding forces when multiple antigens bind to multiple antigen-binding molecules is called avidity. When antibodies (i.e., multivalent) containing multiple antigen-binding domains bind to multiple epitopes, the binding force (affinity) works synergistically, so avidity is higher than avidity.

[0188] Binding activityThe following exemplifies methods for confirming epitope binding using test antigen-binding molecules containing an antigen-binding domain against IL-6R. However, methods for confirming epitope binding using test antigen-binding molecules containing antigen-binding domains against antigens other than IL-6R can also be appropriately performed based on the following exemplifications.

[0189] For example, whether the tested antigen binding molecule containing the antigen binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed by the following operation. For the above purpose, a linear peptide comprising the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. The peptide can be chemically synthesized. Alternatively, the region encoding the amino acid sequence equivalent to the extracellular domain in the cDNA of IL-6R can be obtained by genetic engineering technology. Next, the binding activity of the linear peptide comprising the amino acid sequence constituting the extracellular domain and the tested antigen binding molecule containing the antigen binding domain for IL-6R is evaluated. For example, by ELISA using an immobilized linear peptide as an antigen, the binding activity of the antigen binding molecule to the peptide can be evaluated. Alternatively, based on the level of inhibition caused by the linear peptide in the binding of the antigen binding molecule to IL-6R expressing cells, the binding activity to the linear peptide can also be clearly determined. Through these tests, the binding activity of the antigen binding molecule to the linear peptide can be clearly determined.

[0190] Alternatively, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a three-dimensional epitope can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. Examples include situations where a test antigen-binding molecule containing an antigen-binding domain for IL-6R strongly binds to IL-6R-expressing cells when in contact with the cells, but the antigen-binding molecule does not substantially bind to an immobilized linear peptide containing the amino acid sequence that constitutes the extracellular domain of IL-6R. Here, "substantially not binding" means a binding activity of less than 80%, typically less than 50%, preferably less than 30%, and particularly preferably less than 15% of the binding activity to human IL-6R-expressing cells.

[0191] Examples of methods for measuring the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain to IL-6R-expressing cells include the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, evaluation can be performed using the principles of ELISA or FACS (fluorescence activated cell sorting) using IL-6R-expressing cells as antigens.

[0192] In the ELISA format, the binding activity of the test antigen binding molecule containing the antigen binding domain for IL-6R to IL-6R expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzyme reaction. That is, the test polypeptide complex is added to an ELISA plate fixed 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, a dilution series of the test antigen binding molecule is prepared, and the antibody binding titer relative to IL-6R expressing cells is determined, thereby comparing the binding activity of the test antigen binding molecule to IL-6R expressing cells.

[0193] Binding of test antigen-binding molecules to antigens expressed on the surface of cells suspended in a buffer solution, etc. can be detected by flow cytometry. Examples of known flow cytometry instruments include the following.

[0194] FACSCanto TM II

[0195] FACSAria TM

[0196] FACSArray TM

[0197] FACSVantage TM SE

[0198] FACSCalibur TM (Both are trade names of BD Biosciences)

[0199] EPICS ALTRA HyPerSort

[0200] Cytomics FC 500

[0201] EPICS XL-MCL ADC EPICS XL ADC

[0202] Cell Lab Quanta / Cell Lab Quanta SC (both trade names of Beckman Coulter)

[0203] For example, an example of a preferred method for determining the binding activity of a test antigen binding molecule containing an antigen binding domain for IL-6R to an antigen can include the following method. First, cells expressing IL-6R are reacted with the test antigen binding molecule and stained with a secondary antibody labeled with FITC that recognizes the test antigen binding molecule. The test antigen binding molecule is diluted with a suitable preferred buffer, whereby the antigen binding molecule is prepared to a desired concentration for use. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. Then, fluorescence intensity and cell number are measured using FACSCalibur (BD). The amount of antibody binding to the cell is reflected as the fluorescence intensity, i.e., the geometric mean, obtained by analyzing using CELL QUEST Software (BD). That is, by obtaining the geometric mean, the binding activity of the test antigen binding molecule represented by the binding amount of the test antigen binding molecule can be measured.

[0204] Whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be tested by cross-blocking assays, for example. For example, a competition ELISA assay is a preferred cross-blocking assay.

[0205] Specifically, in a cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is incubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to the IL-6R protein in the wells is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule competing 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 wells coated with IL-6R protein.

[0206] The amount of the tested antigen binding molecules bound to the wells via the IL-6R protein can be easily determined by pre-labeling the antigen binding molecules. For example, biotin-labeled antigen binding molecules are determined using avidin peroxidase conjugates and suitable substrates. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen binding molecules can be labeled with other detectable or measurable markers. Specifically, radiolabeled or fluorescent labels are known.

[0207] If a competing antigen-binding molecule can block the binding of a test antigen-binding molecule containing an antigen-binding domain for IL-6R by at least 20%, preferably at least 20-50%, and even more preferably at least 50%, as compared to the binding activity obtained in a control experiment performed in the absence of the candidate competing antigen-binding molecule complex, then the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competing antigen-binding molecule, or competes for binding to the same epitope.

[0208] When identifying the structure of the epitope bound by a test antigen-binding molecule containing an antigen-binding domain for IL-6R, whether the test antigen-binding molecule and a control antigen-binding molecule share the same epitope can be evaluated by comparing the binding activity of the two antigen-binding molecules toward a peptide obtained by introducing amino acid mutations into the peptide constituting the epitope.

[0209] As the method for this determination binding activity, for example, in the aforementioned ELISA format, the binding activity of the linear peptide having introduced a mutation can be measured by comparing the tested antigen binding molecules and the control antigen binding molecules. As the method beyond the ELISA, after the tested antigen binding molecules and the control antigen binding molecules are flowed through the post, the antigen binding molecules eluted in the eluent are quantitatively measured, and the binding activity of the mutant peptide bound to the post can also be measured. It is well known that the method for adsorbing the mutant peptide on the post is known as the form of a fusion peptide with GST.

[0210] In addition, when the identified epitope is a three-dimensional epitope, whether the tested antigen binding molecule and the control antigen binding molecule share the same epitope can be evaluated by the following method. First, prepare cells expressing IL-6R and cells expressing IL-6R in which a mutation is introduced into the epitope. These cells are suspended in a suitable buffer such as PBS, and the tested antigen binding molecule and the control antigen binding molecule are added to the obtained cell suspension. Then, they are washed with a suitable buffer and an antibody labeled with FITC that can recognize the tested antigen binding molecule and the control antigen binding molecule is added to the obtained cell suspension. The fluorescence intensity and cell number of the cells stained with the labeled antibody are measured by FACSCalibur (BD company). The concentration of the tested antigen binding molecule and the control antigen binding molecule is appropriately diluted by a suitable buffer, and thus can be prepared as the desired concentration for use. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. The binding amount of the labeled antibody to the cell is reflected as the fluorescence intensity, i.e., the geometric mean, obtained by analyzing using CELLQUEST Software (BD company). That is, by obtaining the geometric mean, the binding activities of the test and control antigen-binding molecules, represented by the amount of labeled antibody bound, can be measured.

[0211] In this method, for example, "not substantially binding to mutant IL-6R expressing cells" can be judged 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 a FACSCalibur as a flow cytometer is used for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The comparative value (ΔGeo-Mean) is calculated by the following formula 1 from the geometric mean in the presence and absence of the polypeptide complex, thereby determining the ratio of increase in fluorescence intensity caused by the binding of the antigen-binding molecule.

[0212] (Formula 1)

[0213] ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0214] The geometric mean comparative value (ΔGeo-Mean value of mutant IL-6R molecules) reflecting the amount of binding of the test antigen-binding molecule to mutant IL-6R-expressing cells obtained by analysis is compared with the ΔGeo-Mean comparative value reflecting the amount of binding of the test antigen-binding molecule to IL-6R-expressing cells. In this case, the concentrations of the test antigen-binding molecule used to calculate the ΔGeo-Mean comparative value for mutant IL-6R-expressing cells and IL-6R-expressing cells are particularly preferably prepared to be the same or substantially the same. Antigen-binding molecules whose recognition of an epitope in IL-6R is predetermined are used as control antigen-binding molecules.

[0215] A test antigen-binding molecule is considered to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value of the test antigen-binding molecule relative to mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% less than the ΔGeo-Mean comparison value of the test antigen-binding molecule relative to 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). By comparing the comparison values, as long as they can be considered substantially identical, it can be assessed that the test antigen-binding molecule and the control antigen-binding molecule have the same epitope.

[0216] target tissue

[0217] The term "target tissue" used in this specification refers to the tissue of the cell comprising the antigen to which the antigen-binding molecules of the present invention are present and which is combined according to the concentration of low-molecular-weight compounds, and refers to the tissue that the combination of the antigen-binding molecules and the membrane-type molecules expressed in the cell or the combination of the soluble molecules present in the tissue brings positive pharmacological effects to the body comprising the tissue. At this time, "positive pharmacological effects" refer to the alleviation, alleviation, improvement or healing of the symptoms caused by the pathological site comprising the target tissue to the body comprising the tissue. As a scheme for the non-limiting mechanism of this pharmacological effect, for example, in the case of the symptoms caused by malignant tumors such as cancer, cytotoxicity and proliferation inhibition for cancer cells and immune activation in cancerous tissues can be exemplified. As a scheme for this non-limiting mechanism, for example, in the case of inflammatory diseases, blocking activity and immunosuppression of the effects of inflammatory cytokines in inflammatory tissues can be exemplified.

[0218] Cancer tissue-specific compounds

[0219] The term "cancer tissue specific compound (cancer tissue specific compound)" used in this specification refers to a compound that is differentially present in cancer tissue compared to non-cancerous tissue. In this specification, the term "cancer" is generally used to refer to a malignant neoplasm, which can be metastatic or non-metastatic. For example, as non-limiting examples of cancers produced by epithelial tissues such as the digestive tract and skin, there can be exemplified: brain tumors, skin cancer, head and neck cancer, esophageal cancer, lung cancer, gastric cancer, duodenal cancer, breast cancer, prostate cancer, cervical cancer, endometrial cancer, pancreatic cancer, liver cancer, colorectal cancer, colon cancer, bladder cancer, and ovarian cancer. In addition, as non-limiting examples of sarcomas produced by non-epithelial tissues (stroma) such as muscle, there can be exemplified: osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Furthermore, non-limiting examples of hematological tumors originating from hematopoietic organs include malignant lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma, leukemias including acute myelocytic leukemia or chronic myelocytic leukemia, and acute lymphatic leukemia or chronic lymphatic leukemia, and multiple myeloma. The term "neoplastic" as used broadly in this specification also refers to any newly generated pathological tissue tumor. In the present invention, a neoplasm causes the formation of a tumor, which is characterized in part by angiogenesis. Neoplasms can be benign, such as hemangiomas, gliomas, teratomas, etc., or malignant, such as carcinomas, sarcomas, gliomas, astrocytomas, neuroblastomas, retinoblastomas, etc.

[0220] The term "cancerous tissue" refers to tissue containing at least one cancer cell. Thus, for example, if cancerous tissue contains cancer cells and blood vessels, it refers to all cell types that contribute to the formation of a tumor mass containing cancer cells and endothelial cells. In this specification, a tumor refers to a foci of tumor tissue. The term "tumor" is generally used to refer to either benign or malignant neoplasms.

[0221] For example, in several embodiments, a cancer tissue-specific compound can be a compound defined by qualitative cancer tissue specificity, such as being present in cancer tissue but not in non-cancerous tissue, or being absent in cancer tissue but present in non-cancerous tissue. In other embodiments, a cancer tissue-specific compound can be a compound defined by quantitative cancer tissue specificity, such as being present in cancer tissue at a different concentration (e.g., a higher concentration or a lower concentration) compared to non-cancerous tissue. For example, a cancer tissue-specific compound can be present at any differential concentration. Generally, however, a cancer tissue specific compound can increase cancer tissue specificity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 100-fold. 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 The cancer tissue specific compound is preferably present at a concentration that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to at least 100% (i.e., absent). Preferably, the cancer tissue specific compound is differentially present at a statistically significant concentration (i.e., a p value of less than 0.05 and / or a q value of less than 0.10, as determined using either a Welch's t-test or a Wilcoxon rank sum test). As a non-limiting embodiment of the cancer tissue-specific compound, the following compounds can be exemplified, which are cancer tissue-specific metabolites produced by unique metabolic activities in cancer cells, immune cells, and stromal cells contained in the cancer tissue as described below (cancer tissue-specific metabolites; cancer cell-specific metabolites, metabolites specific to immune cells infiltrating cancer tissue, and cancer stromal cell-specific metabolites).

[0222] The term "non-natural compound" used in this specification refers to a chemical substance of non-natural origin and its metabolite. As one embodiment of the invention, it is a chemical substance of non-natural origin and its metabolite that has the property of accumulating in target tissues after being administered from outside the body to the body. Examples of non-natural compounds include (1) Capecitabine (Xeloda) and 5-FU (fluorouracil) as its metabolite, and (2) TH-302 and brominated isophosphoramide 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). In addition, it is also known that TH-302 is converted into Br-IPM by reduction under hypoxic conditions such as those around cancer tissue (Duan JX, et al. J Med Chem. 2008). For example, when capecitabine (xeloda) is administered, it is metabolized to 5-FU by cancer-specific metabolic enzymes, etc., and thus the concentration of 5-FU in the cancer local area becomes high (Desmoulin F. et al. Drug Metab Dispos. 2002), so it is believed that by using 5-FU as a switch antibody, it is possible to selectively bind to the target antigen only in the cancer local area. In addition, in addition to metabolic enzymes, it is believed that molecules generated by cancer-specific hypoxic environments and acidic environments can also be used as switches. For example, TH-302 (Duan JX, et al. J Med Chem. 2008) is metabolized to Br-IPM under hypoxic conditions, and thus it is believed that using Br-IPM as a switch antibody can selectively bind to the target antigen only in the cancer local area. For example, as a method of administering a non-natural compound to the body, oral administration, eye drops, transdermal administration, nasal administration, intravenous administration, pulmonary administration and other known administration methods can be cited, but are not limited thereto.

[0223] As used herein, the term "non-natural compound" encompasses, in addition to chemical substances and their metabolites that have the property of accumulating in target tissues, chemical substances that can control the action of antibodies by, for example, administering the non-natural compound as a switch, for example, orally, thereby controlling the action of an antibody through oral administration. Specifically, a switch antibody that binds to a specific antigen is administered invasively, such as by intravenous or subcutaneous administration, using a non-natural compound as a switch. Subsequently, an exogenous compound acting as a switch is administered non-invasively, such as orally, thereby controlling the action of the antibody. Examples of such compounds include, but are not limited to, ATPγS and kynurenine metabolites.

[0224] Antibody drugs have a long half-life, so the duration of their effects can be a disadvantage when side effects occur. However, if the effects of antibodies can be controlled through non-invasive administration, such as oral administration of non-natural compounds, as described above, the drug's effects can be terminated by stopping administration of the switch molecule when side effects occur. Furthermore, by pre-administering a switch antibody, the switch molecule can be administered only when symptoms of a disease occur, allowing the pharmacological effects to be exerted only when necessary through non-invasive administration, such as oral administration.

[0225] The "antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a non-natural compound" of the present invention can exert positive pharmacological effects when administered to an organism.

[0226] Cancer tissue-specific metabolites

[0227] The term "metabolism" refers to the chemical changes that occur in the tissues of organisms, including "assimilation" and "dissimilation". Assimilation refers to the biosynthesis or accumulation of molecules, and dissimilation refers to the decomposition of molecules. "Metabolites" are intermediates or products resulting from the metabolism of substances. "Primary metabolites" refer to metabolites that are directly related to the growth or reproduction process of cells or organisms; "secondary metabolites" refer to products such as antibiotics or pigments that are not directly related to their growth or reproduction process, but are the result of the metabolism of substances that are not directly related to the biosynthesis and common life phenomena in cells or organisms. Metabolites can be metabolites of "biopolymers" or "low molecular weight" metabolites. "Biopolymers" are macromolecules containing more than one repeating unit. Biopolymers are commonly found in biological systems and include molecules with a molecular weight of approximately 5,000 or more that form structures such as cells of organisms and the intercellular matrix and interstitial matrix adhering thereto, particularly polysaccharides (such as carbohydrates), peptides (this term is used to include polypeptides and proteins), and polynucleotides, as well as their analogs, such as compounds composed of amino acid analogs or non-amino acid groups, or compounds containing amino acid analogs or non-amino acid groups.

[0228] The term "low molecule" used in this specification refers to a chemical substance of natural origin or a chemical substance of non-natural origin other than the "biopolymer" present in the body. Preferably, it is a target tissue-specific compound or a non-natural compound, but it is not limited thereto. As a non-limiting embodiment of the cancer tissue-specific metabolites described in this specification, low molecular weight metabolites specific to cancer cells (Eva Gottfried, Katrin Peter and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132) can be appropriately cited. Furthermore, it also includes metabolites produced in large quantities by immune cells infiltrating cancer tissue or metabolites produced in large quantities by stromal cells (cancer stromal cells or cancer stromal fibroblasts (CAF)) that support the survival and / or growth of cancer cells. As infiltrating immune cells, dendritic cells, suppressive dendritic cells, suppressive T cells, exhausted T cells (exhausted T cell), myeloma derived suppressor cells (myeloma derived suppressor cell, MDSC) and the like can be exemplified. Furthermore, the metabolites of the present invention also include compounds that are released from within cells (cancer cells, immune cells, stromal cells) present in cancer tissues to the extracellular space when cells die due to apoptosis, necrosis, or the like.

[0229] In order to identify cancer cell-specific metabolites, in addition to analysis 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), or analysis at the proteome level (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15, Hood et al. (Mol. Cell. Proteomi cs (2005) 4, 1741-1753), a metabolic analysis centered on metabolic analysis is also preferably used. That is, in order to identify metabolites in a test sample, a metabolic analysis using high pressure liquid chromatography (HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol. Recognit. (1997) 10, 182-187), mass spectrometry (Gates and Sweeley (Clin. Chem. (1978) 24, 1663-1673) (GC / MS and LC / MS)) and ELISA, etc., alone or in combination, can be used.

[0230] These studies have elucidated the heterogeneity within tumors caused by changes in the concentration gradients of metabolites (such as glucose or oxygen) and growth factors that enable cancer cells to grow under low oxygen tension conditions (Dang and Semenza (Trends Biochem. Sci. (1999) 24, 68-72)). In these studies, cell line models were used to understand the changes in energy utilization pathways caused by different degrees of tumor malignancy (Vizan et al. (Cancer Res. (2005) 65, 5512-5515). As a non-limiting example of the components of the metabolomics platform technology, there is the sample extraction, separation, detection, spectroscopic analysis, data normalization, description of class-specific metabolites, pathway mapping, confirmation, and functional characterization of candidate metabolites described in Lawton et al. (Pharmacogenomics (2008) 9, 383). These methods can identify cancer cell-specific metabolites in the desired cancer tissue.

[0231] As a non-limiting embodiment of the cancer tissue-specific compound or cancer tissue-specific metabolite used in the present invention, preferably, at least one compound is selected from the following compounds. The term "at least one compound" means that the antigen-binding activity of the same antigen-binding domain described below is dependent not only on a single cancer tissue-specific compound or cancer tissue-specific metabolite but also on multiple cancer tissue-specific compounds or cancer tissue-specific metabolites.

[0232] (1) Primary metabolites of the glycolysis system or Kreb's cycle, such as lactic acid, succinic acid, and citric acid As a non-limiting embodiment of the cancer tissue-specific compounds used in the present invention, particularly cancer cell-specific metabolites, preferred examples include primary metabolites produced as a result of glucose metabolism, such as lactate, succinate, and citric acid, which are present at higher concentrations in cancer tissues compared to surrounding non-cancerous tissues. A glycolytic phenotype characterized by upregulation (upregulation) of glycolytic enzymes (Embden-Myerhof pathway), such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH), has long been known as the Warburg effect, a characteristic of solid tumors.

[0233] Specifically, in tumor cells, high expression of the M2 isoform of pyruvate kinase, which is required for glycolysis under anaerobic conditions, rather than the M1 isoform, is believed to play a beneficial role in the growth of tumor cells in vivo (Christofk et al. (Nature (2008) 452, 230-233). The pyruvate produced by pyruvate kinase is subject to feedback inhibition by lactate, which is produced as a result of the equilibrium reaction of lactate dehydrogenase (LDH) under anaerobic conditions. This feedback inhibition is said to promote mitochondrial respiration (Krebs cycle) and inhibit cell growth, and therefore, upregulation of LDH, hexokinase, and glucose transporters (GLUTs) is believed to play an important role in the growth of tumor cells (Fantin et al. (Cancer) Cell (2006) 9, 425-434)). Glucose is metabolized through the glycolysis system, and its final metabolite lactic acid is transported to the periphery of the tumor together with protons. As a result, it is said that the pH of the tissue surrounding the tumor changes to an acidic condition. It is known that lactic acid, the final product of the glycolysis system, and succinic acid and citric acid generated by the promotion of respiration in mitochondria accumulate in cancer tissues (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). As a non-limiting embodiment of the cancer tissue-specific compounds used in the present invention, in particular, cancer cell-specific metabolites, preferably, primary metabolites generated by the metabolism of such a glycolysis system, namely lactic acid, succinic acid, citric acid, etc., can be cited. In addition, it is also known that due to cell death, succinic acid present in high concentrations in cells leaks out of the cells (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is believed that the concentration of succinic acid will increase in cancer tissues where cell death frequently occurs.

[0234] (2) Amino acids such as alanine, glutamic acid, and aspartic acidIn addition to the glucose metabolism mentioned above, amino acid metabolism is also known to be altered in tumor cells, which require a continuous supply of essential and non-essential amino acids required for the biosynthesis of biopolymers under anaerobic conditions. Glutamine, the most widely distributed amino acid in the body, contains two nitrogen atoms in its side chain and acts as a nitrogen transporter. Tumor cells with increased rates of glutamine uptake are thought to act as glutamine traps. This increase in glutamine uptake and conversion to glutamate and lactate is called "glutaminolysis" and is considered 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 and an increase in glutamate concentrations (Droge et al. (Immunobiology (1987) 174, 473-479). Subsequently, through the transfection of lung cancer tissue 13 Metabolic studies of C radiolabeled glucose in 13 C-labeled succinate, 13 C-labeled alanine, 13 C-labeled glutamate, and 13 A correlation was observed between the concentrations of C-labeled citric acid. Preferred examples of the cancer tissue-specific compound used in the present invention, but not limited to, include alanine, glutamic acid, and aspartic acid, which accumulate at high concentrations in cancer tissues through glutaminolysis and the like.

[0235] (3) Metabolites of amino acids such as kynurenine

[0236] Indoleamine 2,3-dioxygenase (IDO) is a tryptophan metabolic enzyme that is highly expressed in many cancers, including melanoma, colon cancer, and renal cancer (Uyttenhove et al. (Nat. Med. (2003) 9, 1269-127), and two isoforms are known to exist (Lob et al. (Cancer Immunol. Immunother. (2009) 58, 153-157)). IDO catalyzes the conversion of tryptophan to kynurenine (shown in Chemical Formula 1) and is the first enzyme in the de novo synthesis pathway of nicotinamide nucleotide (NAD). In addition, in gliomas that do not express IDO, kynurenine is converted to tryptophan by the liver. IDO is expressed in dendritic cells infiltrating cancerous tissues, which also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). Furthermore, IDO is expressed in myeloid-derived suppressor cells (MDSCs) in cancerous tissues, which also produce kynurenine (Yu et al. (J. Immunol. (2013) 190, 3783-3797)).

[0237] [Chemistry 1]

[0238]

[0239] Kynurenine is known to suppress allogeneic T cell responses (Frumento et al. (J. Exp. Med. (2002) 196, 459-468), and a mechanism has been proposed in which tumor cells evade antitumor immune responses through this suppression and promote glioma cell proliferation through an autocrine proliferation mechanism in which kynurenine acts as an endogenous ligand for the aryl hydrocarbon receptor expressed in gliomas (Opitz et al. (supra)). Kynurenine is converted to anthranilic acid by kynureninase (shown in [Chemical Formula 2]) and to 3-hydroxykynurenine by kynurenine 3-hydroxylase (shown in [Chemical Formula 3]). Both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, a precursor for the formation of NAD.

[0240] [Chemistry 2]

[0241]

[0242] [Chemistry 3]

[0243]

[0244] Kynurenine is converted to kynurenic acid by kynurenine aminotransferase (shown in [Chemical 4]). Preferred examples of cancer tissue-specific compounds, particularly cancer cell-specific metabolites, used in the present invention include kynurenine and its metabolites, i.e., metabolites of amino acids such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid.

[0245] [Chemistry 4]

[0246]

[0247] (4) Prostaglandin E2 and other arachidonic acid metabolites Prostaglandin E2 (PGE2) ([chemical 5]) is a metabolite of arachidonic acid of the prostaglandins of the prostaglandins synthesized by cyclooxygenase (COX)-1 / 2 and thromboxane (Warner and Mitchell (FASEB J. (2004) 18,790-804)). Promote the proliferation of PGE2 colon cancer cells, and inhibit its apoptosis (Sheng et al. (Cancer Res. (1998) 58,362-366)). Known in many cancer cells, the expression of cyclooxygenase is variable. That is, mainly find that COX-1 is constitutively expressed in almost all tissues, in contrast, COX-2 is induced by certain inflammatory cytokines and oncogenes in tumors (Warner and Mitchell (above)). It has also been reported that the overexpression of COX-2 and the poor prognosis of breast cancer (Denkert et al. (Clin.Breast Cancer Research, 2003). Cancer (2004) 4, 428-433), and the rapid progression of ovarian cancer (Denker et al. (Mod. Pathol. (2006) 19, 1261-1269). In addition, suppressor T cells infiltrating cancer tissues also produce prostaglandin E2 (Curr. Med. Chem. (2011) 18, 5217-5223). Low molecular weight prostaglandins and leukotrienes, metabolites of arachidonic acid, are known to act as stimulatory factors that control the autocrine and / or paracrine growth of cancer (Nat. Rev. Cancer (2 012)12(11)782-792). As a non-limiting embodiment of the cancer tissue-specific compound used in the present invention, in particular, a cancer cell-specific metabolite or an immune cell-specific metabolite infiltrating cancer tissue, preferably, a metabolite of arachidonic acid such as prostaglandin E2 can be mentioned. In addition to prostaglandin E2, thromboxane A2 (TXA2) is produced more frequently in cancer tissues such as colorectal cancer (J. Lab. Clin. Med. (1993) 122, 518-523), and can be preferably mentioned as a non-limiting embodiment of the metabolite of arachidonic acid in the present invention.

[0248] [Chemistry 5]

[0249]

[0250] (5) Adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and other NucleosideIt is known that when cancer cells undergo cell death, a large amount of ATP leaks out of the cell. Therefore, the ATP concentration in cancer tissue is significantly higher than that in normal tissue (PLoS One. (2008) 3, e2599). Various cell types release adenine nucleotides in the form of ATP, ADP, and AMP. It is metabolized by extracellular enzymes such as eco-5'-nucleotidase (CD73) on the cell surface (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. A significant increase in extracellular adenosine concentration has been reported in hypoxic tissues found in solid cancers (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 is expressed in breast cancer (Canbolat et al. (Breast Cancer)). Increased activity has been observed in cancer cells (Cancer Res. Treat. (1996) 37, 189-193), gastric cancer (Durak et al. (Cancer Lett. (1994) 84, 199-202), pancreatic cancer (Flocke and Mannherz (Biochim. Biophys. Acta (1991) 1076, 273-281), and glioblastoma (Bardot et al. (Br. J. Cancer (1994) 70, 212-218)). Accumulation of adenosine in cancer tissues has been proposed to be caused by increased intracellular adenosine production due to dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick and Willis (Biochem. J. (1989) 261, 541-550). Furthermore, suppressor T cells infiltrating cancer tissues also express ATPase. , thereby producing adenosine (Proc. Natl. Acad. Sci. (2006) 103 (35), 13132-13137, Curr. Med. Chem. (2011) 18, 5217-5223). It is believed that the produced adenosine makes the cancer tissue into an immunosuppressive environment through adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011), 18, 5217-23). ​​As a non-limiting embodiment of the cancer tissue-specific compound used in the present invention, ATP, ADP, AMP, or adenosine, which are accumulated in high concentrations in cancer tissues through the metabolism of purine nucleotides such as ATP, can be preferably mentioned. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, so that inosine accumulates in high concentrations.

[0251] (6) Uric acid

[0252] Uric acid is a product of the metabolic pathway of purine nucleosides in the body and is released outside cells such as blood or interstitial cavities. In addition, it has been clarified in recent years that it is released by dead cells present in lesions such as cancerous tissue (Nat.Med. (2007) 13, 851-856). As a non-limiting scheme of the cancer tissue-specific compound used in the present invention, it is also preferred to enumerate uric acid accumulated in cancer tissue with high concentrations by the metabolism of purine nucleotides such as ATP.

[0253] (7) 1-Methylnicotinamide The enzyme nicotinamide N-methyltransferase is known to be highly expressed in many human cancer tissues. When this enzyme produces the stable metabolite 1-methylnicotinamide from nicotinamide, it consumes the methyl group of S-adenosylmethionine (SAM), which serves as a methyl donor. Therefore, it is proposed that high expression of nicotinamide N-methyltransferase contributes to tumorigenesis by impairing DNA methylation capacity accompanied by 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 outside of cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). As a non-limiting embodiment of the cancer tissue-specific compound used in the present invention, 1-methylnicotinamide, which accumulates at high concentrations in cancer tissues through the metabolism of this nicotinamide, is also preferably mentioned.

[0254] Inflammatory tissue-specific compounds

[0255] The term "inflammatory tissue-specific compound" as used herein refers to a compound that is differentially present in inflammatory tissue compared to non-inflammatory tissue. In this specification, "inflammatory tissue" refers to, for example, the following illustrative examples.

[0256] Joints in rheumatoid arthritis and osteoarthritis

[0257] Lungs (alveoli) in bronchial asthma and COPD

[0258] Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis

[0259] Fibrotic tissue in fibrosis of the liver, kidney, and lung

[0260] Tissues that have undergone rejection during organ transplantation

[0261] Arteriosclerosis, blood vessels in heart failure, heart (myocardium)

[0262] Visceral fat in metabolic syndrome

[0263] Skin tissue in dermatitis such as atopic dermatitis

[0264] Spinal nerves in herniated disc and chronic low back pain.

[0265] Inflammatory tissue-specific metabolites

[0266] Inflammatory tissue-specific metabolites refer to metabolites produced in large quantities by immune cells infiltrating the inflammatory tissue, and metabolites specifically produced in large quantities by normal cells damaged in the inflammatory tissue. Examples of infiltrating immune cells include effector T cells, mature dendritic cells, neutrophils, granulocytes (mast cells), basophils, etc. In addition, metabolites in the present invention also include compounds released from cells (immune cells, normal cells) present in the inflammatory tissue when cell death occurs due to apoptosis, necrosis, etc.

[0267] As a non-limiting embodiment of the inflammatory tissue-specific compound or inflammatory tissue-specific metabolite used in the present invention, preferably, at least one compound is selected from the following compounds. The term "at least one compound" means that the antigen-binding activity of the same antigen-binding domain described below is dependent not only on a single inflammatory tissue-specific compound or inflammatory tissue-specific metabolite, but also on multiple inflammatory tissue-specific compounds or inflammatory tissue-specific metabolites.

[0268] (1) Prostaglandin E2 and other arachidonic acid metabolites It is known that in rheumatoid arthritis and osteoarthritis, the concentration of PGE2 is high (Eur.J.Clin.Pharmacol.(1994)46,3-7.、Clin.Exp.Rheumatol.(1999)17,151-160、Am.J.Vet.Res.(2004)65,1269-1275.).As the inflammatory tissue specific compound used in the present invention, particularly inflammatory cell specific metabolite, the non-limiting embodiment of the immune cell specific metabolite that infiltrates into inflammatory tissue, preferably can enumerate the metabolites of arachidonic acid such as this prostaglandin E2.

[0269] (2) Adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and other NucleosideIt is known that the concentration of ATP is high in lung cells that have inflammation caused by bronchial asthma (Nat. Med. (2007) 13, 913-919). In addition, it is also known that the concentration of ATP is high in lung cells that have inflammation caused by COPD (Am. J. Respir. Crit. Care Med. (2010) 181, 928-934). In addition, adenosine concentrations have been observed to be high in the joint fluid of patients with rheumatoid arthritis (Journal of Pharmaceutical and Biomedical Analysis (2004) 36877-882). It is also known that the concentration of ATP is high in tissues that have undergone rejection reactions 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). Furthermore, 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 a non-limiting embodiment of the inflammatory tissue-specific compound used in the present invention, preferred examples include ATP, ADP, AMP, or adenosine, which accumulate at high concentrations in inflamed tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is degraded into inosine by adenosine deaminase, so that inosine accumulates at a high concentration.

[0270] (3) Uric acid

[0271] Uric acid is a product of the metabolic pathway of purine nucleosides in the body and is released outside cells such as blood or interstitial cavities. In addition, it has been clearly shown in recent years that uric acid released from cells undergoing necrosis promotes inflammatory response (J. Clin. Invest. (2010) 120 (6), 1939-1949). As a non-limiting embodiment of the inflammatory tissue-specific compound used in the present invention, it is also preferred to cite uric acid that accumulates in high concentrations in inflammatory tissues through the metabolism of purine nucleotides such as ATP.

[0272] Antigen binding domain

[0273] As used herein, the term "antigen-binding domain" may refer to a domain of any structure as long as it binds to the target antigen. Preferred examples of such domains include the variable regions of antibody heavy and light chains, the approximately 35 amino acid A domain contained in the cell membrane protein Avimer (International Publications WO 2004 / 044011 and WO 2005 / 040229), Adnectins containing a 10Fn3 domain that binds to proteins in the glycoprotein fibronectin expressed on the cell membrane (International Publication WO 2002 / 032925), Affibodies using as a scaffold the IgG binding domain of the 58 amino acid three-helix bundle of Protein A (International Publication WO 1995 / 001937), and DARPins (Designed Ankyrin Repeats), which are regions of ankyrin repeats (ARs) that are exposed on the molecular surface and have a structure in which a turn consisting of 33 amino acid residues overlaps with two antiparallel helices and a loop. proteins) (International Publication WO2002 / 020565); anticalins, which are four loop regions on one side of a barrel structure in which eight antiparallel chains are twisted in the central direction, which is highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO2003 / 029462); and a concave region within a horseshoe-shaped structure formed by repeated overlapping leucine-rich repeat (LRR) units of the variable lymphocyte receptor (VLR) that lacks an immunoglobulin structure and is an acquired immune system of jawless fish such as lampreys and hagfishes (International Publication WO2008 / 016854).

[0274] Preferred examples of the antigen-binding domains of the present invention include those comprising the variable regions of the heavy and light chains of antibodies. Preferred examples of such antigen-binding domains include "scFv (single-chain Fv)", "single-chain antibodies", "Fv", "scFv2 (single-chain Fv2)", "Fab", and "F(ab')2".

[0275] The antigen-binding domains in the antigen-binding molecules of the present invention may bind to the same epitope. Here, the same epitope may exist in, for example, a protein comprising the amino acid sequence described in SEQ ID NO: 1. Alternatively, the antigen-binding domains in the antigen-binding molecules of the present invention may bind to different epitopes. Here, different epitopes may exist in, for example, a protein comprising the amino acid sequence described in SEQ ID NO: 1.

[0276] Specificity

[0277] Specificity refers to the state in which a molecule on one side of a specifically bound molecule does not substantially bind to molecules other than one or more of its binding target molecules. In addition, it can also be used for situations in which an antigen-binding domain is specific for a specific epitope among multiple epitopes contained in a certain antigen. In addition, when the epitope bound by the antigen-binding domain is contained in multiple different antigens, the antigen-binding molecule having this antigen-binding domain can bind to various antigens containing this epitope. Here, substantially not binding means that the binding activity of the specific binding molecule to molecules other than the aforementioned target molecules is determined by the method described in the above-mentioned binding activity, and the binding activity is shown to be less than 80% of the binding activity to the aforementioned target molecules, generally less than 50%, preferably less than 30%, and particularly preferably less than 15%.

[0278] Cytotoxicity

[0279] In one non-limiting embodiment of the present invention, there is provided an antigen-binding domain containing an antigen-binding activity that changes according to the concentration of a low molecular weight compound (e.g., a cancer tissue-specific compound, an inflammatory tissue-specific compound, or a metabolite thereof), and having cytotoxicity to cells expressing membrane-type molecules on their cell membranes, and a pharmaceutical composition containing the antigen-binding molecule as an active ingredient. In the present invention, cytotoxicity can include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and cytotoxicity caused by T cells. In the present invention, CDC activity means cytotoxicity caused by the complement system. On the other hand, ADCC activity means the activity of immune cells, etc., which bind to the Fc region of an antigen-binding molecule containing an antigen-binding domain that binds to a membrane-type molecule expressed on the cell membrane of a target cell through the Fcγ receptor expressed on the immune cell, thereby causing damage to the target cell. Whether an antigen-binding molecule of interest has ADCC activity or CDC activity can be determined by known methods (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, ed. Coligan et al. (1993)).

[0280] Specifically, first, effector cells, complement solution, and target cells are prepared.

[0281] (1) Preparation of effector cells Spleen was removed from CBA / N mice and spleen cells were isolated in RPMI1640 medium (Invitrogen). The spleen cells were washed with the same medium containing 10% fetal bovine serum (FBS, HyClone) and the concentration was adjusted to 5 × 10 6 / mL, thereby preparing effector cells.

[0282] (2) Preparation of complement solution

[0283] The complement solution can be prepared by diluting Baby Rabbit Complement (CEDARLANE) 10-fold with a culture medium containing 10% FBS (Invitrogen).

[0284] (3) Preparation of target cells

[0285] Antigen-expressing cells were incubated with 0.2 mCi of 51The target cells were radiolabeled by culturing them with Cr-sodium chromate (GE Healthcare Bio-Sciences) in DMEM medium containing 10% FBS at 37°C for 1 hour. After radiolabeling, the cells were washed three times with RPMI1640 medium containing 10% FBS and the cell concentration was adjusted to 2×10 5 / mL, thereby preparing the target cells.

[0286] ADCC activity or CDC activity can be measured by the method described below. During the determination of ADCC activity, each 50 μl of target cells added to a 96-well U-bottom culture plate (Becton Dickinson) was reacted with the antigen binding molecules at room temperature for 15 minutes. Then, the culture plate with 100 μl of effector cells was allowed to stand for 4 hours in a carbon dioxide incubator. The final concentration of the antigen binding molecule can be set to a concentration of, for example, 0 or 10 μg / ml. After standing, a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company) was used to measure the radioactivity of 100 μl supernatants recovered from each well. Using the measured value, cytotoxicity (%) can be calculated based on the formula of (AC) / (BC)x 100. A represents the radioactivity (cpm) of each sample, B represents the radioactivity (cpm) of the sample to which 1% NP-40 (nacalai tesque) has been added, and C represents the radioactivity (cpm) of the sample containing only target cells.

[0287] On the other hand, during the determination of CDC activity, the target cells and antigen binding molecules of each 50 μl added to a 96-well flat-bottom culture plate (Becton Dickinson) were reacted on ice for 15 minutes. Then, the culture plate to which 100 μl of complement solution was added was left to stand for 4 hours in a carbon dioxide incubator. The final concentration of the antigen binding molecules can be set to a concentration of, for example, 0 or 3 μg / mL. After standing, a gamma counter was used to measure the radioactivity of the 100 μl supernatants recovered by each well. Cytotoxicity can be calculated in the same manner as the ADCC activity assay.

[0288] In addition, the modified antigen binding molecules modifiers of cell-damaging substances such as chemotherapeutic drugs, toxic peptides or radioactive chemicals described later also can be suitably used as cytotoxic antigen binding molecules of the present invention.This modified antigen binding molecules (hereinafter referred to as antigen binding molecules drug conjugates) can be obtained by chemically modifying gained antigen binding molecules.In addition, as the modification method of antigen binding molecules, the method established in fields such as antibody-drug conjugates can be suitably used.In addition, the modified antigen binding molecules of toxic peptides can be obtained by being connected in the gene frame of the gene encoding the toxic peptides and the antigen binding molecules of the present invention in suitable host cells after expressing, from the culture fluid of this cell, separating and obtaining.

[0289] Neutralizing activity

[0290] In the non-limiting scheme of the present invention, there is provided a pharmaceutical composition for inducing immune response, said composition containing antigen binding molecules as an effective ingredient, said antigen binding molecules containing the antigen binding domains that antigen binding activity changes according to the concentration of cancer tissue specific compound, and there is neutralizing activity to the membrane type molecule. In the non-limiting another scheme of the present invention, there is provided a pharmaceutical composition for inducing immune response, said composition containing antigen binding molecules as an effective ingredient, said antigen binding molecules containing the antigen binding domains that antigen binding activity changes according to the concentration of low molecular weight compounds (for example, cancer tissue specific compound, inflammatory tissue specific compound, their metabolites etc.), and except having cytotoxicity to the cells expressing membrane type molecules on its cell membrane, there is also neutralizing activity to the membrane type molecule. Generally, neutralizing activity refers to the activity of the biological activity of the part that suppresses viruses or toxins etc. to cell with biological activity. That is, the substance with neutralizing activity refers to the receptor binding to which the part or the part is combined, and suppresses the material of the combination of the part and receptor. The receptor blocked by neutralizing activity with the combination of the part can not be played by the receptor. When the antigen-binding molecule is an antibody, an antibody with such neutralizing activity is generally referred to as a neutralizing antibody. The neutralizing activity of a test substance can be determined by comparing the biological activity in the presence of a ligand with that in the absence or presence of the test substance.

[0291] For example, IL-6 represented by SEQ ID NO: 27 is a preferred primary ligand for the IL-6 receptor. The IL-6 receptor, a type I membrane protein whose amino-terminal extracellular domain forms a heterotetramer with the gp130 receptor, which dimerizes in response to IL-6 (Heinrich et al. (Biochem. J. (1998) 334, 297-314)). This heterotetramer formation activates Jak, which binds to the gp130 receptor. Jak undergoes autophosphorylation and phosphorylation of the receptor. The phosphorylation sites on the receptor and Jak function as binding sites for molecules belonging to the Stat family with SH2 residues, such as Stat3, MAP kinase, PI3 / Akt, other SH2 residue-bearing proteins, or adaptors. Stat bound to the gp130 receptor is then phosphorylated by Jak. Phosphorylated Stat forms dimers and migrates to the nucleus, regulating the transcription of target genes. Jak or Stat can also participate in signaling cascades through other types of receptors. Deregulated IL-6 signaling cascades are observed in autoimmune disease pathologies or inflammation, multiple myeloma, prostate cancer, and other cancers. Stat3, which can function as an oncogene, is constantly activated in many cancers. In prostate cancer and multiple myeloma, crosstalk occurs between the signaling cascade from the IL-6 receptor and that from members of the epidermal growth factor receptor (EGFR) family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46(2), 55-66)).

[0292] The signal cascade in this cell is different according to the cell type, so the target molecule can be suitably set according to the target cell of interest, and is not limited to the above-mentioned factor. By measuring the activation of the signal in vivo, neutralization activity can be evaluated. In addition, the transcriptional induction effect on the target gene in the downstream of the signal cascade in the organism can also be used as an index to detect the activation of the signal in the organism. The change in the transcriptional activity of the target gene can be detected by the principle of reporter assay (reporter assay). Specifically, a reporter gene such as GFP (Green Fluorescence Protein, green fluorescent protein) or luciferase is configured downstream of the transcription factor or promoter region of the target gene, and its reporter activity is measured, and thus the change in transcriptional activity can be measured in the form of reporter activity. The assay kit for the activation of the signal in the organism can suitably use a commercially available product (for example, Mercury Pathway Profiling Luciferase System (Clontech) etc.).

[0293] Furthermore, as a method for measuring the neutralizing activity of receptor ligands such as the EGF receptor family that acts on the signal cascade that usually works in the direction of promoting cell proliferation, the neutralizing activity of the antigen binding molecules can be evaluated by measuring the proliferation activity of the target cells. For example, as a method for evaluating or measuring the inhibitory effect of the neutralizing activity of anti-HB-EGF antibodies on the proliferation of cells whose proliferation is promoted by growth factors of the EGF family, such as HB-EGF, the following method can be suitably used. As a method for evaluating or measuring the cell proliferation inhibitory activity in vitro, a method for measuring the neutralizing activity of [ 3 A method for measuring the uptake of [H]-labeled thymidine as an indicator of DNA replication ability. A simpler method is the pigment exclusion method or the MTT method, which measures the ability to exclude pigments such as trypan blue from cells under a microscope. The latter method utilizes the ability of living cells to convert MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a tetrazolium salt, into a blue formazan. The ability of the product. More specifically, the antibody to be tested is added to the culture medium of the tested cells together with the ligand. After a certain period of time, an MTT solution is added to the culture medium and allowed to stand for a certain period of time to allow MTT to enter the cells. As a result, the yellow compound MTT is converted into a blue compound by the succinate dehydrogenase in the mitochondria of the cells. The blue product is dissolved and its absorbance is measured after color development, thereby serving as an indicator of the number of viable cells. In addition to MTT, reagents such as MTS, XTT, WST-1, and WST-8 are also commercially available (nacalai tesque, etc.) and can be used appropriately. When measuring activity, a binding antibody with the same isotype as the anti-HB-EGF antibody but without the cell proliferation inhibitory activity can be used as a control antibody, and the activity is determined by whether the anti-HB-EGF antibody shows stronger cell proliferation inhibitory activity than the control antibody.

[0294] Cells suitable for evaluating activity include, for example, the ovarian cancer cell line RMG-1, whose proliferation is promoted by HB-EGF, and mouse Ba / F3 cells transformed with a vector expressing a gene encoding a fusion protein hEGFR / mG-CSFR, which is an in-frame fusion of the extracellular domain of human EGFR and the intracellular domain of mouse G-CSF receptor. Thus, those skilled in the art can appropriately select cells for evaluating activity and use them to measure the aforementioned cell proliferation activity.

[0295] Antibody

[0296] In this specification, antibodies refer to natural immunoglobulins or immunoglobulins produced by partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma and serum where they exist naturally or from the culture supernatant of hybridoma cells that produce antibodies, or can be partially or completely synthesized by using methods such as genetic recombination. As examples of antibodies, preferably, immunoglobulin isotypes and subclasses of these isotypes can be mentioned. As human immunoglobulins, 9 types (isotypes) are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. IgG1, IgG2, IgG3, and IgG4 among these isotypes can be included in the antibodies of the present invention. As human IgG1, human IgG2, human IgG3, and human IgG4 constant regions, multiple allotype sequences formed by genetic polymorphism are recorded in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, but any one of them can be used in the present invention. In particular, the amino acid sequence at positions 356-358 of human IgG1, as represented by EU numbering, can be either DEL or EEM. Furthermore, multiple allotype sequences resulting from genetic polymorphisms in the human Igκ (kappa) constant region and human Igλ (lambda) constant region are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these allotypes may be used in the present invention.

[0297] Methods for producing antibodies with desired binding activity are well known to those skilled in the art. The following examples illustrate methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies). Antibodies that bind to antigens other than IL-6R can also be appropriately produced according to the following examples.

[0298] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Anti-IL-6R antibodies are preferably prepared from mammalian monoclonal antibodies. Mammalian monoclonal antibodies include those produced by hybridomas and those produced using genetic engineering techniques using host cells transformed with expression vectors containing antibody genes. The monoclonal antibodies of the present invention include both humanized antibodies and chimeric antibodies.

[0299] Hybridomas producing monoclonal antibodies can be prepared using known techniques, for example, as follows. Specifically, a mammal is immunized using the IL-6R protein as a sensitizing antigen according to conventional immunization methods. The resulting immune cells are fused with known parental cells using conventional cell fusion methods. Subsequently, the monoclonal antibody-producing cells are screened using conventional screening methods to select hybridomas producing anti-IL-6R antibodies.

[0300] Specifically, monoclonal antibodies are prepared, for example, as follows. First, the IL-6R gene whose nucleotide sequence is disclosed in SEQ ID NO: 2 is expressed to obtain the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody production. Specifically, the gene sequence encoding IL-6R is inserted into a known expression vector, and appropriate host cells are transformed therewith. The desired human IL-6R protein is purified from the host cells or the culture supernatant by known methods. To obtain soluble IL-6R from the culture supernatant, a soluble IL-6R protein described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), i.e., a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, is expressed instead of the IL-6R protein represented by SEQ ID NO: 1. Alternatively, purified native IL-6R protein can be used as a sensitizing antigen.

[0301] The purified IL-6R protein can be used as a sensitizing antigen for immunizing mammals. In addition, a partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can also be obtained by chemical synthesis based on the amino acid sequence of human IL-6R. Alternatively, it can be obtained by integrating a portion of the IL-6R gene into an expression vector and expressing it. Furthermore, it can also be obtained by degrading the IL-6R protein using a protease. The region and size of the IL-6R peptide used as a partial peptide are not particularly limited to a specific scheme. For a preferred region, any sequence can be selected from the amino acid sequence corresponding to amino acids 20 to 357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids constituting the peptide serving as the sensitizing antigen is preferably 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.

[0302] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be preferably utilized to produce a fusion protein for use as a sensitizing antigen. A vector expressing the fusion protein can be prepared by in-frame fusing genes encoding two or more desired polypeptide fragments and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9:47-9:58 (1989) Cold Spring Harbor Lab. press). Methods for obtaining IL-6R for use as a sensitizing antigen and immunization methods using it are also described in detail in International Publications WO2003 / 000883, WO2004 / 022754, and WO2006 / 006693.

[0303] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, and is preferably selected in consideration of its compatibility with the parent cells used for cell fusion. In general, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0304] The animals are immunized with the sensitizing antigen according to known methods. For example, as a common method, immunization is performed by injecting the sensitizing antigen into the peritoneal cavity or subcutaneously of the mammal. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline is mixed with a common adjuvant, such as Freund's complete adjuvant, as needed, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. In addition, an appropriate carrier can be used for immunization with the sensitizing antigen. In particular, when using a partial peptide with a small molecular weight as the sensitizing antigen, it is sometimes desirable to immunize the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0305] Alternatively, hybridomas producing the desired antibody can be produced using DNA immunization as follows. DNA immunization refers to an immunization method in which a sensitizing antigen is expressed in an immunized animal administered with a vector DNA constructed to express a gene encoding an antigenic protein, thereby imparting immune stimulation. Compared to conventional immunization methods in which protein antigens are administered to the immunized animal, DNA immunization is expected to offer the following advantages.

[0306] -Can maintain the structure of membrane proteins such as IL-6R and impart immune stimulation

[0307] - No need to purify immune antigens

[0308] In order to obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to the immunized animal. The DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the immunized animal. As an expression vector, commercially available expression vectors such as pcDNA3.1 can be preferably used. As a method for administering the vector to the body, commonly used methods can be adopted. For example, gold particles adsorbed with the expression vector are introduced into the cells of the immunized animal individual using a gene gun, thereby performing DNA immunization. Furthermore, the preparation of antibodies that recognize IL-6R can also be prepared by the method described in International Publication WO2003 / 104453.

[0309] After the mammal is immunized in this manner and the increase in the titer of antibodies binding to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells are spleen cells.

[0310] As the cell fused with the aforementioned immune cell, mammalian myeloma cells can be used.Myeloma cells preferably possess the appropriate selection marker for screening.Selection marker refers to the property that gives cells the ability to survive (or die) under specific culture conditions. Among the selection markers, it is known that hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter referred to as HGPRT deficiency) or thymidine kinase deficiency (hereinafter referred to as TK deficiency) etc. The cells with HGPRT, TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). The cells of HAT sensitivity can not select to carry out DNA synthesis in culture medium in HAT and can die, but if fused with normal cells, the salvage pathway of normal cells can be utilized to continue the synthesis of DNA, therefore HAT selection culture medium also can breed.

[0311] HGPRT-deficient and TK-deficient cells can each be selected by a culture medium containing 6-thioguanine, 8-azaguanine (hereinafter referred to as 8AG), or 5' bromodeoxyuridine. Normal cells with these pyrimidine analogs integrated into their DNA will die. Cells lacking the above enzymes that cannot integrate these pyrimidine analogs can survive in the selection culture medium. In addition, the selection marker known as G418 resistance can confer resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) through the neomycin resistance gene. Various myeloma cells preferred in cell fusion are well known.

[0312] As such myeloma cells, for example, 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),269-2 70), 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.

[0313] Basically, cell fusion of the aforementioned immune cells and myeloma cells is performed according to a known method, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0314] More specifically, for example, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and further, to improve fusion efficiency, an auxiliary agent such as dimethyl sulfoxide can be added as desired.

[0315] The usage ratio of immunocyte and myeloma cell can be set arbitrarily.For example, relative to myeloma cell, preferably make immunocyte be 1 to 10 times.As the culture fluid for aforementioned cell fusion, use RPMI1640 culture fluid, MEM culture fluid and the common culture fluid for such cell culture that are suitable for the propagation of aforementioned myeloma cell strain, for example, and then can preferably add serum supplementation such as fetal calf serum (FCS).

[0316] For cell fusion, the aforementioned immune cells and myeloma cells are thoroughly mixed in the aforementioned culture medium at the prescribed amounts. A PEG solution (e.g., with an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, typically at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Subsequently, the appropriate culture medium listed above is added sequentially, and repeated centrifugation and supernatant removal can be performed to remove cell fusion agents that may be detrimental to hybridoma growth.

[0317] The hybridoma obtained in this way can be selected by culturing with common selection culture fluid, for example HAT culture fluid (culture fluid containing hypoxanthine, aminopterin and thymidine). The culture using the above-mentioned HAT culture fluid is sustainable enough for the time (usually, the sufficient time is several days to several weeks) of cell death beyond the desired hybridoma. Then, by common limiting dilution method, screening and monocloning of the hybridoma producing the desired antibody are implemented.

[0318] The hybridoma obtained in this way can be selected by using the selection culture fluid corresponding to the selection marker possessed by the myeloma for cell fusion.For example, the cell with HGPRT, TK defect can be selected by cultivating with HAT culture fluid (culture fluid containing hypoxanthine, aminopterin and thymidine).That is, when using the myeloma cell of HAT sensitivity in cell fusion, in HAT culture fluid, the cell that has successfully fused with the normal cell can selectively be bred.The cultivation using above-mentioned HAT culture fluid can continue the time that the cell (non-fused cell) beyond the desired hybridoma (enzyme A) is enough to die.Particularly, conventionally, desired hybridoma can be selected by cultivating for several days to several weeks.Then, by common limiting dilution method, the screening and the monoclonal production of the hybridoma of desired antibody can be implemented.

[0319] The screening and monocloning of the desired antibody can preferably be carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that can use laser analysis to analyze cells in contact with fluorescent antibodies and measure the fluorescence emitted by each cell to determine the binding of the antibody to the cell surface.

[0320] To screen hybridomas producing the monoclonal antibodies of the present invention using FACS, cells expressing IL-6R are first prepared. Preferred cells used for screening are mammalian cells forced to express IL-6R. By using untransformed mammalian cells as a control host cell, it is possible to selectively detect antibody binding activity to cell-surface IL-6R. In other words, by selecting hybridomas that produce antibodies that bind to cells forced to express IL-6R but not to host cells, hybridomas producing IL-6R monoclonal antibodies can be obtained.

[0321] Alternatively, the binding activity of antibodies to immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of the hybridoma is brought into contact with the immobilized cells in the wells, and antibodies bound to the immobilized cells are detected. When the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas selected through these screenings that produce the desired antibody with binding ability to the antigen can be cloned by limiting dilution methods, etc.

[0322] The monoclonal antibody-producing hybridoma thus prepared can be subcultured in a conventional culture medium or stored in liquid nitrogen for a long period of time.

[0323] The hybridoma can be cultured according to conventional methods, and the desired monoclonal antibody can be obtained from its culture supernatant. Alternatively, the hybridoma can be administered to a mammal compatible with the hybridoma and allowed to proliferate, and the monoclonal antibody can be obtained from its ascites. The former method is suitable for obtaining high-purity antibodies.

[0324] It is also possible to suitably use antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas. The cloned antibody gene is integrated into an appropriate vector and introduced into a host, thereby expressing the antibody encoded by the gene. Methods for the isolation of antibody genes, their introduction into vectors, and transformation of host cells have been established by, for example, Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). In addition, methods for producing recombinant antibodies as described below are also known.

[0325] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody can be obtained from hybridoma cells producing the anti-IL-6R antibody. To this end, total RNA is typically extracted from the hybridoma. For example, the following method can be used to extract mRNA from cells.

[0326] -Guandanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299)

[0327] -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0328] The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Bioscience). Alternatively, a kit for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience), is commercially available. Using this kit, mRNA can be obtained from hybridomas. cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Kogyo Co., Ltd.). In addition, for the synthesis and amplification of cDNA, a 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) using a SMART RACE cDNA Amplification Kit (manufactured by Clontech) and PCR can be appropriately adopted. Furthermore, during the synthesis of such cDNA, suitable restriction enzyme sites as described below can be introduced into both ends of the cDNA.

[0329] The target cDNA fragment is purified from the resulting PCR product and then ligated with the vector DNA. This recombinant vector is then produced and introduced into E. coli, for example. After colonies are selected, the desired recombinant vector can be prepared from the E. coli that formed these colonies. Furthermore, whether the recombinant vector has the base sequence of the target cDNA can be confirmed using known methods, such as the dideoxynucleotide chain termination method.

[0330] In order to obtain the gene encoding the variable region, it is convenient to utilize the 5'-RACE method, wherein primers for variable region gene amplification are used. First, RNA extracted from hybridoma cells is used as a template to synthesize cDNA to obtain a 5'-RACE cDNA library. Commercially available test kits such as the SMART RACE cDNA amplification kit can be suitably used in the synthesis of the 5'-RACE cDNA library.

[0331] The antibody gene is amplified by PCR using the resulting 5'-RACE cDNA library as a template. Based on the known antibody gene sequence, primers for mouse antibody gene amplification can be designed. These primers have different base sequences depending on the subclass of immunoglobulin. Therefore, it is ideal to use commercially available kits such as the Iso Strip Mouse Monoclonal Antibody Subclass Detection Kit (Roshiu Daignos Test) to predetermine the subclass.

[0332] Specifically, for example, to obtain genes encoding mouse IgG, the following primers can be used, which are capable of amplifying genes encoding the heavy chains γ1, γ2a, γ2b, and γ3, and the light chains κ and λ. To amplify IgG variable region genes, 3' primers that anneal to a region corresponding to the constant region near the variable region are typically used. The 5' primers used are those included with the 5' RACE cDNA library preparation kit.

[0333] Using the PCR product obtained by such amplification, an immunoglobulin composed of a combination of heavy and light chains can be reconstructed. The binding activity of the reconstructed immunoglobulin against IL-6R is used as an indicator to screen for the desired antibody. For example, when obtaining an antibody against IL-6R, it is further preferred that the antibody binds specifically to IL-6R. Antibodies that bind to IL-6R can be screened, for example, as follows:

[0334] Step (1) contacting an antibody comprising a V region encoded by a cDNA obtained from a hybridoma with cells expressing IL-6R;

[0335] Step (2), detecting the binding of cells expressing IL-6R to the antibody; and

[0336] Step (3), selecting an antibody that binds to cells expressing IL-6R.

[0337] Methods for detecting the binding of antibodies to IL-6R-expressing cells are well known. Specifically, the binding of antibodies to IL-6R-expressing cells can be detected using methods such as FACS as described above. To evaluate the binding activity of antibodies, fixed samples of IL-6R-expressing cells can be used as appropriate.

[0338] As the screening method of the antibody with binding activity as index, panning method can also be suitably adopted, which utilizes phage vector. When obtaining antibody gene in the form of the library of the subclass of heavy chain and light chain from polyclonal antibody expression cell group, it is advantageous to utilize the screening method of phage vector. The gene encoding the variable region of heavy chain and light chain can form single-chain Fv (scFv) by being connected with appropriate linker sequence. By inserting the gene encoding scFv into phage vector, phage expressing scFv on the surface can be obtained. After this phage contacts with desired antigen, reclaim the phage with antigen, thereby can reclaim the DNA of the scFv with target binding activity. By repeating this operation as required, scFv with desired binding activity can be concentrated.

[0339] After obtaining the target cDNA encoding the V region of the anti-IL-6R antibody, the cDNA is digested by a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes will recognize and digest base sequences that appear less frequently in the base sequences that constitute the antibody gene. Furthermore, in order to insert one copy of the digested fragment into the vector in the correct direction, it is preferred to insert a restriction enzyme that can provide sticky ends. The cDNA encoding the V region of the anti-IL-6R antibody digested as described above is inserted into an appropriate expression vector, thereby obtaining an antibody expression vector. At this time, as long as the gene encoding the antibody constant region (C region) and the gene encoding the aforementioned V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to a constant region and a variable region having different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the aforementioned 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' side of an expression vector containing DNA encoding the desired antibody constant region. Both vectors, digested with the same combination of restriction enzymes, are then in-frame fused to construct a chimeric antibody expression vector.

[0340] In order to produce anti-IL-6R monoclonal antibodies, the antibody gene is integrated into an expression vector in a manner such that expression is regulated by an expression regulatory region. The expression regulatory region used to express the antibody includes, for example, an enhancer and a promoter. In addition, a suitable signal sequence can be added to the amino acid terminus to secrete the expressed antibody outside the cell. In the examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) was used as a 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 outside the cell as a mature polypeptide. Next, the expression vector is used to transform an appropriate host cell, thereby obtaining a recombinant cell expressing the DNA encoding the anti-IL-6R antibody.

[0341] To express antibody genes, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is incorporated into separate expression vectors. By co-transfecting the same host cell with vectors containing both H and L chains, antibody molecules containing both H and L chains can be expressed. Alternatively, DNA encoding both H and L chains can be incorporated into a single expression vector and used to transform host cells (see International Publication No. WO 1994 / 011523).

[0342] A variety of combinations of host cells and expression vectors for producing antibodies by introducing isolated antibody genes into appropriate hosts are known. These expression systems can be used to isolate the antigen-binding domains of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be suitably used. Specifically, animal cells can exemplify the following cells.

[0343] (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 carrying sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1))

[0344] (2) Amphibian cells: Xenopus oocytes, etc.

[0345] (3) Insect cells: sf9, sf21, Tn5, etc.

[0346] Alternatively, as plant cells, an antibody gene expression system based on cells derived from Nicotiana (Nicotiana tabacum) and other tobacco plants is known. For transformation of plant cells, callus cultured cells can be suitably used.

[0347] Furthermore, the following cells can be used as fungal cells.

[0348] Yeast: yeasts of the genus Saccharomyces, such as Saccharomyces serevisiae, and yeasts of the genus Pichia, such as Pichia pastoris;

[0349] - Filamentous fungi: Aspergillus niger and other Aspergillus species.

[0350] In addition, antibody gene expression systems utilizing prokaryotic cells are also known. For example, when using bacterial cells, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be suitably utilized. 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.

[0351] In addition to the above-mentioned host cells, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, the antibody gene can be constructed as a fusion gene by in-frame insertion into a gene encoding a protein inherently produced in milk. As a protein secreted into milk, for example, goat beta-casein can be used. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The desired antibody can be obtained in the form of a fusion protein with a milk protein in the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones can be given to the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0352] When the antigen binding molecules described in this specification sheets are administered to a human, the antigen binding domains in the antigen binding molecules may be derived from recombinant antibodies that have been artificially modified for purposes such as reducing heterologous antigenicity for humans. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies may be manufactured using known methods.

[0353] The variable region of the antibody used to make the antigen-binding domains of the antigen-binding molecules described in this specification is generally composed of three complementary determining regions (CDRs) clamped by four framework regions (FRs). CDRs are regions that substantially determine the binding specificity of an antibody. The amino acid sequence of CDRs is rich in diversity. On the other hand, the amino acid sequence constituting FRs often shows a high degree of identity even between antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of a certain antibody can be transplanted to other antibodies by transplanting CDRs.

[0354] Humanized antibodies are also referred to as reshaped human antibodies. Specifically, it is well known that the CDR of an animal other than a human, such as a mouse antibody, is transplanted into a human antibody. The common genetic recombination method for obtaining humanized antibodies is also known. Specifically, as a method for transplanting the CDR of a mouse antibody into the FR of a person, it is well known that for example Overlap Extension PCR is used. In Overlap Extension PCR, a base sequence encoding the mouse antibody CDR to be transplanted is added to the primers for synthesizing the human antibody FR. Primers are prepared respectively for the four FRs. Generally, for transplanting mouse CDR into human FR, it is considered advantageous to select human FRs with high homology to mouse FRs in terms of maintaining CDR function. That is, it is generally preferred to use the following human FRs, which comprise an amino acid sequence with high homology to the adjacent FR amino acid sequence of the mouse CDR to be transplanted.

[0355] In addition, the base sequences to be connected are designed to connect in-frame with each other. Human FRs are synthesized separately by each primer. As a result, products with DNA encoding mouse CDRs added to each FR are obtained. The base sequences encoding mouse CDRs in each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using human antibody genes as templates are annealed to each other, and a complementary strand synthesis reaction is performed. Through this reaction, human FRs are connected via the mouse CDR sequences.

[0356] The V region gene obtained by finally connecting 3 CDRs and 4 FRs is amplified in its full length by annealing with its 5' end and 3' end and adding a primer with an appropriate restriction enzyme recognition sequence. The DNA obtained as described above and the DNA encoding the human antibody C region are inserted into an expression vector in a manner of performing in-frame fusion, thereby making a human antibody expression vector. After the integration vector is introduced into the host to establish recombinant cells, the recombinant cells are cultivated and the humanized antibody is produced in the culture of the cultured cells by expressing the DNA encoding the humanized antibody (referring to European Patent Publication EP239400 and International Publication WO1996 / 002576).

[0357] By qualitatively or quantitatively measuring and evaluating the antigen binding activity of the humanized antibody prepared as described above, human antibody FRs can be appropriately selected so that the CDRs form a good antigen binding site when connected by CDRs. As needed, the amino acid residues of the FRs can also be replaced in a manner such that the human antibody CDRs are reconstructed to form a suitable antigen binding site. For example, the PCR method used to transplant mouse CDRs into human FRs can be applied to introduce amino acid sequence mutations into the FRs. Specifically, partial base sequence mutations can be introduced into primers that anneal to the FRs. Base sequence mutations are introduced into the FRs synthesized by such primers. By measuring and evaluating the antigen binding activity of mutant antibodies that have undergone amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Cancer Res., (1993) 53, 851-856).

[0358] Alternatively, desired human antibodies can be obtained by DNA immunization using transgenic animals having a full repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) as immunized animals.

[0359] And then, it is also known to use human antibody library to obtain human antibody by panning method.For example, the V region of human antibody is expressed on the surface of phage by phage display method in the form of single-chain antibody (scFv).The phage of scFv that expresses and binds to antigen can be selected.By analyzing the gene of the phage selected, the DNA sequence of the human antibody V region that encodes and binds to antigen can be determined.After determining the DNA sequence of the scFv that binds to antigen, after the V region sequence and the desired human antibody C region sequence are fused in frame, an appropriate expression vector is inserted, and thus expression vector can be made.This expression vector is imported into the suitable expression cell of above-mentioned enumeration, by making the gene expression of this human antibody of encoding, this human antibody can be obtained. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0360] Except phage display method, as the technology using human antibody library to obtain human antibody by panning, also known use the technology of cell-free translation system, the technology of cell or virus surface presentation antigen binding molecules, the technology of use emulsion etc.For example, as the technology using cell-free translation system, can use: by the removal of terminator codon etc. and via ribosome formation mRNA and the protein complex of translated ribosome display method, utilize compounds such as puromycin and make gene sequence and translated protein covalently bound cDNA display method, mRNA display method or use the CIS display method etc. that form gene and the protein complex of translated for the conjugated protein of nucleic acid.In addition, as the technology of cell or virus surface presentation antigen binding molecules, except phage display method, can also use E.coli display method, gram-positive bacteria display method, yeast display method, mammalian cell display method, virus display method etc.As the technology using emulsion, can use based on in emulsion, comprise gene and translation related molecule in vitro virus display method etc. These methods are already known (Nat Biotechnol. 2000 Dec; 18(12): 1287-92, Nucleic Acids Res. 2006; 34(19): e127, Proc Natl Acad Sci US A. 2004 Mar 2; 101(9): 2806-10, Proc Natl Acad Sci US A. 2004 Jun 22; 101(25): 9193-8, Protein Eng Des Sel. 2008 Apr; 21(4): 247-55, Proc Natl Acad Sci US A. 2000 Sep 26; 97(20): 10701-5, MAbs. 2010 Sep-Oct; 2(5): 508-18, Methods Mol Biol. 2012; 911: 183-98).

[0361] In addition, as a method for obtaining antibody genes, in addition to the above, the method of B cell cloning (identification and cloning of coding sequences of each antibody, isolation thereof, and use for constructing expression vectors for preparing each antibody (particularly IgG1, IgG2, IgG3 or IgG4)) described in Bernasconi et al. (Science (2002) 298, 2199-2202) or International Publication WO2008 / 081008 can also be appropriately used.

[0362] In addition, as a non-limiting embodiment of the antibody of the present invention, there can be mentioned: a chimeric antigen receptor (CAR) obtained by integrating a fusion of an antibody or a fragment thereof that recognizes an antigen other than a T cell receptor and a T cell signaling domain into a T cell, and a T cell incorporating the chimeric antigen receptor, but the present invention is not limited thereto.

[0363] EU number and Kabat number

[0364] According to the method used in the present invention, the amino acid positions assigned to CDRs and FRs of antibodies follow the Kabat convention (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 the Kabat numbering, and the amino acids in the constant region are represented according to the EU numbering based on the Kabat amino acid positions.

[0365] Antigen-binding domain whose binding activity varies depending on the concentration of low-molecular-weight compounds

[0366] As low molecular weight compound, for example, target tissue specific compound or non-natural compound can be enumerated.The following examples illustrate the selection method of the antigen binding domains of target tissue specific compound dependence, but the selection method of the antigen binding domains of the low molecular weight compound dependence other than the target tissue specific compound, etc. can also be suitably implemented according to the following example.In order to obtain the antigen binding activity that changes according to the concentration of the target tissue specific compound, the method shown in one of the above-mentioned binding activity items can be suitably adopted.As a non-limiting scheme, several specific examples are illustrated below.For example, in order to confirm that the antigen binding domains (or the antigen binding molecules comprising the domain) in the absence of the target tissue specific compound are compared to the binding activity of antigen, the antigen binding domains (or the antigen binding molecules comprising the domain) in the presence of the compound are increased to the binding activity of antigen, and the antigen binding domains (or the antigen binding molecules comprising the domain) in the absence of the target tissue specific compound and in the presence or in the presence of low concentration and in the presence of high concentration are compared to the binding activity of antigen. In a different non-limiting embodiment, for example, to confirm that the antigen-binding activity of an antigen-binding domain (or an antigen-binding molecule comprising such a domain) is higher in the presence of a high concentration of a target tissue-specific compound than in the presence of a low concentration of the compound, the antigen-binding activities of the antigen-binding domain (or an antigen-binding molecule comprising such a domain) in the presence of a low concentration and a high concentration of the target tissue-specific compound are compared.

[0367] Furthermore, in the present invention, the expression “the antigen-binding activity in the presence of a target tissue-specific compound is higher than the antigen-binding activity in the absence of the compound” may also be expressed as “the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the absence of a target tissue-specific compound is lower than the antigen-binding activity in the presence of the compound.” In addition, in the present invention, the expression “the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the absence of a target tissue-specific compound is lower than the antigen-binding activity in the presence of the compound” may also be expressed as “the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the absence of a target tissue-specific compound is weaker than the antigen-binding activity in the presence of the compound.”

[0368] Furthermore, in the present invention, the expression “the antigen-binding activity of the target tissue-specific compound in the presence of a high concentration is higher than the antigen-binding activity of the compound in the presence of a low concentration” may also be expressed as “the antigen-binding activity of the target tissue-specific compound of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the presence of a low concentration is lower than the antigen-binding activity of the compound in the presence of a high concentration”. In addition, in the present invention, the expression “the antigen-binding activity of the target tissue-specific compound of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the presence of a low concentration is lower than the antigen-binding activity of the compound in the presence of a high concentration” may also be expressed as “the antigen-binding activity of the target tissue-specific compound of the antigen-binding domain (or an antigen-binding molecule comprising the domain) in the presence of a low concentration is weaker than the antigen-binding activity of the compound in the presence of a high concentration”.

[0369] Conditions other than the concentration of the target tissue-specific compound when measuring antigen binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under the conditions of HEPES buffer and 37°C. For example, Biacore (GE Healthcare) etc. can be used for measurement. For the determination of the binding activity of an antigen-binding domain (or an antigen-binding molecule comprising the domain) to an antigen, when the antigen is a soluble molecule, the antigen is loaded as an analyte on a chip to which an antigen-binding domain (or an antigen-binding molecule comprising the domain) is fixed, thereby evaluating the binding activity to the soluble molecule; when the antigen is a membrane molecule, the antigen-binding domain (or an antigen-binding molecule comprising the domain) is loaded as an analyte on a chip to which an antigen is fixed, thereby evaluating the binding activity to the membrane molecule.

[0370] As long as the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule of the present invention (or an antigen-binding molecule comprising such a domain) in the absence of a target tissue-specific compound is weaker than the antigen-binding activity in the presence of the target tissue-specific compound, the ratio of the antigen-binding activity in the absence of the compound to the antigen-binding activity in the presence of the compound is not particularly limited. Preferably, the ratio of the KD (dissociation constant) in the absence of the target tissue-specific compound to the KD in the presence of the antigen, i.e., KD (in the absence of the compound) / KD (in the presence of the compound), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (in the absence of the compound) / KD (in the presence of the compound) value is not particularly limited and can be any value such as 400, 1000, or 10,000 as long as it can be prepared by the skills of those skilled in the art. When no antigen-binding activity is observed in the absence of the target tissue-specific compound, the upper limit is an infinite value.

[0371] As long as the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule of the present invention (or an antigen-binding molecule comprising such a domain) in the presence of a target tissue-specific compound at a low concentration is weaker than the antigen-binding activity in the presence of a high concentration of the target tissue-specific compound, the ratio of the antigen-binding activity of the compound in the presence of a low concentration to the antigen-binding activity of the compound in the presence of a high concentration is not particularly limited. Preferably, the ratio of the KD (dissociation constant) of the target tissue-specific compound in the presence of a low concentration to the KD in the presence of a high concentration, i.e., KD (in the presence of a low compound concentration) / KD (in the presence of a high compound concentration) is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (in the presence of a low compound concentration) / KD (in the presence of a high compound concentration) is not particularly limited and can be any value, such as 400, 1000, or 10,000, as long as it can be prepared by the skills of those skilled in the art. When no antigen-binding activity is observed in the presence of low concentrations of the target tissue-specific compound, the upper limit is an infinite value.

[0372] As the value of antigen-binding activity, KD (dissociation constant) can be used when the antigen is a soluble molecule, while the apparent KD (apparent dissociation constant) can be used when the antigen is a membrane-bound molecule. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scatchard plots, flow cytometry, etc.

[0373] In addition, as another indicator representing the ratio of the antigen-binding activity of the antigen-binding domain of the present invention (or antigen-binding molecule comprising the domain) in the absence of a target tissue-specific compound to the antigen-binding activity in the presence of the compound, for example, the dissociation rate constant kd (dissociation rate constant) can also be used appropriately. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator representing the ratio of binding activity, the ratio of the kd (dissociation rate constant) relative to the antigen in the absence of the target tissue-specific compound to the kd (dissociation rate constant) in the presence of the compound, i.e., the value of kd (in the absence of the compound) / kd (in the presence of the compound) is preferably 2 or more, more preferably 5 or more, more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (in the absence of the compound) / kd (in the presence of the compound) is not particularly limited and can be any value such as 50, 100, or 200 as long as it can be made by the technical common sense of those skilled in the art. When no antigen-binding activity is observed in the absence of the target tissue-specific compound, since dissociation does not occur, the upper limit is an infinite value.

[0374] Alternatively, as another indicator representing the ratio of the antigen-binding activity of the target tissue-specific compound of the present invention (or antigen-binding molecule comprising such a domain) in the presence of a low concentration of the compound to the antigen-binding activity in the presence of a high concentration, the dissociation rate constant, kd (dissociation rate constant), may be suitably used. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator representing the ratio of binding activity, the ratio of the kd (dissociation rate constant) relative to the antigen in the presence of a low concentration of the target tissue-specific compound to the kd (dissociation rate constant) in the presence of a high concentration of the compound, i.e., kd (in the presence of low compound concentration) / kd (in the presence of high compound concentration), is preferably 2 or greater, more preferably 5 or greater, even more preferably 10 or greater, and even more preferably 30 or greater. The upper limit of the value of Kd (in the presence of low compound concentration) / kd (in the presence of high compound concentration) is not particularly limited and can be any value, such as 50, 100, or 200, as long as it is within the technical knowledge of those skilled in the art. When no antigen-binding activity is observed in the presence of a low concentration of a target tissue-specific compound, dissociation does not occur, so the upper limit is an infinite value.

[0375] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble molecule, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane-bound molecule. Kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods well known to those skilled in the art, for example, using Biacore (GE healthcare), flow cytometers, etc. In addition, in the present invention, when measuring the antigen-binding activity of an antigen-binding domain (or an antigen-binding molecule comprising the domain) at a certain concentration of a target tissue-specific compound, the conditions other than the concentration of the compound are preferably set to be the same.

[0376] For example, as one embodiment provided by the present invention, an antigen-binding domain (or antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound can be obtained by screening for antigen-binding domains (or antigen-binding molecules) comprising the following steps (a) to (c).

[0377] Step (a): obtaining the antigen-binding activity of the antigen-binding domain (or antigen-binding molecule) in the absence of a target tissue-specific compound;

[0378] Step (b): obtaining the antigen-binding activity of the antigen-binding domain (or antigen-binding molecule) in the presence of a target tissue-specific compound;

[0379] Step (c): Selecting an antigen-binding domain (or antigen-binding molecule) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound.

[0380] For example, as one embodiment provided by the present invention, an antigen-binding domain (or antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by screening for antigen-binding domains (or antigen-binding molecules) comprising the following steps (a) to (c).

[0381] Step (a): obtaining the antigen-binding activity of the antigen-binding domain (or antigen-binding molecule) in the presence of a low concentration of a target tissue-specific compound;

[0382] Step (b): obtaining the antigen-binding activity of the antigen-binding domain (or antigen-binding molecule) in the presence of a high concentration of a target tissue-specific compound;

[0383] Step (c): Selecting an antigen-binding domain (or antigen-binding molecule) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound.

[0384] Furthermore, as one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound can be obtained by screening of antigen-binding domains (or antigen-binding molecules) or libraries thereof comprising the following steps (a) to (c).

[0385] Step (a): contacting the antigen-binding domain (or antigen-binding molecule) or a library thereof in the presence of a target tissue-specific compound with an antigen;

[0386] Step (b): placing the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a) above in the absence of the compound;

[0387] Step (c): Isolating the antigen-binding domain (or antigen-binding molecule) dissociated in the above step (b).

[0388] Furthermore, as one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by screening of antigen-binding domains (or antigen-binding molecules) or libraries thereof comprising the following steps (a) to (c).

[0389] Step (a): contacting the antigen-binding domain (or antigen-binding molecule) or a library thereof in the presence of a high concentration of a target tissue-specific compound with an antigen;

[0390] Step (b): placing the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a) above in the presence of a low concentration of the compound;

[0391] Step (c): Isolating the antigen-binding domain (or antigen-binding molecule) dissociated in the above step (b).

[0392] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of such a compound can be obtained by screening of antigen-binding domains (or antigen-binding molecules) or libraries thereof comprising the following steps (a) to (d).

[0393] Step (a): contacting the library of antigen-binding domains (or antigen-binding molecules) with an antigen in the absence of a target tissue-specific compound;

[0394] Step (b): selecting an antigen-binding domain (or antigen-binding molecule) that did not bind to the antigen in the aforementioned step (a);

[0395] Step (c): allowing the antigen-binding domain (or antigen-binding molecule) selected in step (b) to bind to the antigen in the presence of the compound;

[0396] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (c).

[0397] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by screening of antigen-binding domains (or antigen-binding molecules) or libraries thereof comprising the following steps (a) to (d).

[0398] Step (a): contacting the library of antigen binding domains (or antigen binding molecules) with an antigen in the presence of a low concentration of a target tissue-specific compound;

[0399] Step (b): selecting an antigen-binding domain (or antigen-binding molecule) that did not bind to the antigen in the aforementioned step (a);

[0400] Step (c): allowing the antigen-binding domain (or antigen-binding molecule) selected in step (b) to bind to the antigen in the presence of a high concentration of the compound;

[0401] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (c).

[0402] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (c).

[0403] Step (a): contacting the library of antigen-binding domains (or antigen-binding molecules) with an antigen-immobilized column in the presence of a target tissue-specific compound;

[0404] Step (b): eluting the antigen-binding domain (or antigen-binding molecule) bound to the column in step (a) above from the column in the absence of the compound;

[0405] Step (c): Isolating the antigen-binding domain (or antigen-binding molecule) eluted in the aforementioned step (b).

[0406] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by a screening method comprising the following steps (a) to (c).

[0407] Step (a): contacting the library of antigen-binding domains (or antigen-binding molecules) with an antigen-immobilized column in the presence of a high concentration of a target tissue-specific compound;

[0408] Step (b): eluting the antigen-binding domain (or antigen-binding molecule) bound to the column in step (a) above from the column in the presence of a low concentration of the compound;

[0409] Step (c): Isolating the antigen-binding domain (or antigen-binding molecule) eluted in the aforementioned step (b).

[0410] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (d).

[0411] Step (a): passing the library of antigen-binding domains (or antigen-binding molecules) through a column immobilized with an antigen in the absence of a target tissue-specific compound;

[0412] Step (b): Recovering the antigen-binding domain (or antigen-binding molecule) that was not bound to the column and eluted in the aforementioned step (a);

[0413] Step (c): allowing the antigen-binding domain (or antigen-binding molecule) recovered in step (b) to bind to the antigen in the presence of the compound;

[0414] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (c).

[0415] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by a screening method comprising the following steps (a) to (d).

[0416] Step (a): passing the library of antigen-binding domains (or antigen-binding molecules) through a column immobilized with antigen in the presence of a low concentration of a target tissue-specific compound;

[0417] Step (b): Recovering the antigen-binding domain (or antigen-binding molecule) that was not bound to the column and eluted in the aforementioned step (a);

[0418] Step (c): allowing the antigen-binding domain (or antigen-binding molecule) recovered in step (b) to bind to the antigen in the presence of a high concentration of the compound;

[0419] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (c).

[0420] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (d).

[0421] Step (a): contacting the library of antigen-binding domains (or antigen-binding molecules) with an antigen in the presence of a target tissue-specific compound;

[0422] Step (b): obtaining the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a);

[0423] Step (c): placing the antigen-binding domain (or antigen-binding molecule) obtained in the aforementioned step (b) in the absence of the compound;

[0424] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) whose antigen-binding activity in step (c) is weaker than the reference selected in step (b).

[0425] Furthermore, as one embodiment of the present invention, an antigen-binding domain (or an antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound can be obtained by a screening method comprising the following steps (a) to (d).

[0426] Step (a): contacting the library of antigen binding domains (or antigen binding molecules) with an antigen in the presence of a high concentration of a target tissue-specific compound;

[0427] Step (b): obtaining the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a);

[0428] Step (c): placing the antigen-binding domain (or antigen-binding molecule) obtained in the above step (b) in the presence of a low concentration of the compound;

[0429] Step (d): Isolating the antigen-binding domain (or antigen-binding molecule) whose antigen-binding activity in step (c) is weaker than the reference selected in step (b).

[0430] It should be noted that the aforementioned steps can be repeated more than two times. Therefore, according to the present invention, there is provided an antigen-binding domain (or antigen-binding molecule comprising such a domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound, or an antigen-binding domain (or antigen-binding molecule comprising such a domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than that in the presence of a high concentration of the compound, obtained by a screening method further comprising repeating steps (a) to (c) or (a) to (d) more than two times in the aforementioned screening method. The number of repetitions of steps (a) to (c) or (a) to (d) is not particularly limited and is generally within 10 times.

[0431] In the screening methods of the present invention, target tissue-specific compounds can be compounds whose target tissue specificity is defined by being present in target tissue at a different concentration (e.g., a higher concentration or a lower concentration) than in non-target tissues. For example, target tissue-specific compounds can be differentially present at any concentration. However, in general, target tissue-specific compounds can be present at a concentration that increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 100-fold, or at least 100-fold. 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 The concentration of the drug is present at a level of 100 times or more, up to infinity (i.e., it does not exist in non-target tissues).

[0432] The threshold value for distinguishing between low concentration and high concentration can be appropriately set for each compound. For example, in a non-limiting embodiment of the threshold value of ATP or adenosine, the low concentration condition can be appropriately set from 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, or 0 M as the threshold value. The high concentration condition can be at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 10 times, or at least 10 times the respective threshold values. 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 In addition, in a non-limiting embodiment of PGE2, as a threshold value, the low concentration condition can be suitably set in a value of 10pM, 1pM, 100fM, 10fM, 1fM or 0M. Corresponding to the set threshold value, the high concentration condition can be suitably set in a value of at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 10 times, or more. 3 times, at least 10 4 times, at least 10 5 times, at least 10 6Furthermore, in a non-limiting embodiment of kynurenine, as a threshold value, the low concentration condition can be appropriately set at a value of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or 0 M. Corresponding to the set threshold value, the high concentration condition can be at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 10 times, or more of each threshold value. 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 The value of multiple can be set appropriately.

[0433] The antigen-binding activity of an antigen-binding domain (or antigen-binding molecule) can be measured by methods well known to those skilled in the art. For conditions other than the concentration of the target tissue-specific compound, those skilled in the art can appropriately determine. The antigen-binding activity of an antigen-binding domain (or antigen-binding molecule) can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate: Dissociation rate constant), or apparent kd (Apparent dissociation: Apparent dissociation rate constant). They can be measured by methods well known to those skilled in the art, for example, Biacore (GE healthcare), Scatchard plot, FACS, etc. can be used.

[0434] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the presence of a target tissue-specific compound is higher than that in the absence of the compound has the same meaning as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the absence of the target tissue-specific compound is lower than that in the presence of the compound.

[0435] Furthermore, in the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the presence of a high concentration of a target tissue-specific compound is higher than that in the presence of a low concentration of the compound has the same meaning as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the absence of the target tissue-specific compound is lower than that in the presence of the compound.

[0436] As long as the antigen-binding activity in the absence of the target tissue-specific compound is lower than the antigen-binding activity in the presence of the compound, the difference between the antigen-binding activity in the presence of the compound and the antigen-binding activity in the absence is not particularly limited. The antigen-binding activity in the presence of the preferred compound is more than 2 times the antigen-binding activity in the absence, more preferably more than 10 times, more preferably more than 40 times. The upper limit of the difference in antigen-binding activity is not particularly limited. As long as the technology of those skilled in the art can be made, it can be any value such as 400 times, 1000 times, or 10,000 times. When no antigen-binding activity is observed in the absence of the target tissue-specific compound, the upper limit is an infinite numerical value.

[0437] The antigen-binding domains (or antigen-binding molecules comprising the domains) of the present invention screened by the aforementioned screening methods can be any antigen-binding domains (or antigen-binding molecules), for example, the above-mentioned antigen-binding domains (or antigen-binding molecules) can be screened. For example, antigen-binding domains (or antigen-binding molecules) with native sequences can be screened, or antigen-binding domains (or antigen-binding molecules) with substituted amino acid sequences can be screened.

[0438] library

[0439] According to a certain embodiment, the antigen-binding domain of the present invention (or an antigen-binding molecule containing the domain) can be obtained from a library, which mainly comprises a plurality of antigen-binding molecules, the sequences of the plurality of antigen-binding molecules being different from each other, and the antigen-binding domain thereof containing at least one amino acid residue that changes the binding activity of the antigen-binding molecule to the antigen according to the concentration of the low-molecular compound. As a non-limiting embodiment of the low-molecular compound, a target tissue-specific compound or a non-natural compound can be cited. Examples of target tissue-specific compounds include: (1) primary metabolites of the glycolytic system or the Kreb's cycle such as lactic acid, succinic acid, and citric acid; (2) amino acids such as alanine, glutamic acid, or aspartic acid; (3) kynurenine and its metabolites, i.e., metabolites of amino acids such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid; (4) metabolites of arachidonic acid such as prostaglandin E2; and (5) nucleosides having a purine ring structure such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). The following example illustrates a library primarily comprising a plurality of antigen-binding molecules whose sequences differ from one another and whose antigen-binding domains contain at least one amino acid residue that varies the antigen-binding activity of the antigen-binding molecule in response to adenosine and / or ATP, which are target tissue-specific compounds. However, libraries of antigen-binding molecules whose antigen-binding activity varies depending on the concentration of low-molecular-weight compounds other than adenosine and / or ATP can also be appropriately applied according to the following examples.

[0440] As used herein, a "library" refers to a collection of multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, each having a different sequence, or nucleic acids or polynucleotides encoding these molecules or polypeptides. The sequences of the multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules contained in a library are not a single sequence, but rather antigen-binding molecules or fusion polypeptides containing antigen-binding molecules having different sequences.

[0441] As a scheme of the "library" in this specification, it is possible to provide not only a library that can effectively obtain antigen-binding molecules that bind to the target antigen in the presence of low molecular weight molecules and do not bind to the target antigen in the absence of low molecular weight molecules (low molecular weight dependence), but also a library that can effectively obtain antibodies that bind to the target antigen in the absence of low molecular weight molecules and do not bind to the target antigen in the presence of low molecular weight molecules (low molecular weight anti-dependence).

[0442] In one embodiment of the present invention, a fusion polypeptide of an antigen-binding molecule of the present invention and a heterologous polypeptide can be prepared. In a certain embodiment, the fusion polypeptide can be obtained by fusing with at least a portion of a viral coat protein, for example, a viral coat protein selected from pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI, and mutants thereof.

[0443] In a certain embodiment, the antigen binding molecules of the present invention can be ScFv, Fab fragments, F(ab)2 or F(ab')2. In another embodiment, a library is provided that mainly comprises a plurality of fusion polypeptides whose sequences are different from each other, and the fusion polypeptides are fusion polypeptides of these antigen binding molecules and heterologous polypeptides. Specifically, a library is provided that mainly comprises a plurality of fusion polypeptides whose sequences are different from each other, and the fusion polypeptides are fusion polypeptides obtained by fusion of these antigen binding molecules with viral coat proteins, for example, fusion polypeptides obtained by fusion of at least a portion of viral coat proteins selected from pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and mutants thereof. The antigen binding molecules of the present invention can also include a dimerization domain. In a certain embodiment, the aforementioned dimerization domain can be present between the heavy chain or light chain variable region of an antibody and at least a portion of a viral coat protein. The dimerization domain can include at least one dimerization sequence and / or one or more sequences comprising cysteine ​​residues. The dimerization domain can preferably be connected to the C-terminus of the heavy chain variable region or constant region. The dimerization domain can assume various structures, depending on whether the antibody variable region is prepared as a fusion polypeptide component with a viral coat protein component (without an amber stop codon following the dimerization domain), or whether the antibody variable region is prepared primarily without viral coat protein components (e.g., with an amber stop codon following the dimerization domain). When the antibody variable region is prepared primarily as a fusion polypeptide component with a viral coat protein component, bivalent presentation is achieved through one or more disulfide bonds and / or a single dimerization sequence.

[0444] In this specification, the term "sequences differ from each other" in describing a plurality of antigen-binding molecules with different sequences means that the sequences of the antigen-binding molecules in the library differ from each other. That is, the number of sequences that differ from each other in the library reflects the number of independent clones with different sequences in the library, and is sometimes also referred to as the "library size". A typical phage display library has 10 6 to 10 12 By using well-known techniques such as ribosome display, the library size can be amplified to 10 14However, the actual number of phage particles used in panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess is also called the "library equivalent number", which means that there can be 10 to 10,000 clones with the same amino acid sequence. Therefore, the term "sequences are different from each other" in the present invention means that the sequences of the antigen-binding molecules in the library excluding the library equivalent number are different from each other, and more specifically means that there are 10 to 10,000 antigen-binding molecules with different sequences. 6 to 10 14 molecules, preferably 10 7 to 10 12 molecules, more preferably 10 8 to 10 11 , particularly preferably 10 8 to 10 10 .

[0445] In addition, the term "multiple" in the record of the library mainly comprising multiple antigen binding molecules in the present invention, for example, antigen binding molecules of the present invention, fusion polypeptide, polynucleotide molecules, vector or virus, generally refers to two or more sets of these substances. For example, as long as more than two substances are different from each other in a specific form, it is represented that the substance has more than two kinds. As an example, the mutant amino acid observed at the specific amino acid position in the amino acid sequence can be enumerated. For example, when, except for flexible residues or except for the specific mutant amino acid at the hypervariable amino acid position exposed to the surface, when two or more antigen binding molecules of the present invention having substantially the same sequence, preferably the same sequence, exist, then the antigen binding molecules of the present invention exist multiple. In other examples, when, except for the base encoding flexible residues or except for the base encoding the specific mutant amino acid at the hypervariable amino acid position exposed to the surface, when two or more polynucleotide molecules of the present invention having substantially the same sequence, preferably the same sequence, exist, then the polynucleotide molecules of the present invention exist multiple.

[0446] Furthermore, the expression "mainly comprising" in the description of the library of the present invention as mainly comprising a plurality of antigen-binding molecules reflects the number of antigen-binding molecules whose antigen-binding activity varies depending on the concentration conditions of low-molecular-weight compounds (e.g., target tissue-specific compounds) among the number of independent clones with different sequences in the library. Specifically, it is preferred that there are at least 10 antigen-binding molecules in the library that exhibit such binding activity. 4 Furthermore, it is more preferred that the antigen-binding domain of the present invention can be composed of at least 10 antigen-binding molecules that exhibit such binding activity. 5 It is further preferred that the antigen-binding domain of the present invention can be obtained from a library of at least 10 antigen-binding molecules that exhibit such binding activity. 6It is particularly preferred that the antigen-binding domain of the present invention can be obtained from a library of at least 10 antigen-binding molecules that exhibit such binding activity. 7 It is also preferred that the antigen-binding domain of the present invention can be obtained from a library of at least 10 antigen-binding molecules that exhibit such binding activity. 8 In other expressions, it can also be suitably expressed as the ratio of antigen-binding molecules whose antigen-binding activity of the antigen-binding domain differs depending on the presence or absence of adenosine and / or ATP, among the number of independent clones with different sequences in the library. Specifically, the antigen-binding domain of the present invention can be composed of antigen-binding molecules that exhibit such binding activity accounting for 10% of the number of independent clones with different sequences in the library. -6 %% to 80%, preferably 10 -5 % to 60%, more preferably 10 -4 % to 40% of the library is obtained. The case of fusion polypeptides, polynucleotide molecules or vectors is the same as above and can be expressed by the number of molecules or the ratio of all molecules. In addition, the case of viruses is the same as above and can be expressed by the number of virus individuals or the ratio of all individuals. As a non-limiting embodiment of the present invention, when there is one antigen that binds to multiple antigen-binding molecules, the number of antigen-binding molecules that show such binding activity in the library is preferably at least 10 molecules, 100 molecules, 1000 molecules, 10 4 molecules, 10 5 molecules, 10 6 molecules, 10 7 molecules or 10 8 In addition, it is more preferred that the antigen-binding domains of the present invention be obtained from a library in which at least 10 molecules of antigen-binding molecules showing such binding activity are present. It is further preferred that the antigen-binding domains of the present invention be obtained from a library in which at least 100 molecules of antigen-binding molecules showing such binding activity are present. It is particularly preferred that the antigen-binding domains of the present invention be obtained from a library in which at least 1000 molecules of antigen-binding molecules showing such binding activity are present.

[0447] As one embodiment of the present invention, there is provided a library produced by a method comprising the following steps (a) and (b):

[0448] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0449] (i) one or more amino acid sites that are not involved in binding the low molecular weight compound;

[0450] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs;

[0451] (iii) one or more amino acid positions that are not important for the formation of the canonical structure; and

[0452] Step (b) designing a library comprising: nucleic acids encoding unaltered versions of the antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain, each having an alteration at one or more amino acid positions identified in step (a) and having sequences different from each other.

[0453] In the present invention, "one or more amino acid sites that are not involved in binding to low molecular weight compounds" can be identified by methods such as crystal structure analysis of a complex of a low molecular weight compound and an antibody, stereostructure analysis using NMR, or introduction of amino acid mutations. As a non-limiting embodiment of the present invention, residues of antibodies that are not involved in binding to low molecular weight compounds can be identified based on crystal structure analysis of a complex of a low molecular weight compound and an antibody. Here, "participating in binding to low molecules" refers to a state in which the side chains or main chain atoms of the amino acids forming the H chain or L chain of the antibody form intermolecular interactions with the atoms of the low molecular weight compound at a distance that can have an effect on the binding activity, or a state in which a certain amino acid residue contributes to the binding of the low molecular weight compound (including indirect effects such as stabilizing the stereostructure of the CDR loop to the configuration when the low molecular weight compound is bound), as well as a state that satisfies both of these.

[0454] The "state in which intermolecular interactions are formed" in this specification can be determined, for example, based on the interatomic distances between the non-hydrogen atoms of the side chains or main chains of the amino acids constituting the H chain or L chain of the antibody and the non-hydrogen atoms constituting the low molecular weight compound, according to crystal structure analysis of the complex of the low molecular weight compound and the antibody. For example, the interatomic distance is preferably or More preferably, the interatomic distance is or Within.

[0455] More specifically, the possibility of an interaction occurring can be directly determined based on information about the interatomic distances in the three-dimensional structure, the type of intermolecular interaction formed, and the type of atom. Mutations to amino acid residues such as Ala or Gly can be more accurately determined based on their effect on the activity of low-molecular-weight compounds, but are not limited to these.

[0456] The "state of indirect influence" in this specification can be estimated by, for example, analyzing the configuration of each amino acid residue and the state of intermolecular interaction with surrounding residues based on the three-dimensional structure of the complex between the low molecule and the antibody, and whether the binding of the low molecule is indirectly affected. The introduction of mutations to amino acid residues such as Ala or Gly can be more accurately judged based on the effect on the activity of the low molecule compound.

[0457] As a scheme of the present invention, even if the residue that is accredited as not participating in conjunction with low molecule is replaced by other amino acid, the amino acid that also can maintain the combination with compound with appropriate degree can be selected.Whereby, the library that selected amino acid appears in selected residue can be designed.At this moment, the mode design that mainly comprises multiple antigen binding molecules is formed in that the residue that is accredited as not participating in conjunction with low molecule compound is replaced by amino acid whose set of antigen binding molecules is different from each other.

[0458] In another embodiment, the amino acid site not involved in binding to the low molecular weight compound may be determined as an amino acid site other than one or more amino acid sites selected from the amino acid sites involved in binding to the low molecular weight compound.

[0459] As a non-limiting embodiment of the present invention, "one or more amino acid sites not involved in binding low-molecular-weight compounds" can be identified by introducing amino acid mutations. For example, variable region amino acids are comprehensively altered, and the binding of each variant to low-molecular-weight compounds is measured using known methods such as Biacore. The binding activity (affinity) of each variant to low-molecular-weight compounds is calculated as a KD value. This KD value is compared with the KD value of the unaltered variant of the parent sequence, and altered positions that exhibit binding above a certain threshold are identified as amino acid sites not involved in binding low-molecular-weight compounds. For example, the binding activity (affinity) of each variant for low molecules is calculated as a KD value based on the results of measurement using a known method such as Biacore. As heavy chain sites, those whose low molecule binding ability is not reduced to 1 / 100, 1 / 50, 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 of the binding ability of the unmodified form due to the modification, and as light chain sites, those whose low molecule binding ability is not reduced to 1 / 100, 1 / 50, 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 of the binding ability of the unmodified form due to the modification, are determined to be amino acid sites not involved in binding to low molecular weight compounds. However, the present invention is not limited to the above criteria. Alternatively, instead of comparing the KD value with the unmodified form of the parental sequence, the binding activity (affinity) of each variant to low molecules is calculated as a KD value, and those with a binding ability of at least 10mM, 1mM, 100uM, 10uM, 1uM, 100nM, 10nM, 1nM, 100pM, 10pM, or 1pM as heavy chain sites, and those with a binding ability of at least 10mM, 1mM, 100uM, 10uM, 1uM, 100nM, 10nM, 1nM, 100pM, 10pM, or 1pM as light chain sites are determined to be amino acid sites not involved in binding to low molecular weight compounds, but are not limited to the above criteria.

[0460] The low-molecular-weight binding activity of the unmodified and modified forms can be measured by appropriately selecting from methods known to those skilled in the art (Biacore, ELISA, ECL, etc.).

[0461] In another embodiment, the amino acid site not involved in binding to the low molecular weight compound may be determined as an amino acid site other than one or more amino acid sites selected from the amino acid sites involved in binding to the low molecular weight compound.

[0462] The "library comprising nucleic acids encoding multiple variants of the antigen-binding domains or antigen-binding molecules comprising the antigen-binding domains, each of which has a different encoding sequence" in the present invention includes, but is not particularly limited to, designing a library comprising multiple variants of the antigen-binding domains or antigen-binding molecules comprising the antigen-binding domains in which amino acids at specific sites are changed to desired amino acids using known library technologies such as NNK and TRIMLibrary (Gonzalez-Munoz A et al. MAbs 2012, Lee CV et al. J Mol Biol. 2004, Knappik A. et al. J Mol Biol. 2000, Tiller T et al. MAbs 2013).

[0463] The term "one or more amino acids" in the present invention does not particularly limit the number of amino acids, and may include two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty amino acids.

[0464] In the present invention, "amino acid sites with diverse amino acid occurrence frequencies" refer to amino acid sites at which two or more types of amino acids are confirmed as being present at a frequency of 1% or more in the antibody repertoire of the animal species to which the parent antibody (parent antigen-binding domain) belongs.

[0465] The "parent antigen-binding domain" in the present invention refers to an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound and serves as a template for library preparation, or an antigen-binding domain having low-molecular-weight compound-binding activity.

[0466] The "antibody repertoire of the animal species to which the parent antigen-binding domain belongs" in the present invention refers to the components of the antibody gene sequence genetically confirmed in the animal species from which the parent antigen-binding domain originates. As an example, and not limited thereto, when the parent antigen-binding domain is from a human, the antibody repertoire of the animal species to which the parent antigen-binding domain belongs refers to the components of the antibody gene sequence genetically confirmed in humans; and when the parent antigen-binding domain is from a rabbit, the antibody repertoire of the animal species to which the parent antigen-binding domain belongs refers to the components of the antibody gene sequence genetically confirmed in rabbits. Sequences that are not actually expressed as antibodies due to the presence of stop / start codons or frameshifts, even if present genetically, are not included.

[0467] When the parent antigen-binding domain is derived from a non-human animal, it can be humanized according to conventional methods, which are widely known to those skilled in the art (European Patent Publication No. EP239400, International Publications No. WO1996 / 002576, WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735, WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388, Cancer Research International, 1995 / 014439, WO1996 / 015439, WO1997 / 015544, WO1997 / 015639, WO1998 / 015730, WO1999 / 015831, WO1999 / 015914, WO1999 / 020914, WO1999 / 020924, WO1999 / 020936, WO1999 / 020937, WO1999 / 020938 Res., (1993) 53, 851-856, BBRC., (2013) 436 (3): 543-50, etc.). When the parent antigen-binding domain is humanized according to the conventional method and then libraryized, in the "antibody repertoire of the animal species to which the parent antigen-binding domain belongs" in the present invention, both the antigen-binding domain before humanization and the antigen-binding domain after humanization can be treated as the parent antigen-binding domain. Accordingly, the components of the animal species from which the antigen-binding domain before humanization comes and humans can be applied as the same animal species. As an example, but not limited to this, when the antigen-binding domain before humanization comes from a rabbit, the antibody repertoire of the animal species to which the parent antigen-binding domain belongs refers to the components of the antibody gene sequence confirmed in the genes of humans and / or rabbits. Among them, even if present in the gene, sequences that are not actually expressed as antibodies due to the presence of stop / start codons or frameshifts are not included.

[0468] As an example, but not limited to this, the antibody spectrum of the animal species to which the parent antigen-binding domain belongs can be studied by reference to known databases. Sites with diverse amino acid frequency of occurrence are generally present in the CDR region. In one embodiment, when determining the hypervariable positions of known and / or natural antibodies, the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) are effective. In addition, a large number of sequences of human light chains and heavy chains collected and their configurations are provided in multiple databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html), and the information of these sequences and their configurations is useful for determining the hypervariable positions in the present invention.

[0469] In addition, as another scheme, the antibody spectrum of the animal species to which the parent antigen binding domain belongs can also be studied by cloning the antibody gene obtained by the biological species and analyzing the sequence. As an example, it is not limited thereto, the antibody spectrum of the people can be studied by analyzing the sequence of the natural library composed of the antibody gene constructed from normal human lymphocytes and its constituents by the antibody sequence, i.e., natural sequence, which does not contain deviations (Gejima et al. (Human Antibodies (2002) 11,121-129) and Cardoso et al. (Scand. J. Immunol. (2000) 51,337-344)). When studying the constituents, it is desirable to analyze at least 100, preferably 200, and more preferably 400 sequences.

[0470] It is not limited thereto, and the antibody repertoire of the animal species to which the parent antigen-binding domains in the present invention belong is more preferably a subclass of the germline to which the parent antigen-binding domains belong. As examples of frameworks, sequences of fully human framework regions currently known and included in websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ) can be suitably used as sequences of the germline contained in the antigen-binding molecules of the present invention. Germline sequences can be classified into subclasses based on their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798), Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461), and Cox et al. (Nat. Genetics (1994) 7, 162-168). As an example, seven subclasses have been reported for the heavy chain variable region of human antibodies, seven subclasses for Vκ, and ten subclasses for Vλ. Each amino acid position can be studied by analyzing the amino acid composition within the subclass to which the parent antigen-binding domain belongs, but this approach is not particularly limiting.

[0471] In the present invention, the "amino acid sites not important for the formation of the canonical structure" refers to the clustering of the three-dimensional structures of the antibody heavy and light chains, mainly CDR1 and 2, and the structure can be classified according to the subclass of the antibody, the length and sequence of the CDR. In each canonical structure, the residues important for maintaining the structure are already known. By referring to reports such as Chothia et al. (J. Mol. Biol. (1992) 227, 799-817), Al-Lazikani et al. (J. Mol. Biol. (1997) 273, 927-948), and Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798), the canonical structure into which the parent antigen-binding molecule is classified and the residues important for its structure can be determined.

[0472] Furthermore, the presence of residues important for maintaining structure in antigen-binding domains other than antibodies is well known, but the presence of residues is not limited thereto. Structural analysis of prepared mutants can identify amino acid sites in each antigen-binding domain that are not important for forming the canonical structure.

[0473] Another embodiment of the library of the present invention includes the following libraries.

[0474] A library produced by a method comprising the following steps (a) to (d):

[0475] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0476] (i) one or more amino acid sites that are not involved in binding the low molecular weight compound;

[0477] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs;

[0478] (iii) one or more amino acid positions that are not important for the formation of the canonical structure;

[0479] Step (b) preparing multiple variants of the antigen-binding domain or antigen-binding molecule comprising the antigen-binding domain, each having a sequence different from another, with alterations at one or more amino acid positions identified in step (a);

[0480] Step (c) identifying one or more amino acid changes that do not substantially change the low molecular compound binding activity of each variant; and

[0481] Step (d) produces a library comprising nucleic acids encoding unaltered variants and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain identified in step (c) that have altered sequences at one or more amino acids.

[0482] In the "step of producing multiple variants of the antigen-binding domain or antigen-binding molecule comprising the antigen-binding domain having different sequences" of the present invention, among the amino acid positions identified in step (a), the CDR1 and CDR2 positions can be replaced with amino acids with a germline frequency of 10% or greater, 9% or greater, 8% or greater, 7% or greater, 6% or greater, 5% or greater, 4% or greater, 3% or greater, 2% or greater, or 1% or greater, and the CDR3 position can be replaced with an amino acid with a germline frequency of 10% or greater, 9% or greater, 8% or greater, 7% or greater, 6% or greater, 5% or greater, 4% or greater, 3% or greater, 2% or greater, or 1% or greater, to produce the variants, but are not limited to these.

[0483] The term "variants" used in the present invention refers to variants of the antigen-binding domain in which at least one or more amino acids are substituted relative to the unmodified antigen-binding domain of the parent sequence.

[0484] In the present invention, the term "identifying one or more amino acid changes that do not substantially alter the low-molecular-weight compound binding activity of each variant" has the following meanings. For example, the binding of each variant to the low-molecular-weight compound is measured using known methods such as Biacore, and the binding activity (affinity) of each variant to the low-molecular-weight compound is calculated as a KD value. This KD value is compared with the KD value of the unmodified variant as the parent sequence, and altered positions showing binding above a certain threshold are identified as sites suitable for alteration. The amino acid substituted at this site can be identified as an amino acid suitable for libraryization (a flexible residue present in the library), but this is not limiting. Alternatively, instead of comparing the KD value of the unmodified variant as the parent sequence, altered positions showing binding above a certain threshold can be identified as sites suitable for alteration. The amino acid substituted at this site can be identified as an amino acid suitable for libraryization (a flexible residue present in the library), but this is not limiting.

[0485] In the present invention, the term "modified position showing binding higher than a certain standard" when determining amino acids suitable for libraryization has the following meaning. For example, as a result of measurement using a known method such as Biacore, the binding activity (affinity) of each variant to low molecules is calculated as a KD value. Sites in the heavy chain where the low molecule binding ability is not reduced to 1 / 100, 1 / 50, 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 of the binding ability of the unmodified form due to the modification, and sites in the light chain where the low molecule binding ability is not reduced to 1 / 100, 1 / 50, 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 of the binding ability of the unmodified form due to the modification, are determined to be sites suitable for libraryization. Amino acids substituted at these sites can be determined as amino acid positions suitable for libraryization, but the criteria are not limited to the above. Alternatively, the binding activity (affinity) of each variant to low molecules is calculated as a KD value by comparing the KD value with the unmodified variant as the parental sequence. Sites with a binding ability of at least 10 mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or 1 pM for the heavy chain and at least 10 mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or 1 pM for the light chain are determined to be sites that can be modified. The amino acid substituted at this site can be determined as an amino acid position that can be libraryized, but is not limited to the above criteria.

[0486] The low-molecular-weight binding activity of the unmodified and modified forms can be measured by appropriately selecting from methods known to those skilled in the art (Biacore, ELISA, ECL, etc.).

[0487] In another embodiment, the amino acid site not involved in binding to the low molecular weight compound may be determined as an amino acid site other than one or more amino acid sites selected from the amino acid sites involved in binding to the low molecular weight compound.

[0488] The "step of producing a library comprising a nucleic acid encoding an unmodified variant and a plurality of variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain whose sequences are different from each other and in which one or more amino acids identified in step (d) have been altered" in the present invention includes, but is not limited to, constructing the library such that the frequency of occurrence of the amino acids at specific sites of each amino acid identified in step (d) is the same (for example, such that each amino acid appears at 10% when the amino acid composition is 10 types).

[0489] Another embodiment of the library of the present invention includes the following libraries.

[0490] A library produced by a method comprising the following steps 1) and 2):

[0491] Step 1) contacting a library comprising a plurality of antigen-binding molecules having low-molecular-weight compound-binding activity with a low-molecular-weight compound; and

[0492] Step 2) Nucleic acids encoding multiple variants of antigen-binding molecules having low-molecular-weight compound-binding activity are concentrated from the library.

[0493] As another embodiment, the library is a library in which the antigen-binding molecules are antigen-binding molecules comprising the heavy chain variable region and light chain variable region of an antibody, and is produced by a method comprising any one of the following steps 1) to 3).

[0494] Step 1) Concentrating nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound binding activity from the library to design a library, wherein the library comprises nucleic acids encoding one or more variants in which amino acids in the heavy chain variable region are altered;

[0495] Step 2) enriching nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound-binding activity from the library to design a library, wherein the library comprises nucleic acids encoding one or more variants with altered amino acids in the light chain variable region;

[0496] In step 3), nucleic acids encoding antigen-binding molecules enriched from the variable region libraries in steps 1) and 2) are combined to design a library.

[0497] " Concentration " in the present invention refers to the presence ratio of nucleic acid encoding variants with target activity compared to the library before the concentration operation is implemented. As an example, it is not limited thereto, the nucleic acid encoding variants of antigen binding molecules with low molecular weight compound binding activity can be achieved as follows: the presence ratio of nucleic acid encoding variants of antigen binding molecules with low molecular weight compound binding activity is increased by panning. More specifically, as an example, a phage presenting a library containing multiple antigen binding molecules on a surface by a phage display method is contacted with the low molecular weight compound and the phage presenting the molecule without binding activity and the phage presenting the molecule are removed by a washing operation, and only the phage presenting the antigen binding molecules that maintain binding is recovered, thus the presence ratio of nucleic acid encoding variants of antigen binding molecules with low molecular weight compound binding activity can be increased by panning, but it is not limited thereto. More specifically, the presence ratio of nucleic acid encoding variants with target activity is preferably increased by 1.1 times compared to the library before the concentration operation is implemented. More preferably, the library of the present invention can be produced by increasing the abundance ratio of nucleic acids encoding variants having the target activity by 1.2 times or more, 1.5 times or more, 2 times or more, 4 times or more, 10 times or more, 25 times or more, or 100 times or more.

[0498] Method for producing a library

[0499] The present invention also relates to a method for producing a "library" of the various aspects encompassed by the present invention.

[0500] The "library production method" of the present invention is not limited to any specific method exemplified below, and includes any method as long as it can produce the "library" of the present invention.

[0501] Examples of the "library production method" of the present invention include the following methods.

[0502] In addition, each specific matter in the "library production method" exemplified below has the technical meaning described in detail above with respect to the "library" of the present invention.

[0503] (Example 1)

[0504] A method for producing a library comprising the following steps (a) and (b), wherein the library mainly comprises:

[0505] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0506] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0507] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound, or antigen-binding molecules comprising such domains;

[0508] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0509] (i) one or more amino acid sites that are not involved in binding the low molecular weight compound;

[0510] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs;

[0511] (iii) one or more amino acid positions that are not important for the formation of the canonical structure; and

[0512] Step (b) designing a library comprising: nucleic acids encoding unaltered versions of the antigen-binding domain and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain, each having an alteration at one or more amino acid positions identified in step (a) and having sequences different from each other.

[0513] (Example 2)

[0514] A method for producing a library comprising the following steps (a) to (d), wherein the library mainly comprises:

[0515] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0516] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0517] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound, or antigen-binding molecules comprising such domains;

[0518] Step (a) identifying amino acid sites in an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound or an antigen-binding domain having low-molecular-weight compound-binding activity that satisfy any one or more of the following (i) to (iii);

[0519] (i) one or more amino acid sites that are not involved in binding the low molecular weight compound;

[0520] (ii) one or more amino acid positions that have a diversity of amino acid occurrence frequencies in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs;

[0521] (iii) one or more amino acid positions that are not important for the formation of the canonical structure;

[0522] Step (b) preparing multiple variants of the antigen-binding domain or antigen-binding molecule comprising the antigen-binding domain, each having a sequence different from another, with alterations at one or more amino acid positions identified in step (a);

[0523] Step (c) identifying one or more amino acid changes that do not substantially change the low molecular compound binding activity of each variant; and

[0524] Step (d) produces a library comprising nucleic acids encoding unaltered variants and nucleic acids encoding multiple variants of the antigen-binding domain or antigen-binding molecules comprising the antigen-binding domain identified in step (c) that have altered sequences at one or more amino acids.

[0525] (Example 3)

[0526] A method for producing a library comprising the following steps 1) and 2), wherein the library mainly comprises:

[0527] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0528] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0529] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound, or antigen-binding molecules comprising such domains;

[0530] Step 1) contacting a library comprising a plurality of antigen-binding molecules having low-molecular-weight compound-binding activity with a low-molecular-weight compound; and

[0531] Step 2) Nucleic acids encoding multiple variants of antigen-binding molecules having low-molecular-weight compound-binding activity are concentrated from the library.

[0532] (Example 4)

[0533] A method for producing a library comprising any one of the following steps 1) to 3), wherein the library mainly comprises:

[0534] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0535] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0536] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains whose antigen-binding activity changes depending on the concentration of a low-molecular-weight compound, or antigen-binding molecules comprising such domains;

[0537] Step 1) Designing a library by concentrating nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound binding activity from the library described in (Example 3), wherein the library described in (Example 3) comprises nucleic acids encoding one or more variants with altered amino acids in the heavy chain variable region;

[0538] Step 2) Designing a library by concentrating nucleic acids encoding multiple variants of antigen-binding molecules with low molecular weight compound binding activity from the library described in (Example 3), wherein the library described in (Example 3) comprises nucleic acids encoding one or more variants with altered amino acids in the light chain variable region;

[0539] In step 3), nucleic acids encoding antigen-binding molecules enriched from the variable region libraries in steps 1) and 2) are combined to design a library.

[0540] (Example 5)

[0541] The method for producing a library according to any one of (Example 1) to (Example 4) is characterized in that the antigen-binding molecule is a fusion polypeptide of an antigen-binding domain and at least a portion of a viral coat protein.

[0542] (Example 6)

[0543] The method for producing a library described in any one of (Example 1) to (Example 4) above, wherein the aforementioned antigen-binding molecule is an antigen-binding molecule comprising the heavy chain and light chain of an antibody, and the method further comprises the step of designing a synthetic library of the aforementioned heavy chain and / or light chain.

[0544] (Example 7)

[0545] The method for producing the library of the aforementioned (Example 6) is characterized in that the heavy chain and / or light chain of the aforementioned antibody comprises a germline-derived framework sequence.

[0546] (Example 8)

[0547] The method for producing a library according to any one of (Example 1) to (Example 7), wherein the low-molecular compound is a target tissue-specific compound or a non-natural compound.

[0548] (Example 9)

[0549] The method for producing a library according to any one of (Example 1) to (Example 8), wherein the target tissue is cancer tissue or inflammatory tissue.

[0550] (Example 10)

[0551] The method for producing a library described in the aforementioned (Example 9), wherein the cancer tissue-specific compound is at least one compound selected from the group consisting of nucleosides having a purine ring structure, amino acids and their metabolites, lipids and their metabolites, primary metabolites of sugar metabolism, and nicotinamide and its metabolites.

[0552] (Example 11)

[0553] The method for producing a library according to any one of (Example 1) to (Example 10), wherein the low molecular weight compound is kynurenine, adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

[0554] (Example 12)

[0555] The method for producing a library according to any one of (Example 1) to (Example 11), wherein the amino acid site not involved in binding to the low molecular weight compound is a site other than any one or more of the following amino acids:

[0556] H chain: 97, 100c, 101, 94, 95, 100d, 100e, 33, 50, 52, 56, 57, 58, 99, 100, 100a, 54, 55 (Kabat numbering);

[0557] L chain: 49, 55, 95c, 96, 95a, 95b (Kabat numbering).

[0558] Library (other solutions)

[0559] As one embodiment of the library of the present invention capable of obtaining antigen-binding domains whose antigen-binding activity varies depending on the concentration of a low molecular weight compound, the following library can be mentioned.

[0560] A library mainly comprising:

[0561] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0562] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0563] This library is characterized in that the antigen-binding domains or antigen-binding molecules are antigen-binding domains having low-molecular-weight compound-binding activity or antigen-binding molecules comprising an antigen-binding domain.

[0564] As a library of this scheme, it is preferred to have 1.2×10 8 The diversity of the above.

[0565] The term "mainly comprising" in the expression "library mainly comprising a plurality of antigen-binding molecules" in this embodiment reflects the number of antigen-binding molecules having binding activity to low-molecular-weight compounds (e.g., target tissue-specific compounds) among the number of independent clones with different sequences in the library. Specifically, it is preferred that at least 10 antigen-binding molecules exhibiting such binding activity are present in the library. 4 In other expressions, it can also be suitably expressed as the ratio of antigen-binding molecules whose antigen-binding activity of the antigen-binding domain differs depending on the presence or absence of low molecules, among the number of independent clones with different sequences in the library. Specifically, the antigen-binding domain of the present invention can be composed of antigen-binding molecules that exhibit such binding activity accounting for 10% of the number of independent clones with different sequences in the library. -6 %% to 80%, or 10 -5 % to 60%, preferably 10 -4 % to 40%, more preferably 10 -3 % to 40%, more preferably 10 -2 The same applies to fusion polypeptides, polynucleotide molecules, or vectors, where the expression can be expressed by the number of molecules or the ratio of all molecules. Furthermore, the same applies to viruses, where the expression can be expressed by the number of individual viruses or the ratio of all individual viruses.

[0566] As one embodiment of the library of the present invention capable of obtaining antigen-binding domains whose antigen-binding activity varies depending on the concentration of a low molecular weight compound, the following library can be mentioned.

[0567] A library mainly comprising:

[0568] (i) a plurality of antibody molecules that differ in sequence from one another; or

[0569] (ii) nucleic acids encoding a plurality of antibody molecules whose sequences are different from each other,

[0570] The library is characterized in that the antibody molecules are antibody molecules having low molecular weight compound binding activity and have diversity that satisfies any one or more of the following (i) to (vi);

[0571] (i) The diversity of heavy chain CDR1 is more than 13

[0572] (ii) The diversity of heavy chain CDR2 is more than 129

[0573] (iii) The diversity of heavy chain CDR3 is 5 or more

[0574] (iv) The diversity of light chain CDR1 is more than 193

[0575] (v) The diversity of light chain CDR2 is greater than 7

[0576] (vi) The diversity of light chain CDR3 is 17 or more.

[0577] The term "mainly comprising" in the expression "library mainly comprising a plurality of antigen-binding molecules" in this embodiment reflects the number of antigen-binding molecules having binding activity to low-molecular-weight compounds (e.g., target tissue-specific compounds) among the number of independent clones with different sequences in the library. Specifically, it is preferred that at least 10 antigen-binding molecules exhibiting such binding activity are present in the library. 4 In other expressions, it can also be suitably expressed as the ratio of antigen-binding molecules whose antigen-binding activity of the antigen-binding domain differs depending on the presence or absence of low molecules, among the number of independent clones with different sequences in the library. Specifically, the antigen-binding domain of the present invention can be composed of antigen-binding molecules that exhibit such binding activity accounting for 10% of the number of independent clones with different sequences in the library. -6 %% to 80%, or 10 -5 % to 60%, preferably 10 -4 % to 40%, more preferably 10 -3 % to 40%, more preferably 10 -2 The same applies to fusion polypeptides, polynucleotide molecules, or vectors, where the expression can be expressed by the number of molecules or the ratio of all molecules. Furthermore, the same applies to viruses, where the expression can be expressed by the number of individual viruses or the ratio of all individual viruses.

[0578] As one embodiment of the library of the present invention capable of obtaining antigen-binding domains whose antigen-binding activity varies depending on the concentration of a low molecular weight compound, the following library can be mentioned.

[0579] A library mainly comprising:

[0580] (i) multiple antigen-binding domains that differ in sequence from one another, or antigen-binding molecules comprising such domains; or

[0581] (ii) nucleic acids encoding multiple antigen-binding domains whose sequences differ from each other, or antigen-binding molecules comprising such domains;

[0582] The aforementioned antigen-binding domain or antigen-binding molecule is an antigen-binding domain or an antigen-binding molecule containing an antigen-binding domain having low-molecular-weight compound binding activity, and the library is a library for obtaining antigen-binding domains or antigen-binding molecules containing antigen-binding domains whose antigen-binding activity varies depending on the concentration of the low-molecular-weight compound.

[0583] The term "mainly comprising" in the expression "library mainly comprising a plurality of antigen-binding molecules" in this embodiment reflects the number of antigen-binding molecules having binding activity to low-molecular-weight compounds (e.g., target tissue-specific compounds) among the number of independent clones with different sequences in the library. Specifically, it is preferred that at least 10 antigen-binding molecules exhibiting such binding activity are present in the library. 4 In other expressions, it can also be suitably expressed as the ratio of antigen-binding molecules whose antigen-binding activity of the antigen-binding domain differs depending on the presence or absence of low molecules, among the number of independent clones with different sequences in the library. Specifically, the antigen-binding domain of the present invention can be composed of antigen-binding molecules that exhibit such binding activity accounting for 10% of the number of independent clones with different sequences in the library. -6 %% to 80%, or 10 -5 % to 60%, preferably 10 -4 % to 40%, more preferably 10 -3 % to 40%, more preferably 10 -2 The same applies to fusion polypeptides, polynucleotide molecules, or vectors, where the expression can be expressed by the number of molecules or the ratio of all molecules. Furthermore, the same applies to viruses, where the expression can be expressed by the number of individual viruses or the ratio of all individual viruses.

[0584] Amino groups that change the antigen-binding activity of the antigen-binding domain depending on the presence or absence of low molecular weight molecules acidRegarding the method for obtaining (screening method for) templates used in preparing libraries in the present invention (antigen-binding domains whose antigen-binding activity varies depending on the concentration of a low molecular weight compound or antigen-binding domains having low molecular weight compound-binding activity), these antigen-binding domains and the like can be prepared arbitrarily, including pre-existing antigen-binding domains or antibodies, pre-existing libraries (such as phage libraries), hybridomas obtained from immunized animals, antibodies or libraries produced from B cells derived from immunized animals, antibodies or libraries produced from immune cells such as B cells of animals immunized with a conjugate of a highly immunogenic T cell epitope peptide appropriately linked to an adjuvant, such as, but not limited to, adenosine or ATP, as one embodiment of a low molecular weight compound. As a non-limiting example of such a T cell epitope peptide, preferably, there is the p30 helper peptide derived from tetanus toxin (shown in SEQ ID NO: 4, also referred to as Fragment C (FrC)).

[0585] As a non-limiting embodiment of a method for producing the antigen-binding domains or antibodies of the present invention obtained by the aforementioned screening method, a library can be used that comprises: a 35-amino acid component called the A domain contained in the cell membrane protein Avimer present in the body; an Adnectin comprising a 10Fn3 domain that binds to a protein in the cell membrane glycoprotein fibronectin; an Affibody scaffolded by the IgG-binding domain of the 58-amino acid three-helix bundle of Protein A; DARPins (Designed Ankyrin Repeat proteins) containing surface-exposed regions of ankyrin repeats (ARs) having a structure in which a turn consisting of 33 amino acid residues overlaps with two antiparallel helices and a loop; and a neutrophil gelatinase-associated lipocalin-binding protein (NGL). These include the four loop regions on one side of anticalin, a barrel structure with eight antiparallel chains twisted in the central direction, which is highly conserved in lipocalin molecules such as lipocalin (NGAL); and the concave region within a horseshoe-shaped structure composed of repeated overlapping leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR) that lacks an immunoglobulin structure and is the source of the immune system of jawless fish such as lampreys and hagfishes.

[0586] Preferred examples of the antigen-binding domains of the present invention include those containing the variable regions of the heavy and light chains of antibodies. Preferred examples of such antigen-binding domains include "scFv (single-chain Fv)", "single-chain antibodies", "Fv", "scFv2 (single-chain Fv2)", "Fab", "F(ab')2", or IgG, and libraries containing these can also be used.

[0587] Furthermore, as a non-limiting embodiment of a method for preparing the antigen-binding domains or antibodies of the present invention obtained by the aforementioned screening method, the following method can be employed: using the aforementioned library, an antigen-binding domain or antibody having low-molecular-weight compound binding activity is prepared by panning. Examples of libraries that can be used include, but are not limited to, phage display libraries, ribosome display libraries, mRNA display libraries, cDNA display libraries, CIS display libraries, E. coli display libraries, Gram-positive bacteria display libraries, yeast display libraries, mammalian cell display libraries, virus display libraries, and in vitro virus display libraries.

[0588] As one approach for preparing antigen-binding domains or antibodies with low-molecular-weight compound binding activity by panning, low-molecular-weight compounds immobilized on supports such as beads can be used. The immobilized low-molecular-weight compound can be prepared by contacting a low-molecular-weight compound synthesized in a state chemically linked to biotin via a linker with beads or plates immobilized with streptavidin or neutravidin; or by joining a low-molecular-weight compound covalently linked to an adjuvant such as bovine serum albumin (BSA) with beads or plates via hydrophobic interactions, but is not limited thereto. These methods are already known (J Immunol Methods. 2003 Sep; 280 (1-2): 139-55, BMC Biotechnol. 2009 Jan 29; 9: 6. doi: 10.1186 / 1472-6750-9-6). By recovering these immobilized antigen-binding domains or antibodies with low-molecular-weight compound binding activity, antigen-binding domains or antibodies with low-molecular-weight compound binding activity can be prepared.

[0589] Alternatively, as another embodiment of the method for preparing antigen-binding domains or antibodies with low molecular weight compound binding activity by panning, fluorescently labeled low molecular weight compounds, biotin-labeled low molecular weight compounds, and fluorescently labeled streptavidin (or neutravidin or avidin) can be used. After the library presented on the surface of a cell or the like is contacted with the fluorescently labeled low molecular weight compound, or the biotin-labeled low molecular weight compound, and fluorescently labeled streptavidin (or neutravidin or avidin), the antigen-binding domains or antibodies with low molecular weight compound binding activity can be prepared by using fluorescence activated cell sorting (FACS). These methods are already known (Proc Natl Acad Sci US A. 2000 Sep 26; 97 (20): 10701-5.).

[0590] In addition, as a non-limiting embodiment of the method for preparing the antigen-binding domains or antibodies of the present invention obtained by the aforementioned screening methods, pre-existing antigen-binding domains with binding activity to low molecular weight compounds can be used. For example, and not limited to this, using adenosine and / or ATP as an example, molecules belonging to the kinase family with ATP binding activity can be used as antigen-binding domains. Alternatively, molecules belonging to the adenosine deaminase family with adenosine binding activity can be used as antigen-binding domains. By libraryizing the fractions that do not participate in binding to these ATP or adenosine, antigen-binding molecules that bind to the antigen in an ATP or adenosine concentration-dependent manner can be obtained.

[0591] As a method for obtaining an antigen-binding domain using a non-antibody-like protein, although not limited thereto, a library obtained using the loop-forming site of the non-antibody protein, the surface residues of the α-helix, etc. can be used. The construction method of such a library is already known (Nat Biotechnol. 2004 May; 22 (5): 575-82, J Mol Biol. 1998 Dec 11; 284 (4): 1141-51., Nat Biotechnol. 1997 Aug; 15 (8): 772-7). Furthermore, using the library constructed as described above, the technology of obtaining an antigen-binding domain with low-molecular compound binding activity by panning is also known. As an example, the constructed library is expressed on the surface of a phage by phage display. Phage expressing a binding domain that binds to a low-molecular compound linked to bovine serum albumin, biotin, etc. can be selected using beads, antigen tubes (immunotubes), plates, etc. The method for obtaining such a non-antibody-like antigen-binding domain with low molecular compound binding activity is already known (Proc Natl Acad Sci US A. 1999 Mar 2; 96 (5): 1898-903). Furthermore, for these antigen-binding domains with low molecular compound binding activity, although not limited thereto, by analyzing the crystal structure and making mutants to evaluate the binding activity, it is possible to identify amino acid sites that are not involved in binding low molecular compounds and amino acid sites that are not important for the formation of the classical structure (J Mol Biol. 2003 Jul 4; 330 (2): 385-96, Proteins. 2003 Oct 1; 53 (1): 121-9). By introducing diversity into the amino acid sites thus identified, the library described in the present invention can be constructed. Furthermore, as another embodiment of the present invention, a library with limited amino acid sites can also be used. As an example, although not limited thereto, in anticalins, which are four loop regions on one side of a barrel structure in which eight antiparallel chains are twisted in the central direction and which are highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) and are reported as non-antibody-like antigen-binding domains, it is known that the amino acid sites for binding to low molecular weight compounds and the amino acid sites for binding to proteins are different, and it is known that different libraries can be used in which the amino acid sites that can participate in the respective binding are modified (FEBS Lett. 2014 Jan 21; 588(2): 213-8).Therefore, after obtaining the antigen-binding domain with low-molecular-weight compound binding activity from a library capable of obtaining binding domains against low-molecular-weight compounds, diversity is introduced at the amino acid sites used to obtain the antigen-binding domain with protein-binding activity relative to the obtained antigen-binding domain with low-molecular-weight compound binding activity, thereby constructing the library of the present invention. More specifically, it is known that in human lipocalin 2 (Lcn2), amino acid sites V33, L36, I41, Y52, T54, S68, L70, W79, R81, K134, T136, and Y138 can be used as diversity introduction sites for a library for obtaining low molecular weight compound binding domains (JAm Chem Soc. 2009 Mar 18; 131(10): 3565-76). Similarly, amino acid sites A40, L42, E44, K46, D47, Q49, K50, L70, R72, K73, D77, W79, P101, G102, L103, K125, S127, Q128, R130, and Y132 can be used as diversity introduction sites for a library for obtaining protein binding domains (Proc Natl Acad Sci USA. 2009 May 19; 106(20): 8198-203). Therefore, first, a library comprising antigen-binding domains with diversity at amino acid positions V33, L36, I41, Y52, T54, S68, L70, W79, R81, K134, T136, and Y138 is used to obtain antigen-binding domains with low molecular weight compound binding activity. Then, relative to the obtained antigen-binding domains with low molecular weight compound binding activity, diversity is introduced at amino acid positions A40, L42, E44, K46, D47, Q49, K50, R72, K73, D77, P101, G102, L103, K125, S127, Q128, R130, and Y132 to construct the library described in the present invention, but is not limited thereto. Regarding non-antibody-like antigen-binding domains and antibodies other than lipocalin molecules, those skilled in the art can construct the libraries of the present invention by appropriately referring to the above-mentioned library construction methods. Alternatively, antigen-binding domains with binding activity to low-molecular-weight compounds can be utilized. For example, Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), which belongs to the lipocalin family and is known to have binding activity to ATP, ADP, AMP, and adenosine, can be utilized (J Biol Chem. 2000 Apr 28; 275(17):12639-50, Biochemistry. 2002 Mar 19; 41(11):3810-8).Although not limited to this, by analyzing the crystal structure of the antigen-binding domain and preparing mutants to evaluate binding activity, amino acid sites that are not involved in binding to low molecular weight compounds and amino acid sites that are not important for the formation of the canonical structure can be identified. By introducing diversity into the amino acid sites thus identified, the library described in the present invention can be constructed. The existence of antigen-binding domains belonging to the lipocalin family that have binding activity for various low molecular weight compounds such as ATP, ADP, AMP, and adenosine, as well as histamine, serotonin, epinephrine, and norepinephrine is well known (J Biol Chem. 2003 Feb 14; 278(7):4611-7, Expert Rev Clin Immunol. 2007 Jul; 3(4):491-501). By utilizing these, the library described in the present invention using antigen-binding domains that have binding activity for various low molecular weight compounds can be constructed, but the present invention is not limited to this.

[0592] Regarding other non-antibody-like antigen-binding domains and antibodies, those skilled in the art can construct the libraries of the present invention by appropriately referring to the above-mentioned library construction methods.

[0593] As a non-limiting scheme of the present invention, taking adenosine and / or ATP as an example, it is described in detail below, but about the low molecules other than adenosine and / or ATP, it is also possible to be suitable for the following examples. As the amino acid that changes the binding activity of antigen binding molecules to antigen according to the presence or absence of adenosine and / or ATP as described above, the amino acid that forms adenosine and / or ATP binding motif can be exemplified. The position of the amino acid in the antigen-binding domains comprising the aforementioned amino acids is not limited to a specific position, as long as the binding activity of antigen binding domains to antigen is changed according to the presence or absence of adenosine and / or ATP, it can be any position in the heavy chain variable region or light chain variable region that form the antigen-binding domains. That is, the antigen-binding domains of the present invention can be obtained by following libraries, which mainly comprise antigen-binding domains of heavy chains containing amino acids that change the binding activity of antigen binding molecules to antigen according to the presence or absence of adenosine and / or ATP and antigen binding molecules whose sequences are different from each other. In a non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library comprising a heavy chain CDR1, CDR2, and / or CDR3 containing amino acids that vary the binding activity of the antigen-binding molecule to the antigen according to the presence or absence of adenosine and / or ATP, and antigen-binding molecules whose sequences are different from one another. In another non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library comprising a heavy chain FR1, FR2, FR3, and / or FR4 containing amino acids that vary the binding activity of the antigen-binding molecule to the antigen according to the presence or absence of adenosine and / or ATP, and antigen-binding molecules whose sequences are different from one another.

[0594] In addition, in one embodiment of the present invention, the antigen-binding domains of the present invention can be obtained by following library, and the library mainly comprises the amino acid that makes the combination activity of antigen binding molecules to antigen change according to the presence or absence of adenosine and / or ATP in the antigen-binding domains of heavy chain and / or light chain, and the antigen binding molecules of sequence different from each other.In an unrestricted embodiment, the antigen-binding domains of the present invention can be obtained by following library, and the library mainly comprises the amino acid that makes the combination activity of antigen binding molecules to antigen change according to the presence or absence of adenosine and / or ATP in CDR1, CDR2 and / or CDR3 of heavy chain and / or light chain, and the antigen binding molecules of sequence different from each other.In an unrestricted embodiment, the antigen-binding domains of the present invention can be obtained by following library, and the library mainly comprises the amino acid that makes the combination activity of antigen binding molecules to antigen change according to the presence or absence of adenosine and / or ATP in FR1, FR2, FR3 and / or FR4 of heavy chain and / or light chain, and the antigen binding molecules of sequence different from each other.

[0595] As a non-limiting embodiment of such amino acids, there can be exemplified amino acids containing at least one of the amino acids at positions 52, 52a, 53, 96, 100a, or 100c in the heavy chain variable region. Furthermore, as a non-limiting embodiment of such amino acids, there can be exemplified amino acids containing at least one of the amino acids at positions 52, 52a, Arg, 53, 96, Leu, and 100c in the heavy chain variable region.

[0596] For the framework sequence of the light chain and / or heavy chain variable region of antigen binding molecules, as long as the antigen binding domains of heavy chain and / or light chain contain an amino acid that changes the binding activity of antigen binding molecules to antigens according to the presence or absence of adenosine and / or ATP, any framework sequence can be used. The origin of the framework sequence is not limited and can be obtained by any organism or person of non-human animals. Preferably, as any organism, preferably, an organism selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels and non-human primates can be enumerated. In a particularly preferred embodiment, it is desirable that the framework sequence of the light chain and / or heavy chain variable region of antigen binding molecules has the germline framework sequence of people. Therefore, in one embodiment of the present invention, if the framework sequence is completely the sequence of people, it is believed that when giving people (such as the treatment of a disease), the antigen binding molecules of the present invention will not cause an immunogenic reaction substantially or completely. According to the above meaning, "containing germline sequence" of the present invention means that a part of the framework sequence of the present invention is identical with a part of the germline framework sequence of any people. Specifically, the framework sequence of the present invention preferably has at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more homology with the germline sequence. For example, when the heavy chain FR2 sequence of the antigen-binding molecule of the present invention is a sequence obtained by combining the heavy chain FR2 sequences of the germline framework sequences of multiple different people, the antigen-binding molecule is also an antigen-binding molecule of the present invention that "contains germline sequences". In addition, when the framework sequence of the antigen-binding molecule of the present invention is a replaced sequence, the antigen-binding molecule is also an antigen-binding molecule of the present invention that "contains germline sequences". As an example of such a replaced sequence, in particular, a sequence in which a portion of the amino acids in the germline framework sequence of a person is replaced with an amino acid sequence that changes the binding activity of the antigen-binding molecule to the antigen according to the presence or absence of adenosine and / or ATP.

[0597] As the example of framework, for example, it is preferred to enumerate the sequence of the fully human framework region known now, which is included in the websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ). The sequence of these framework regions can be suitably used as the germline sequence contained in the antigen binding molecules of the present invention. Germline sequences can be classified based on their similarity (Tomlinson et al. (J.Mol.Biol. (1992) 227,776-798) Williams and Winter (Eur.J.Immunol. (1993) 23,1456-1461) and Cox et al. (Nat.Genetics (1994) 7,162-168)). Suitable germline sequences can be suitably selected from the Vκ, Vλ, VH of 7 subclasses, which are classified into 7 subclasses.

[0598] The fully human VH sequences are not limited to the following, and preferred examples include: VH1 subclass (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), VH2 subclass (e.g., VH2-5, VH2-26, VH2-70), VH3 subclass (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3- 30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74), VH4 subclass (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61), VH5 subclass (VH5-51), VH6 subclass (VH6-1), VH7 subclass (VH7-4, VH7-81), etc. These are also described in known literature (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)), and those skilled in the art can appropriately design the antigen-binding molecules of the present invention based on their sequence information. Fully human frameworks or sub-regions of frameworks other than these may also be preferably used.

[0599] The fully human Vκ sequence is not limited to the following sequences, and preferably includes, for example: A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, O18 classified as the Vk1 subclass; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, O11 classified as the Vk2 subclass; A11, A27, L2, L6, L10, L16, L20, L25 of the subclass; B3 classified as the Vk4 subclass; B2 classified as the Vk5 subclass (also referred to as Vk5-2 in this specification); A10, A14, A26, etc. classified as the Vk6 subclass (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028), Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022) and Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

[0600] The fully human Vλ sequence is not limited to the following sequences, and preferably includes, for example, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22 classified as the VL1 subclass; V2-1, V2-6, V2-7, and V2- 8, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19; ​​V3-2, V3-3, V3-4 classified as VL3 subclass; V4-1, V4-2, V4-3, V4-4, V4-6 classified as VL4 subclass; V5-1, V5-2, V5-4, V5-6, etc. classified as VL5 subclass (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).

[0601] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used together with the "at least one amino acid residue that varies the antigen-binding activity of the antigen-binding domain according to the presence or absence of adenosine and / or ATP" of the present invention. Examples of fully human frameworks that can be used together with the "at least one amino acid residue that varies the antigen-binding activity of the antigen-binding domain according to the presence or absence of adenosine and / or ATP" of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY, POM, etc. (e.g., Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

[0602] While the present invention is not bound by any particular theory, one reason why the use of germline sequences is expected to eliminate deleterious immune responses in most individuals is as follows. As a result of the affinity maturation step that occurs during normal immune responses, somatic mutations frequently occur in the variable regions of immunoglobulins. These mutations primarily occur near CDRs whose sequences are hypervariable, but also affect residues in the framework regions. These framework mutations are not present in germline genes and are less likely to be immunogenic in patients. On the other hand, the general population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are predicted to be low or non-immunogenic in patients. To maximize the likelihood of immune tolerance, genes encoding the variable regions can be selected from a collection of commonly occurring functional germline genes.

[0603] In order to prepare the antigen-binding molecules of the present invention in which the sequence of the aforementioned variable region sequence, the sequence of the heavy chain variable region or the light chain variable region, or the CDR sequence or the framework sequence contains amino acids that change the antigen-binding activity of the antigen-binding domain depending on the presence or absence of adenosine and / or ATP, known methods such as site-specific mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR can be appropriately used.

[0604] For example, by combining a light chain variable region that has been preselected to contain a CDR sequence and / or framework sequence that contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding domain depending on the presence or absence of adenosine and / or ATP with a heavy chain variable region constructed as a randomized variable region sequence library, a library containing a plurality of mutually different antigen-binding molecules of the present invention can be constructed.

[0605] In addition, it is also possible to design the aforementioned CDR sequences and / or framework sequences that are selected as containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding domain according to the presence or absence of adenosine or ATP to contain various amino acids in the sequence of the heavy and / or light chain variable regions that are residues other than these amino acid residues. In the present invention, such residues are also referred to as flexible residues. As long as the antigen-binding activity of the antigen-binding molecules of the present invention changes according to the conditions of the concentration of the tissue-specific compound, the number and position of the flexible residues are not limited to specific schemes. That is, the CDR sequences and / or FR sequences of the heavy and / or light chains may contain one or more flexible residues. Flexible residues can be identified by crystal structure analysis of a complex of adenosine and / or ATP and an antibody, or by introducing mutations, and whether these residues can be replaced with other amino acids to make a library. For example, based on crystal structure analysis of a complex of adenosine and / or ATP and an antibody, residues of the antibody that are not involved in the binding of adenosine and / or ATP can be identified. Even if the residue that will be identified as not participating in the combination of adenosine and / or ATP is replaced by other amino acids, the combination with the compound can be maintained at an appropriate degree of amino acid. Thereby, a library in which the selected amino acid appears in the selected residue can be designed. In this case, a library mainly comprising a plurality of antigen binding molecules can be designed in such a way that the residue that will be identified as not participating in the combination of adenosine and / or ATP is replaced by an amino acid that is different from each other. That is, by combining each flexible residue that is replaced by an amino acid that is different from each other, the sequence diversity of the antigen binding molecules containing the flexible residue can be brought.

[0606] In addition, it is also possible to design antigen binding molecules comprising these residues in such a way that at least one of the residues identified as participating in the combination of adenosine and / or ATP is selected from the residue and the residue being different from the residue. As a non-limiting scheme of the amino acid involved in the combination of adenosine and / or ATP, any one or more amino acids in the amino acids at positions 52, 52a, 53, 96, 100a or 100c contained in the heavy chain variable region can be exemplified. In addition, as a non-limiting scheme of such amino acids, any one or more amino acids in the amino acids such as Ser at position 52, Ser at position 52a, Arg at position 53, Gly at position 96, Leu at position 100a, Trp at position 100c containing in the heavy chain variable region can be exemplified. For example, when Leu at position 100a is identified as being involved in binding to adenosine and / or ATP, the amino acid residue at position 100a of the antigen-binding molecules contained in the library can be, in addition to Leu, an amino acid residue selected from any flexible residues of His, Met, Leu, Arg, Trp, or Tyr.

[0607] As a non-limiting embodiment of the flexible residues, there can be exemplified the amino acids at positions 31, 32, 33, 35, 50, 55, 56, 57, 58, 59, 95, 96, 97, 98, 99, 100, 100a, and 100b contained in the heavy chain variable region. As another non-limiting embodiment of such amino acids, there can be exemplified the amino acids at positions 26, 27, 27a, 27b, 27c, 28, 29, 31, 32, 50, 51, 52, 53, 54, 55, 89, 90, 91, 92, 93, 94, 95a, 96, and 97 contained in the light chain variable region.

[0608] As a non-limiting embodiment of the flexible residue, the following amino acids contained in the heavy chain variable region can be exemplified:

[0609] The amino acid at position 31 is any one of Asp, Gly, Asn, Ser, Arg, or Thr,

[0610] The amino acid at position 32 is any one of Ala, Phe, His, Asn, Ser, or Tyr,

[0611] The amino acid at position 33 is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg, Trp, Val, Tyr, or Thr,

[0612] The amino acid at position 35 is any one of His, Ser, Thr, Tyr, or Asn,

[0613] The amino acid at position 50 is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Arg, Thr, Trp, Val, Tyr, or Ser,

[0614] The amino acid at position 55 is any one of Ala, Glu, Asp, Gly, Leu, Thr, Ser, Arg, or Asn,

[0615] The amino acid at position 56 is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Thr, Trp, Val, or Tyr,

[0616] The amino acid at position 57 is any one of Ala, Lys, Arg, Thr, or Ile,

[0617] The amino acid at position 58 is any one of Asp, Gly, Phe, His, Ser, Thr, Tyr, or Asn,

[0618] The amino acid at position 59 is either Leu or Tyr,

[0619] The amino acid at position 95 is any one of Ala, Ile, Lys, Met, Leu, Arg, Trp, Val, Tyr, or Phe,

[0620] The amino acid at position 96 is any one of Ala, Asp, Asn, or Ser,

[0621] The amino acid at position 97 is any one of Ala, Asp, Gly, Ile, His, Lys, Met, Leu, Asn, Ser, Val, Tyr, or Arg,

[0622] The amino acid at position 98 is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Met, Leu, Asn, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Lys,

[0623] The amino acid at position 99 is any one of Ala, Glu, Asp, Phe, His, Lys, Asn, Gln, Ser, Arg, Trp, Val, Tyr, or Gly,

[0624] The amino acid at position 100 is any one of Ala, Glu, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asp,

[0625] The amino acid at position 100a is any one of Ala, Phe, Ile, His, Lys, Met, Arg, Trp, Val, or Tyr, or

[0626] The amino acid at position 100b is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asn.

[0627] As a non-limiting embodiment of the flexible residue, the following amino acids contained in the light chain variable region can be exemplified:

[0628] The amino acid at position 26 is any one of Ala, Ser, or Thr,

[0629] The amino acid at position 27 is either Thr or Ser,

[0630] The amino acid at position 27a is any one of Gly, Asn, Thr, or Ser,

[0631] The amino acid at position 27b is either Asn or Asp,

[0632] The amino acid at position 27c is either Ile or Val,

[0633] The amino acid at position 28 is either Asp or Gly,

[0634] The amino acid at position 29 is any one of Ala, Asp, Phe, Ser, Arg, Thr, Tyr, or Gly,

[0635] The amino acid at position 31 is any one of Glu, Asp, Lys, or Asn,

[0636] The amino acid at position 32 is any one of Ala, Asp, Ser, Thr, or Tyr,

[0637] The amino acid at position 50 is any one of Asp, Gly, Lys, Asn, Gln, Ser, Arg, Tyr, or Glu,

[0638] The amino acid at position 51 is any one of Asp, Gly, Lys, Asn, Thr, or Val,

[0639] The amino acid at position 52 is any one of Ala, Asp, Asn, Thr, or Ser,

[0640] The amino acid at position 53 is any one of Glu, Asp, His, Asn, Gln, Ser, Tyr, or Lys,

[0641] The amino acid at position 54 is either Lys or Arg,

[0642] The amino acid at position 55 is either Leu or Pro,

[0643] The amino acid at position 89 is any one of Ala, Gly, Phe, Leu, Asn, Gln, Thr, Val, Tyr, or Ser,

[0644] The amino acid at position 90 is any one of Ala, Leu, Thr, Val, or Ser,

[0645] The amino acid at position 91 is any one of Ala, Asp, Phe, His, Lys, Asn, Ser, Arg, Thr, Trp, Val, or Tyr,

[0646] The amino acid at position 92 is any one of Glu, Asp, Ser, Arg, Thr, Val, Tyr, or Ala,

[0647] The amino acid at position 93 is any one of Ala, Asp, Ile, Asn, Ser, Arg, Thr, Val, Tyr, or Gly,

[0648] The amino acid at position 94 is any one of Ala, Asp, Gly, Ile, Asn, Arg, Thr, or Ser,

[0649] The amino acid at position 95 is any one of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asn,

[0650] The amino acid at position 95a is any one of Ala, Glu, Asp, Gly, Ile, His, Lys, Leu, Gln, Pro, Ser, Arg, Thr, Tyr, or Asn,

[0651] The amino acid at position 96 is any one of Ala, Asp, Gly, Phe, His, Lys, Leu, Asn, Gln, Pro, Ser, Thr, Trp, Tyr, or Val, or

[0652] The amino acid at position 97 is any one of Ala, Gly, Ile, Met, Leu, Ser, or Val.

[0653] As one embodiment of the present invention, when the low molecular weight compound is kynurenine, a flexible residue can be identified by crystal structure analysis of a complex of kynurenine and an antibody, or by introducing mutations, and whether the residue can be replaced with other amino acids to create a library. For example, based on crystal structure analysis of a complex of kynurenine and an antibody, residues of the antibody that are not involved in the binding of kynurenine can be identified. Even if the residue identified as not participating in the binding of kynurenine is replaced with other amino acids, an amino acid that can maintain the binding to the compound to an appropriate degree can be selected. In this way, a library in which the selected amino acid appears in the selected residue can be designed. In this case, a library mainly comprising multiple antigen-binding molecules can be designed in such a way that a collection of antigen-binding molecules in which the residue identified as not participating in the binding of kynurenine is replaced with amino acids that are different from each other is formed. That is, by combining each flexible residue replaced with an amino acid that is different from each other, the sequence diversity of the antigen-binding molecules containing the flexible residue can be brought about.

[0654] In addition, it is also possible to design antigen-binding molecules comprising these residues in such a manner that at least one of the residues identified as participating in the binding of kynurenine is selected from the residue and any residue different from the residue. As a non-limiting embodiment of the amino acid identified as participating in the binding of kynurenine, as the amino acid residue whose side chain participates in binding to kynurenine, any one or more amino acids of the amino acids of the H chain: P97, R100c, D101 (Kabat numbering), and the L chain: H49, F55 (Kabat numbering) can be exemplified. In addition, as the amino acid residue that fully participates in binding to kynurenine in the main chain portion, any one or more amino acids of the amino acids of the H chain: R94, D95, R100c, G100d, A100e can be exemplified. Furthermore, according to the X-ray crystal structure, as the residue that is important in maintaining the structure of the H chain CDR3 in the binding configuration of kynurenine, any one or more amino acids of the amino acids of the H chain: P97, P100b, G100d can be exemplified.

[0655] As a non-limiting embodiment of the flexible residue when the low molecular weight in the present invention is kynurenine, examples include amino acids at positions 24, 26, 27, 28, 29, 30, 31, 32, 33, 50, 51, 52, 52a, 53, 54, 55, 56, 58, 73, 95, 96, 97, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, and 102 contained in the heavy chain variable region. As another non-limiting embodiment of such amino acids, the amino acids at positions 27d, 27e, 28, 29, 32, 46, 49, 50, 51, 52, 53, 54, 55, 92, 93, and 94 contained in the light chain variable region can be exemplified.

[0656] As a non-limiting embodiment of the flexible residue, the following amino acids contained in the heavy chain variable region can be exemplified:

[0657] The amino acid at position 24 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0658] The amino acid at position 26 is any one of Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0659] The amino acid at position 27 is any one of Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0660] The amino acid at position 28 is any one of Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Val, Trp, or Tyr,

[0661] The amino acid at position 29 is any one of Phe, Ile, Leu, Trp, or Tyr,

[0662] The amino acid at position 30 is any one of Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr,

[0663] The amino acid at position 31 is any one of Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr, Val, Trp, or Tyr,

[0664] The amino acid at position 32 is any one of Tyr, Phe, or His,

[0665] The amino acid at position 33 is any one of Ala, Gly, Ile, Lys, Gln, Arg, Ser, Thr, Val, or Trp,

[0666] The amino acid at position 50 is any one of Gly, Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0667] The amino acid at position 51 is any one of Ile, Ala, Gly, Lys, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0668] The amino acid at position 52 is any one of Ile, Ala, Glu, Phe, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0669] The amino acid at position 52a is any one of Pro, Ala, Gly, Ser, Thr, or Trp,

[0670] The amino acid at position 53 is any one of Ile, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0671] The amino acid at position 54 is any one of Phe, Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0672] The amino acid at position 55 is any one of Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0673] The amino acid at position 56 is any one of Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr,

[0674] The amino acid at position 58 is any one of Asn, Ala, Asp, Glu, Phe, Gly, His, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0675] The amino acid at position 73 is any one of Glu, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, or Tyr,

[0676] The amino acid at position 95 is either Asp or Gly,

[0677] The amino acid at position 96 is any one of Ala, Glu, Phe, His, Ile, Lys, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0678] The amino acid at position 97 is any one of Pro, Ala, Asn, or Ser,

[0679] The amino acid at position 98 is any one of Val, Leu, or Thr,

[0680] The amino acid at position 99 is any one of Val, Ala, Asp, Phe, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr, or Tyr,

[0681] The amino acid at position 100 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0682] The amino acid at position 100a is any one of Arg, Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, or Val,

[0683] The amino acid at position 100b is any one of Pro, Ala, Lys, Asn, Gln, Arg, or Ser,

[0684] The amino acid at position 100c is any one of Arg, His, Lys, or Gln,

[0685] The amino acid at position 100d is either Gly or Asn,

[0686] The amino acid at position 100e is any one of Ala, Gly or Ser,

[0687] The amino acid at position 100f is either Phe or Leu, or

[0688] The amino acid at position 102 is any one of Ile, Ala, Asp, Glu, Phe, His, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr.

[0689] As a non-limiting embodiment of the flexible residue, the following amino acids contained in the light chain variable region can be exemplified:

[0690] The amino acid at position 27d is any one of His, Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0691] The amino acid at position 27e is any one of Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr,

[0692] The amino acid at position 28 is any one of Asp, Ala, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0693] The amino acid at position 29 is any one of Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0694] The amino acid at position 32 is any one of Tyr, Ala, Phe, Gly, His, Lys, Leu, Pro, Gln, Arg, Val, or Trp,

[0695] The amino acid at position 46 is any one of Leu, Ile, Met, Asn, or Val,

[0696] The amino acid at position 49 is any one of Tyr, Phe, His, or Trp,

[0697] The amino acid at position 50 is any one of Glu, Ala, Phe, Gly, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val, or Tyr,

[0698] The amino acid at position 51 is any one of Ile, Ala, Asp, Glu, Phe, Gly, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0699] The amino acid at position 52 is any one of Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr, Val, Trp, or Tyr,

[0700] The amino acid at position 53 is any one of Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0701] The amino acid at position 54 is any one of Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr,

[0702] The amino acid at position 55 is any one of Phe, Leu, Met, Arg, or Tyr,

[0703] The amino acid at position 92 is any one of Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp, or Tyr,

[0704] The amino acid at position 93 is any one of Gln, Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, or Tyr, or

[0705] The amino acid at position 94 is any one of Phe, His, Ile, Lys, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr.

[0706] Another non-limiting embodiment of the flexible residue when the low molecular weight residue in the present invention is kynurenine includes amino acids at positions 28, 31, 33, 50, 51, 52, 54, 55, 56, 58, 96, 97, 99, 100, 100a, 100b, and 100c contained in the heavy chain variable region. Another non-limiting embodiment of such amino acids includes amino acids at positions 27d, 27e, 28, 29, 32, 52, 53, 54, 56, 92, and 93 contained in the light chain variable region.

[0707] As one embodiment of the present invention, when the low molecular weight compound is adenosine, a flexible residue can be identified by crystal structure analysis of a complex of adenosine and an antibody, or by introducing mutations, and whether the residue can be replaced with other amino acids to create a library. For example, based on crystal structure analysis of a complex of adenosine and an antibody, residues of the antibody that are not involved in the binding of adenosine can be identified. Even if the residue identified as not involved in the binding of adenosine is replaced with other amino acids, amino acids that can maintain binding to the compound to an appropriate degree can be selected. In this way, a library can be designed in which the selected amino acid appears in the selected residue. In this case, a library mainly containing multiple antigen-binding molecules can be designed in a manner that forms a collection of antigen-binding molecules in which the residue identified as not involved in binding adenosine is replaced with different amino acids. That is, by combining each flexible residue replaced with a different amino acid, sequence diversity of the antigen-binding molecules containing the flexible residue can be brought about.

[0708] In addition, it is also possible to design antigen-binding molecules comprising these residues in such a manner that at least one of the residues identified as participating in the binding of adenosine is selected from any residue selected from the residue and a residue different from the residue. As a non-limiting embodiment of the amino acids identified as participating in the binding of adenosine, any one or more of the amino acids A33, I50, G52, S56, T57, W58, G99, Y100, and T100a (Kabat numbering) can be exemplified in the H chain; and Y95c and N96 (Kabat numbering) can be exemplified in the L chain.

[0709] As a non-limiting embodiment of the flexible residue when the low molecular weight in the present invention is adenosine, examples include amino acids at positions 31, 32, 53, 54, 55, 56, 57, 59, 61, 62, 65, 96, 97, 98, 100, 100a, 101, and 102 contained in the heavy chain variable region. As another non-limiting embodiment of such amino acids, examples include amino acids at positions 28, 29, 32, 93, 94, 95, 95a, 95b, and 95c contained in the light chain variable region.

[0710] As a non-limiting embodiment of the flexible residue, the following amino acids contained in the heavy chain variable region can be exemplified:

[0711] The amino acid at position 31 is any one of Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr,

[0712] The amino acid at position 32 is any one of Tyr, Phe, Gly, His, Ile, Lys, Asn, Pro, Gln, Arg, Thr, Val, or Trp,

[0713] The amino acid at position 53 is any one of Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, or Tyr,

[0714] The amino acid at position 54 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Gln, Ser, Thr, Val, or Tyr,

[0715] The amino acid at position 55 is any one of Ser, Ala, Asp, Glu, Phe, Gly, His, Ile...

Claims

1. A library comprising: (i) a plurality of antigen-binding domains having different sequences from each other or an antigen-binding molecule comprising such antigen-binding domains; or (ii) nucleic acids encoding a plurality of antigen-binding domains having different sequences from each other or antigen-binding molecules comprising such antigen-binding domains; The antigen binding domain comprises an antibody heavy chain variable region and an antibody light chain variable region, The antibody heavy chain variable region comprises: (a1) CDR1 contained in the heavy chain shown in SEQ ID NO: 32 、 CDR2 、 CDR3; or (a2) at position 31 according to Kabat numbering 、 32-bit 、 33 bits 、 50 people 、 51 、 52 bits 、 52a bit 、 53 、 54 bits 、 55 、 56 bits 、 58 bits 、 95 、 96 bits 、 97th 、 98 bits 、 99 digits 、 100 、 100a 、 100b bit 、 100c 、 100d, 100e, 100f 、 The amino acid residues in the positions other than position 102 are identical to those in the CDR contained in the heavy chain shown in SEQ ID NO: 32, and the amino acid residues in the positions selected from position 31 according to the Kabat numbering are identical to those in the CDR contained in the heavy chain shown in SEQ ID NO:

32. 、 32-bit 、 33 bits 、 50 people 、 51 、 52 bits 、 52a bit 、 53 、 54 bits 、 55 、 56 bits 、 58 bits 、 95 、 96 bits 、 97th 、 98 bits 、 99 digits 、 100 、 100a 、 100b bit 、 100c 、 100d, 100e, 100f 、 a CDR having an amino acid residue at one position of position 102 that is different from the CDR contained in the heavy chain set forth in SEQ ID NO: 32, and wherein amino acid position 31 is A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W or Y; amino acid position 32 is F or H; amino acid position 33 is G, I, K, Q, R, S, T, V or W; amino acid position 50 is A, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y; amino acid position 51 is A, G, K, L, Q, R, S, T, V, W or Y; amino acid position 52 is A, E, F, H, K, L, N, P, Q, R , S, T, V, W or Y; amino acid at position 52a is A, G, S, T or W; amino acid at position 53 is A, D, E, F, G, H, K, L, N, P, Q, R, S, T, V, W or Y; amino acid at position 54 is A, D, E, G, H, I, K, L, P, Q, R, S, T, V, W or Y; amino acid at position 55 is A, D, E, F, H, I, K, L, N, P, Q, R, S, T, V, W or Y; amino acid at position 56 is A, D, E, F, G, H, I , K, L, N, P, Q, R, S, V, W or Y; amino acid at position 58 is A, D, E, F, G, H, K, P, Q, R, S, T, V, W or Y; amino acid at position 95 is G; amino acid at position 96 is E, F, H, I, K, N, Q, R, S, T, V, W or Y; amino acid at position 97 is A, N or S; amino acid at position 98 is L or T; amino acid at position 99 is A, D, F, H, I, K, L, N, Q, R, S, T or Y; amino acid at position 100 is D, E, F , G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; the amino acid at position 100a is A, D, E, I, K, L, N, P, Q, S, T or V; the amino acid at position 100b is A, K, N, Q, R or S; the amino acid at position 100c is H, K or Q; the amino acid at position 100d is N; the amino acid at position 100e is G or S; the amino acid at position 100f is L; or the amino acid at position 102 is A, D, E, F, H, L, M, N, Q, R, T, V, W or Y; The light chain variable region comprises: (b1) CDR1 contained in the light chain shown in SEQ ID NO: 33 、 CDR2 、 CDR3; or (b2) at position 27d according to Kabat numbering 、 27e 、 28-bit 、 29th 、 32-bit 、 50 people 、 51 、 52 bits 、 53 、 54 bits 、 55 、 92 bits 、 93 、 The amino acid residues in the positions other than position 94 are identical to those in the CDR contained in the light chain shown in SEQ ID NO: 33, and the amino acid residues in the positions selected from position 27d according to the Kabat numbering are identical to those in the CDR contained in the light chain shown in SEQ ID NO:

33. 、 27e 、 28-bit 、 29th 、 32-bit 、 50 people 、 51 、 52 bits 、 53 、 54 bits 、 55 、 92 bits 、 93 、 a CDR having an amino acid residue at one position of position 94 that is different from the CDR contained in the light chain shown in SEQ ID NO: 33, wherein the amino acid at position 27d is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; the amino acid at position 27e is A, D, E, F, G, H, I, K, L, N, P, Q, R, T, V, W or Y; the amino acid at position 28 is A, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; the amino acid at position 29 is A, D, E, F, H, I, K, L, P, Q, R, S, T, V, W or Y; the amino acid at position 32 is A, F, G, H, K, L, P, Q, R, V or W; the amino acid at position 50 is A, F, G, I, K, L, M, Q, S, T, V or Y; the amino acid at position 51 is A, D, E, F, G, K, L, M, Q or Y.

2. The library of claim 1, wherein the antibody heavy chain variable region further comprises: (c1) the heavy chain framework 1 set forth in SEQ ID NO:87, or having the same amino acid residues as the heavy chain framework 1 set forth in SEQ ID NO:87 at positions other than positions 24, 26, 27, 28, 29 and 30 according to the Kabat numbering, and having the same amino acid residues as the heavy chain framework 1 set forth in SEQ ID NO:87 at one position selected from positions 24, 26, 27, 28, 29 and 30 according to the Kabat numbering. A heavy chain framework 1 having different amino acid residues in the heavy chain framework 1 as shown in NO:87, wherein the amino acid at position 24 is D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; the amino acid at position 26 is A, D, E, F, H, I, K, L, P, Q, R, S, T, V, W or Y; the amino acid at position 27 is A, D, E, F, H, I, K, L, N, P, Q, R, S, T, V, W or Y; the amino acid at position 28 is A, D, E, F, G, H, I, K, L, P, Q, R, S, V, W or Y; the amino acid at position 29 is I, L, W or Y; or the amino acid at position 30 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; (c2) heavy chain framework 2 shown in SEQ ID NO:88; (c3) the heavy chain framework 3 set forth in SEQ ID NO:89, or a heavy chain framework 3 having an amino acid residue identical to that of the heavy chain framework 3 set forth in SEQ ID NO:89 at positions other than position 73 according to the Kabat numbering, and having an amino acid residue different from that of the heavy chain framework 3 set forth in SEQ ID NO:89 at position 73 according to the Kabat numbering, wherein the amino acid at position 73 is A, D, F, G, H, I, K, L, M, P, Q, R, S, T, V or Y; and (c4) Heavy chain framework 4 shown in SEQ ID NO:

90.

3. The library of claim 1 or claim 2, wherein the antibody light chain variable region further comprises: (d1) light chain framework 1 shown in SEQ ID NO: 91; (d2) a light chain framework 2 as set forth in SEQ ID NO:92, or a light chain framework 2 having the same amino acid residues as the light chain framework 2 as set forth in SEQ ID NO:92 at positions other than positions 46 and 49 according to the Kabat numbering, and having an amino acid residue different from the light chain framework 2 as set forth in SEQ ID NO:92 at a position selected from positions 46 and 49 according to the Kabat numbering, wherein the amino acid at position 46 is I, M, N or V; and the amino acid at position 49 is F, H or W; (d3) light chain framework 3 shown in SEQ ID NO:93; and (d4) light chain framework 4 shown in SEQ ID NO:

94.

4. A method for producing an antigen-binding molecule comprising an antigen-binding domain, comprising the following steps (a) to (g): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 1 to 3 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 1 to 3 with an antigen in the absence of a low molecular weight compound; Step (b) selecting the antigen-binding domain that does not bind to the antigen in step (a); Step (c) contacting the antigen-binding domain selected in step (b) with an antigen in the presence of a low molecular weight compound; Step (d) selecting the antigen-binding domain that binds to the antigen in step (c); Step (e) connecting the polynucleotide encoding the antigen-binding domain selected in step (d) to the polynucleotide encoding the polypeptide comprising the Fc region; Step (f) culturing the cells introduced with the vector, wherein the vector is operably linked to the polynucleotide obtained in step (e); and step (g) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (f), The low molecular weight compound is kynurenine.

5. A method for producing an antigen-binding molecule comprising an antigen-binding domain, comprising the following steps (a) to (e): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 1 to 3 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 1 to 3 with an antigen in the presence of a low molecular weight compound; Step (b) using a low molecular weight compound at a lower concentration than that in step (a) to dissociate and recover the antigen binding domain; Step (c) ligating the polynucleotide encoding the antigen-binding domain recovered in step (b) to a polynucleotide encoding a polypeptide comprising an Fc region; Step (d) culturing the cells introduced with the vector, wherein the vector is operably linked to the polynucleotide obtained in step (c); and step (e) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (d), The low molecular weight compound is kynurenine.

6. The method of claim 4 or 5, further comprising the following steps (a) to (b): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 1 to 3 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 1 to 3 with a low molecular weight compound; and Step (b) selects the antigen-binding domain recovered in step (a).

7. A library comprising: (i) a plurality of antigen-binding domains having different sequences from each other or an antigen-binding molecule comprising such antigen-binding domains; or (ii) nucleic acids encoding a plurality of antigen-binding domains having different sequences from each other or antigen-binding molecules comprising such antigen-binding domains; The antigen binding domain comprises an antibody heavy chain variable region and an antibody light chain variable region, The antibody heavy chain variable region comprises: (a1) CDR1 contained in the heavy chain variable region shown in SEQ ID NO: 85 、 CDR2 、 CDR3; or (a2) at position 31 according to Kabat numbering 、 32-bit 、 53 、 54 bits 、 55 、 56 bits 、 57 、 59 、 61 bits 、 62-bit 、 65 bits 、 96 bits 、 97th 、 98 bits 、 100 、 100a 、 101st 、 The amino acid residues in the positions other than position 102 are identical to the CDRs contained in the heavy chain variable region set forth in SEQ ID NO: 85, and the amino acid residues in the positions selected from position 31 according to the Kabat numbering are identical to the CDRs contained in the heavy chain variable region set forth in SEQ ID NO:

85. 、 32-bit 、 53 、 54 bits 、 55 、 56 bits 、 57 、 59 、 61 bits 、 62-bit 、 65 bits 、 96 bits 、 97th 、 98 bits 、 100 、 100a 、 101st 、 a CDR having an amino acid residue at position 102 that is different from the CDR contained in the heavy chain variable region shown in SEQ ID NO: 85, The amino acid at position 31 is A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W or Y; the amino acid at position 32 is F, G, H, I, K, N, P, Q, R, T, V or W; the amino acid at position 53 is D, E, F, G, I, K, L, P, Q, R, S, T, V or Y; the amino acid at position 54 is E, F, G, H, I, L, Q, S, T, V , or Y; amino acid at position 55 is A, D, E, F, G, H, I, K, L, Q, R, T, V, or Y; amino acid at position 56 is A, D, E, F, G, H, I, K, L, N, Q, R, T, or V; amino acid at position 57 is A, I, K, L, N, Q, R, S, or V; amino acid at position 59 is A, D, E, F, G, H, I, K, L, Q, R, S, T, V, or W; The amino acid at position 61 is A, F, H, K, L, N, P, Q, R, T, V, W or Y; the amino acid at position 62 is A, D, E, F or G; the amino acid at position 65 is A, D, E, F, I, K, L, N, Q, R, T, V or W; the amino acid at position 96 is A, D, E, F, G, H, K, L, N, Q, S, T, V, W or Y; the amino acid at position 97 is A, D, E, G, H , I, K, L, N, Q, R, S, T, V, W or Y; amino acid at position 98 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S or T; amino acid at position 100 is F; amino acid at position 100a is S or V; amino acid at position 101 is A, E, F, G, H, I, K, L, N, Q, R, S, T, V, W or Y; or amino acid at position 102 is D or N; The antibody light chain variable region comprises: (b1) CDR1 contained in the light chain variable region shown in SEQ ID NO: 86 、 CDR2 、 CDR3; or (b2) at position 28 according to Kabat numbering 、 29th 、 32-bit 、 93 、 94 bits 、 95 、 95a 、 95b 、 The amino acid residues in the positions other than position 95c are identical to the CDRs contained in the light chain variable region set forth in SEQ ID NO: 86, and the amino acid residues in the positions selected from position 28 according to the Kabat numbering are identical to the CDRs contained in the light chain variable region set forth in SEQ ID NO:

86. 、 29th 、 32-bit 、 93 、 94 bits 、 95 、 95a 、 95b 、 A CDR having an amino acid residue at one position of position 95c that is different from the CDR contained in the light chain variable region shown in SEQ ID NO: 86, The amino acid at position 28 is A, F, H, K, N, S, T, V or Y; the amino acid at position 29 is A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, W or Y; the amino acid at position 32 is A, D, F, G or H; the amino acid at position 93 is D, E, F, G, H, L, Q, R, S, T, V or Y; the amino acid at position 94 is A, D, E, F, G, H, I, K, L, P, Q, R, S, The amino acid at position 95 is A, D, E, F, G, H, I, K, L, N, P, Q, R, T, V, W or Y; the amino acid at position 95a is A, D, E, F, H, I, K, L, N, P, Q, R, V, W or Y; the amino acid at position 95b is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or Y; or the amino acid at position 95c is F, H, K, L, N or V.

8. The library of claim 7, wherein the antibody heavy chain variable region further comprises: (c1) the heavy chain framework contained in the heavy chain variable region shown in SEQ ID NO: 85; or (c2) The heavy chain framework contained in the heavy chain variable region shown in SEQ ID NO:

30.

9. The library of claim 7 or claim 8, wherein the antibody light chain variable region further comprises: (d1) the light chain framework contained in the light chain variable region shown in SEQ ID NO: 86; or (d2) The light chain framework contained in the light chain variable region shown in SEQ ID NO:

31.

10. A method for producing an antigen-binding molecule comprising an antigen-binding domain, comprising the following steps (a) to (g): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 7 to 9 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 7 to 9 with an antigen in the absence of a low molecular weight compound; Step (b) selecting the antigen-binding domain that does not bind to the antigen in step (a); Step (c) contacting the antigen-binding domain selected in step (b) with an antigen in the presence of a low molecular weight compound; Step (d) selecting the antigen-binding domain that binds to the antigen in step (c); Step (e) connecting the polynucleotide encoding the antigen-binding domain selected in step (d) to the polynucleotide encoding the polypeptide comprising the Fc region; Step (f) culturing the cells introduced with the vector, wherein the vector is operably linked to the polynucleotide obtained in step (e); and step (g) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (f), in, The low molecular compound is adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

11. A method for producing an antigen-binding molecule comprising an antigen-binding domain, comprising the following steps (a) to (e): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 7 to 9 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 7 to 9 with an antigen in the presence of a low molecular weight compound; Step (b) using a low molecular weight compound at a lower concentration than that in step (a) to dissociate and recover the antigen binding domain; Step (c) ligating the polynucleotide encoding the antigen-binding domain recovered in step (b) to a polynucleotide encoding a polypeptide comprising an Fc region; Step (d) culturing the cells introduced with the vector, wherein the vector is operably linked to the polynucleotide obtained in step (c); and step (e) recovering the antigen-binding molecules from the culture medium of the cells cultured in step (d), in, The low molecular compound is adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

12. The method of claim 10 or 11, further comprising the following steps (a) to (b): Step (a) contacting a library comprising a plurality of antigen-binding domains or antigen-binding molecules according to any one of claims 7 to 9 or a plurality of antigen-binding domains or antigen-binding molecules encoded by a nucleic acid according to any one of claims 7 to 9 with a low molecular weight compound; and Step (b) selects the antigen-binding domain recovered in step (a). 13 . An antigen-binding molecule produced by the method of any one of claims 4 to 6 and 10 to 12.

14. A pharmaceutical composition comprising the antigen-binding molecule of claim 13.

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