Tissue-specific antigen-binding molecules

Abstract

The present invention provides a pharmaceutical composition useful for treating a disease caused by a target tissue, its active ingredient, a screening method for the pharmaceutical composition and the active ingredient, and a manufacturing method. [Solution] The present invention provides an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound. The antigen-binding molecule comprises an Fc region contained in a constant region of a specific sequence, and an FcγR-binding altered Fc region whose binding activity to Fcγ receptors is higher than the binding activity to Fcγ receptors of the Fc region of native human IgG. Use of the antigen-binding molecule enables target tissue-specific treatment of various diseases caused by target tissues.

Description

[Technical Field]

[0001] The present invention provides antigen-binding molecules comprising an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound, methods for producing and screening for the antigen-binding molecules, and pharmaceutical compositions comprising the antigen-binding molecules. [Background technology]

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

[0003] Approved antibody-based cancer therapeutics include Rituxan, which targets the CD20 antigen, cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC and other mechanisms. It is known that the cytotoxic activity of ADCC and other mechanisms depends on the number of antigens expressed on the target cells of the therapeutic antibody (Non-Patent Document 4). Therefore, a high expression level of the target antigen is preferable from the perspective of the efficacy of the therapeutic antibody. However, even if the expression level of the antigen is high, if the antigen is expressed in normal tissues, ADCC and other cytotoxic activities will be exerted against normal cells, resulting in serious side effects. Therefore, it is preferable that the antigen targeted by a therapeutic antibody as a cancer therapeutic drug is specifically expressed on cancer cells. For example, antibody molecules against the EpCAM antigen, which is known as a cancer antigen, were thought to be promising as cancer treatment drugs, but the EpCAM antigen is also known to be expressed in the pancreas, and in fact, in clinical trials, it has been reported that administration of anti-EpCAM antibodies causes the side effect of pancreatitis due to cytotoxic activity against the pancreas (Non-Patent Document 5).

[0004] Following the success of antibody pharmaceuticals that exert cytotoxic activity through ADCC activity, second-generation improved antibody molecules that exert potent cytotoxic activity have been reported, such as by enhancing ADCC activity by removing fucose from the N-glycosylation chains in the Fc region of native human IgG1 (Non-Patent Document 6) and by enhancing binding to FcγRIIIa through amino acid substitution in the Fc region of native human IgG1 (Non-Patent Document 7). As antibody pharmaceuticals that exert cytotoxic activity against cancer cells by mechanisms other than the above-mentioned NK cell-mediated ADCC activity, improved antibody molecules that exert even more potent cytotoxic activity have also been reported, such as antibody drug conjugates (ADCs) in which antibodies are conjugated with drugs that have potent cytotoxic activity (Non-Patent Document 8), and small molecule antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to the cancer cells (Non-Patent Document 9).

[0005] While these antibody molecules exhibit more potent cytotoxic activity, they can also exert cytotoxic activity against cancer cells with low antigen expression, they also exert cytotoxic activity against normal tissues with low antigen expression. In fact, compared to cetuximab, a natural human IgG1 directed against the EGFR antigen, EGFR-BiTE, a bispecific antibody directed against CD3 and EGFR, exerts potent cytotoxic activity against cancer cells by recruiting T cells to cancer cells, thereby exerting antitumor effects. However, because EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE was administered to cynomolgus monkeys (Non-Patent Document 10). Furthermore, bivatuzumab mertansine, an ADC consisting of mertansine conjugated to an antibody directed against CD44v6, which is highly expressed in cancer cells, has been shown to cause severe skin and liver toxicity in clinical trials, due to the CD44v6 expression in normal tissues (Non-Patent Document 11).

[0006] When using an antibody that can exert strong cytotoxic activity even against cancer cells with low antigen expression, the target antigen must be expressed in an extremely cancer-specific manner, but the number of cancer antigens that are expressed in an extremely cancer-specific manner is thought to be limited, as HER2, the target antigen of Herceptin, and EGFR, the target antigen of cetuximab, are also expressed in normal tissues. Therefore, although cytotoxic activity against cancer can be strengthened, side effects due to cytotoxic effects on normal tissues can become a problem.

[0007] Recently, it has been shown that ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, prolongs overall survival in metastatic melanoma (Non-Patent Document 12). However, because ipilimumab systemically inhibits CTLA4, while tumor immunity is enhanced, it also causes serious autoimmune disease-like side effects due to systemic immune activation, which has become a problem (Non-Patent Document 13).

[0008] On the other hand, antibody drugs for diseases other than cancer are known to exert therapeutic effects by inhibiting inflammatory cytokines in inflammatory and autoimmune diseases (Non-Patent Document 14). For example, Remicade and Humira, which target TNF, and Actemra, which targets IL-6R, exert high therapeutic effects against rheumatoid arthritis, but it is also known that systemic neutralization of these cytokines can cause side effects such as infections (Non-Patent Document 15).

[0009] Various technologies applicable to second-generation antibody drugs have been developed, including those that improve effector function, antigen-binding ability, pharmacokinetics, and stability, or reduce the risk of immunogenicity (Non-Patent Document 16). However, few technologies have been reported that enable antibody drugs to specifically act on target tissues to resolve the side effects described above. For example, pH-dependent antibodies have been reported for lesion sites such as cancerous tissues and inflammatory tissues, taking advantage of the acidic pH conditions in these target tissues (Patent Documents 1 and 2). However, the decrease in pH (i.e., increase in hydrogen ion concentration) in cancerous tissues and inflammatory tissues compared to normal tissues is slight, making it difficult to generate antibodies that detect and act on even small increases in hydrogen ion concentration, which are extremely small in molecular weight. Furthermore, normal tissues such as osteoclast bone resorption lacunae and tissues other than the target lesion may also have acidic pH, posing many challenges to the use of pH conditions as an environmental factor specific to the lesion site. On the other hand, a method has been reported in which antibodies that exhibit antigen-binding activity only upon cleavage by proteases expressed in lesion sites such as cancerous tissues and inflammatory tissues (Patent Document 3). However, because antibody cleavage by proteases is irreversible, it has been thought that the problem is that antibodies cleaved at the lesion site can return to normal tissues via the bloodstream and bind to antigens there as well. Furthermore, the cancer specificity of such proteases is also thought to be an issue. Therefore, no technology is known that can exert its efficacy while avoiding side effects by acting reversibly at the lesion site, i.e., cancer or inflammatory sites, without acting systemically in normal tissues or blood. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. WO2003 / 105757 [Patent Document 2] International Publication No. WO2012 / 033953 [Patent Document 3] International Publication No. WO2010 / 081173

Non-licensed literature

[0011] [Non-licensed document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-licensed document 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-licensed document 3] Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327

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[0012] The present invention has been made in view of the above circumstances, and an object thereof is to provide a pharmaceutical composition useful for treating diseases caused by target tissues, and an active ingredient thereof, as well as a method for screening and producing said pharmaceutical composition and said active ingredient. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to achieve the above-mentioned objectives and have created an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound. Furthermore, the present inventors have found that the antigen-binding molecule or a pharmaceutical composition comprising the antigen-binding molecule is useful for treating diseases caused by target tissues, and that the antigen-binding molecule is useful for treating diseases caused by target tissues, including administering the antigen-binding molecule, and that the antigen-binding molecule is useful in producing pharmaceuticals for treating diseases caused by target tissues. Furthermore, the present inventors have created methods for screening and producing the antigen-binding molecule, thereby completing the present invention.

[0014] That is, the present invention provides the following: (1) An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound; (2) The antigen-binding molecule according to (1), wherein the target tissue is a cancer tissue. (3) The antigen-binding molecule according to (2), wherein the cancer tissue-specific compound is a cancer cell-specific metabolite, an immune cell-specific metabolite infiltrating cancer tissue, or a stromal cell-specific metabolite in cancer tissue. (4) The antigen-binding molecule according to (1), wherein the target tissue is an inflammatory tissue. (5) The antigen-binding molecule according to (4), wherein the inflamed tissue-specific compound is a metabolite specific to immune cells infiltrating inflamed tissue or a metabolite specific to normal cells damaged in inflamed tissue. (6) The antigen-binding molecule according to (1), wherein the target tissue-specific metabolite 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. (7) The antigen-binding molecule according to (6), wherein the target tissue-specific metabolite is at least one compound selected from adenosine, adenosine triphosphate, inosine, alanine, glutamic acid, aspartic acid, kynurenine, prostaglandin E2, succinic acid, citric acid, and 1-methylnicotinamide. (8) The antigen-binding molecule according to any one of (1) to (7), wherein the antigen is a membrane-type molecule. (9) The antigen-binding molecule according to any one of (1) to (8), which is an antigen-binding molecule having neutralizing activity. (10) The antigen-binding molecule according to any one of (1) to (9), which is an antigen-binding molecule having cytotoxic activity. (11) The antigen-binding molecule according to any one of (1) to (10), which comprises an Fc region. (12) The antigen-binding molecule according to (11), wherein the Fc region is an Fc region contained in a constant region set forth in SEQ ID NO: 5, 6, 7, or 8. (13) The antigen-binding molecule according to (11), wherein the Fc region comprises an FcγR-binding modified Fc region having higher Fcγ receptor-binding activity than that of the Fc region of native human IgG. (14) In the amino acid sequence of the FcγR binding altered Fc region, positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, and 249 (EU numbering), 250th, 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st , 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 31st 5th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 3 The antigen-binding molecule according to (13), wherein at least one or more amino acids selected from the group consisting of positions 35, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 are different from the amino acids in the Fc region of native human IgG. (15) The amino acid sequence of the FcγR binding altered Fc region, represented by EU numbering; The amino acid at position 221 is either Lys or Tyr; the amino acid at position 222 is Phe, Trp, Glu, or Tyr; the amino acid at position 223 is Phe, Trp, Glu, or Lys; the amino acid at position 224 is Phe, Trp, Glu, or Tyr; The amino acid at position 225 is either Glu, Lys, or Trp; the amino acid at position 227 is Glu, Gly, Lys, or Tyr; the amino acid at position 228 is Glu, Gly, Lys, or Tyr; The amino acid at position 230 is Ala, Glu, Gly, or Tyr; the amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 232 is Glu, Gly, Lys, or Tyr; the amino acid at position 233 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 234 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 235 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 236 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 238 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 239 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 240 is Ala, Ile, Met, or Thr; the amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or Tyr; the amino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The amino acid at position 244 is His; The amino acid at position 245 is Ala, the amino acid at position 246 is Asp, Glu, His, or Tyr; the amino acid at position 247 is Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The amino acid at position 249 is Glu, His, Gln, or Tyr; The amino acid at position 250 is either Glu or Gln, The amino acid at position 251 is Phe, the amino acid at position 254 is either Phe, Met, or Tyr; The amino acid at position 255 is either Glu, Leu, or Tyr; The amino acid at position 256 is either Ala, Met, or Pro; the amino acid at position 258 is Asp, Glu, His, Ser, or Tyr; The amino acid at position 260 is either Asp, Glu, His, or Tyr; the amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; the amino acid at position 263 is Ala, Ile, Met, or Thr; the amino acid at position 264 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 265 is Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 266 is Ala, Ile, Met, or Thr; the amino acid at position 267 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 268 is Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp; the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 270 is Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 271 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 272 is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 273 is either Phe or Ile; the amino acid at position 274 is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 275 is either Leu or Trp, the amino acid at position 276 is any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 279 is Ala, the amino acid at position 280 is Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr; the amino acid at position 281 is Asp, Lys, Pro, or Tyr; The amino acid at position 282 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 283 is Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; the amino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or Tyr; the amino acid at position 285 is Asp, Glu, Lys, Gln, Trp, or Tyr; The amino acid at position 286 is Glu, Gly, Pro, or Tyr; the amino acid at position 288 is Asn, Asp, Glu, or Tyr; the amino acid at position 290 is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr; the amino acid at position 291 is Asp, Glu, Gly, His, Ile, Gln, or Thr; the amino acid at position 292 is Ala, Asp, Glu, Pro, Thr, or Tyr; the amino acid at position 293 is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 294 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 296 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the amino acid at position 297 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 298 is Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 300 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; the amino acid at position 301 is Asp, Glu, His, or Tyr; The amino acid at position 302 is Ile; the amino acid at position 303 is Asp, Gly, or Tyr; the amino acid at position 304 is Asp, His, Leu, Asn, or Thr; The amino acid at position 305 is Glu, Ile, Thr, or Tyr; the amino acid at position 311 is Ala, Asp, Asn, Thr, Val, or Tyr; The amino acid at position 313 is Phe; The amino acid at position 315 is Leu; The amino acid at position 317 is Glu or Gln, the amino acid at position 318 is any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 322 is any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 323 is Ile; the amino acid at position 324 is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 325 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 326 is Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 327 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 328 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 330 is any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 332 is Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 333 is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr; the amino acid at position 334 is Ala, Glu, Phe, Ile, Leu, Pro, or Thr; the amino acid at position 335 is Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 336 is either Glu, Lys, or Tyr; The amino acid at position 337 is either Glu, His, or Asn; the amino acid at position 339 is Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr; The amino acid at position 376 is either Ala or Val; The amino acid at position 377 is either Gly or Lys; The amino acid at position 378 is Asp, The amino acid at position 379 is Asn, The amino acid at position 380 is either Ala, Asn, or Ser; The amino acid at position 382 is either Ala or Ile; The amino acid at position 385 is Glu; The amino acid at position 392 is Thr; The amino acid at position 396 is Leu, The amino acid at position 421 is Lys; The amino acid at position 427 is Asn, The amino acid at position 428 is either Phe or Leu; The amino acid at position 429 is Met; The amino acid at position 434 is Trp, The amino acid at position 436 is Ile, or the amino acid at position 440 is Gly, His, Ile, Leu, or Tyr; The antigen-binding molecule according to (14), which contains at least one amino acid selected from the group consisting of: (16) The antigen-binding molecule according to (11), wherein the Fc region is an Fc region modified so that the composition of the sugar chains attached to position 297 (EU numbering) of the Fc region is such that the proportion of Fc region having a fucose-deficient sugar chain attached thereto is increased, or the proportion of Fc region having a bisecting N-acetylglucosamine attached thereto is increased. (17) The antigen-binding molecule according to any one of (11), (13) to (16), wherein the Fc region has an enhanced FcRn-binding activity under an acidic pH range condition compared to the binding activity of the Fc region represented by any one of SEQ ID NOs: 5, 6, 7, or 8. (18) The Fc region is selected from the amino acid sequences of the Fc region contained in the constant region of SEQ ID NO: 5, 6, 7, or 8, and is selected from the amino acid sequences of positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 3 18. The antigen-binding molecule of (17), wherein the Fc region is substituted with at least one amino acid selected from the group consisting of positions 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447. (19) The Fc region is an amino acid sequence of the Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8, represented by EU numbering; The amino acid at position 238 is Leu, The amino acid at position 244 is Leu, The amino acid at position 245 is Arg; The amino acid at position 249 is Pro, The amino acid at position 250 is either Gln or Glu, or The amino acid at position 251 is Arg, Asp, Glu, or Leu; the amino acid at position 252 is Phe, Ser, Thr, or Tyr; The amino acid at position 254 is either Ser or Thr; The amino acid at position 255 is Arg, Gly, Ile, or Leu; the amino acid at position 256 is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr; the amino acid at position 257 is Ala, Ile, Met, Asn, Ser, or Val; The amino acid at position 258 is Asp, The amino acid at position 260 is Ser; The amino acid at position 262 is Leu, The amino acid at position 270 is Lys. The amino acid at position 272 is either Leu or Arg, the amino acid at position 279 is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 283 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The amino acid at position 285 is Asn, The amino acid at position 286 is Phe, The amino acid at position 288 is either Asn or Pro, The amino acid at position 293 is Val; The amino acid at position 307 is Ala, Glu, Gln, or Met; the amino acid at position 311 is Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp; The amino acid at position 309 is Pro, The amino acid at position 312 is Ala, Asp, or Pro; The amino acid at position 314 is either Ala or Leu, The amino acid at position 316 is Lys; The amino acid at position 317 is Pro, The amino acid at position 318 is either Asn or Thr, the amino acid at position 332 is Phe, His, Lys, Leu, Met, Arg, Ser, or Trp; The amino acid at position 339 is either Asn, Thr, or Trp; The amino acid at position 341 is Pro, The amino acid at position 343 is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The amino acid at position 375 is Arg; the amino acid at position 376 is Gly, Ile, Met, Pro, Thr, or Val; The amino acid at position 377 is Lys. The amino acid at position 378 is Asp, Asn, or Val; Amino acid at position 380 is Ala, Asn, Ser, or Thr the amino acid at position 382 is Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 385 is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; the amino acid at position 386 is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; The amino acid at position 387 is Ala, Arg, His, Pro, Ser, or Thr; The amino acid at position 389 is Asn, Pro, or Ser; The amino acid at position 423 is Asn; The amino acid at position 427 is Asn, The amino acid at position 428 is Leu, Met, Phe, Ser, or Thr the amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The amino acid at position 431 is either His or Asn, the amino acid at position 433 is Arg, Gln, His, Ile, Lys, Pro, or Ser; the amino acid at position 434 is Ala, Gly, His, Phe, Ser, Trp, or Tyr; the amino acid at position 436 is Arg, Asn, His, Ile, Leu, Lys, Met, or Thr; The amino acid at position 438 is Lys, Leu, Thr, or Trp; The amino acid at position 440 is Lys, or the amino acid at position 442 is Lys, and the amino acid at position 308 is Ile, Pro, or Thr; The antigen-binding molecule according to (18), wherein the amino acid sequence is at least one amino acid selected from the group consisting of: (20) The antigen-binding molecule according to any one of (1) to (19), wherein the antigen-binding domains are multispecific or multiparatopic antigen-binding domains. (21) The antigen-binding molecule according to (20), wherein the antigen to which at least one of the antigen-binding domains binds is a membrane-type molecule expressed on the cell membrane of a cancer cell, and the antigen to which at least one of the antigen-binding domains binds is a membrane-type molecule expressed on the cell membrane of an effector cell. (22) The antigen-binding molecule according to (21), wherein the effector cell is an NK cell, a macrophage, or a T cell. (23) The antigen-binding molecule according to (21) or (22), wherein the membrane molecule expressed on the cell membrane of the effector cell is a polypeptide constituting a TCR, CD2, CD3, CD28, CD44, CD16, CD32, CD64, or NKG2D. (24) The antigen-binding molecule according to (20), wherein the antigen to which at least one of the antigen-binding domains binds is a membrane molecule expressed on the cell membrane of a cancer cell, and the antigen to which at least one of the antigen-binding domains binds is a cytotoxic substance. (25) The antigen-binding molecule according to any one of (20) to (24), wherein the antigen-binding molecule is an antibody fragment. (26) The antigen-binding molecule according to any one of (1) to (24), wherein the antigen-binding molecule is an antibody. (27) The antigen-binding molecule according to any one of (1) to (7), wherein the antigen is a soluble molecule. (28) The antigen-binding molecule according to (27), which is an antigen-binding molecule having neutralizing activity. (29) The antigen-binding molecule according to (27) or (28), which comprises an Fc region. (30) The antigen-binding molecule according to (29), wherein the Fc region is an Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8. (31) The antigen-binding molecule of (29), wherein the Fc region has an FcRn-binding activity under an acidic pH range condition that is enhanced compared to the FcRn-binding activity of the Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8. (32) The Fc region is selected from the amino acid sequences of the Fc region contained in the constant region of SEQ ID NO: 5, 6, 7, or 8, including positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, and 318 (EU numbering); The antigen-binding molecule of (31), wherein the Fc region has a substitution of at least one amino acid selected from the group consisting of positions 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447. (33) The Fc region is an amino acid sequence of the Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8, represented by EU numbering; The amino acid at position 238 is Leu, The amino acid at position 244 is Leu, The amino acid at position 245 is Arg; The amino acid at position 249 is Pro, The amino acid at position 250 is either Gln or Glu, or The amino acid at position 251 is Arg, Asp, Glu, or Leu; the amino acid at position 252 is Phe, Ser, Thr, or Tyr; The amino acid at position 254 is either Ser or Thr; The amino acid at position 255 is Arg, Gly, Ile, or Leu; the amino acid at position 256 is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr; the amino acid at position 257 is Ala, Ile, Met, Asn, Ser, or Val; The amino acid at position 258 is Asp, The amino acid at position 260 is Ser; The amino acid at position 262 is Leu, The amino acid at position 270 is Lys. The amino acid at position 272 is either Leu or Arg, the amino acid at position 279 is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 283 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The amino acid at position 285 is Asn, The amino acid at position 286 is Phe, The amino acid at position 288 is either Asn or Pro, The amino acid at position 293 is Val; The amino acid at position 307 is Ala, Glu, Gln, or Met; the amino acid at position 311 is Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp; The amino acid at position 309 is Pro, The amino acid at position 312 is Ala, Asp, or Pro; The amino acid at position 314 is either Ala or Leu, The amino acid at position 316 is Lys; The amino acid at position 317 is Pro, The amino acid at position 318 is either Asn or Thr, the amino acid at position 332 is Phe, His, Lys, Leu, Met, Arg, Ser, or Trp; The amino acid at position 339 is either Asn, Thr, or Trp; The amino acid at position 341 is Pro, The amino acid at position 343 is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The amino acid at position 375 is Arg; the amino acid at position 376 is Gly, Ile, Met, Pro, Thr, or Val; The amino acid at position 377 is Lys. The amino acid at position 378 is Asp, Asn, or Val; Amino acid at position 380 is Ala, Asn, Ser, or Thr the amino acid at position 382 is Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 385 is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; the amino acid at position 386 is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; The amino acid at position 387 is Ala, Arg, His, Pro, Ser, or Thr; The amino acid at position 389 is Asn, Pro, or Ser; The amino acid at position 423 is Asn; The amino acid at position 427 is Asn, The amino acid at position 428 is Leu, Met, Phe, Ser, or Thr the amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The amino acid at position 431 is either His or Asn, the amino acid at position 433 is Arg, Gln, His, Ile, Lys, Pro, or Ser; the amino acid at position 434 is Ala, Gly, His, Phe, Ser, Trp, or Tyr; the amino acid at position 436 is Arg, Asn, His, Ile, Leu, Lys, Met, or Thr; The amino acid at position 438 is Lys, Leu, Thr, or Trp; The amino acid at position 440 is Lys, or the amino acid at position 442 is Lys, and the amino acid at position 308 is Ile, Pro, or Thr; The antigen-binding molecule according to (32), wherein the amino acid sequence is at least one amino acid selected from the group consisting of: (34) The antigen-binding molecule of (29), wherein the Fc region has an FcRn-binding activity under a neutral pH range condition that is enhanced compared to the FcRn-binding activity of the Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8. (35) The Fc region is selected from the amino acid sequences of the Fc region contained in the constant region of SEQ ID NO: 5, 6, 7, or 8, and is selected from the amino acid sequences of positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 3 3. The antigen-binding molecule of (34), wherein the Fc region has a substitution of at least one amino acid selected from the group consisting of positions 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436. (36) The Fc region is an amino acid sequence of the Fc region contained in the constant region set forth in SEQ ID NO: 5, 6, 7, or 8, represented by EU numbering; The amino acid at position 237 is Met; The amino acid at position 248 is Ile; The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; The amino acid at position 252 is either Phe, Trp, or Tyr; The amino acid at position 254 is Thr; The amino acid at position 255 is Glu; the amino acid at position 256 is Asp, Asn, Glu, or Gln; the amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His; The amino acid at position 265 is Ala, The amino acid at position 286 is either Ala or Glu, The amino acid at position 289 is His; The amino acid at position 297 is Ala, The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, the amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg; the amino acid at position 311 is Ala, His, or Ile; The amino acid at position 312 is either Ala or His; The amino acid at position 314 is either Lys or Arg; the amino acid at position 315 is Ala, Asp, or His; The amino acid at position 317 is Ala, The amino acid at position 332 is Val; The amino acid at position 334 is Leu, The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is either Asp or His; The amino acid at position 386 is Pro, The amino acid at position 387 is Glu; The amino acid at position 389 is either Ala or Ser, The amino acid at position 424 is Ala, the amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys; The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, or The amino acid at position 436 is His, Ile, Leu, Phe, Thr, or Val; The antigen-binding molecule according to (35), wherein the amino acid sequence is at least one amino acid selected from the group consisting of: (37) The antigen-binding molecule according to any one of (29) and (31) to (36), wherein the Fc region has higher binding activity to inhibitory Fcγ receptors than to activating Fcγ receptors. (38) The antigen-binding molecule according to (37), wherein the inhibitory Fcγ receptor is human FcγRIIb. (39) The antigen-binding molecule according to (37) or (38), wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). (40) The antigen-binding molecule of any one of (37) to (39), wherein the Fc region contains an amino acid at position 238 or 328 (EU numbering) that is different from the amino acid in the Fc region of native human IgG. (41) The antigen-binding molecule of (40), wherein the amino acid at position 238, according to EU numbering, of the Fc region is Asp, or the amino acid at position 328, according to EU numbering, is Glu. (42) The amino acid sequence of the Fc region, represented by EU numbering: The amino acid at position 233 is Asp, The amino acid at position 234 is either Trp or Tyr, the amino acid at position 237 is Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr; The amino acid at position 239 is Asp, the amino acid at position 267 is either Ala, Gln or Val; The amino acid at position 268 is either Asn, Asp, or Glu; The amino acid at position 271 is Gly; the amino acid at position 326 is any of Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, and Thr; The amino acid at position 330 is either Arg, Lys, or Met; The amino acid at position 323 is Ile, Leu, or Met; or The amino acid at position 296 is Asp, The antigen-binding molecule according to (40) or (41), wherein the amino acid sequence is at least one amino acid selected from the group consisting of: (43) The antigen-binding molecule according to any one of (27) to (42), wherein the antigen-binding molecule is an antibody. (44) A method for producing the antigen-binding molecule according to any one of (1) to (43), comprising selecting an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound. (45) A method for screening an antigen-binding molecule according to any one of (1) to (43), comprising selecting an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound. (46) A pharmaceutical composition comprising the antigen-binding molecule according to any one of (1) to (43). This provides: [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows that a small molecule switch antibody does not bind to an antigen in a normal environment where small molecules are not present, but binds to the antigen in target tissues where small molecules are present at high concentrations. [Figure 2]This is a diagram showing that a small molecule fulfills its switching function by being sandwiched between a small molecule antibody and an antigen complex. In the absence of the small molecule, the interaction between the antibody and antigen is insufficient and the antibody cannot bind to the antigen, but in the presence of the small molecule, it becomes sandwiched between the antibody and antigen, allowing the antibody to bind to the antigen. [Figure 3] 1 shows the results of ELISA for antibody binding to human IL-6, with the vertical axis representing absorbance values ​​used to evaluate the binding activity of each antibody to human IL-6 in the presence or absence of each small molecule. [Figure 4] This is a sensorgram of the interaction between A11 and 4 μmol / L human IL-6 in the presence and absence of 100 μmol / L kynurenine. [Figure 5] This graph shows the change in binding response to A11 immobilized on a CM5 sensor chip when 1 μmol / L of IL-6 was allowed to interact for 60 seconds as an analyte. The vertical axis represents the change in response (RU) before and after IL-6 interaction, and the horizontal axis represents the kynurenine concentration (μmol / L) in the solution at that time. [Figure 6] This graph shows the response to H01 immobilized on a CM5 sensor chip when 1 μmol / L of IL-6 was allowed to interact for 60 seconds as an analyte. The vertical axis shows the change in response (RU) before and after IL-6 interaction, and the horizontal axis shows the kynurenine concentration (μmol / L) in the solution at that time. [Figure 7] This graph shows the response to IL-6 immobilized on a CM5 sensor chip after 60 seconds of interaction with 0.1 μmol / L A11 as an analyte. The vertical axis represents the change in response (RU) before and after interaction with A11, and the horizontal axis represents the kynurenine concentration (μmol / L) in the solution. [Figure 8]This graph shows the dissociation of A11 from IL-6 immobilized on a CM5 sensor chip in the presence of 100 μmol / L kynurenine after interaction of A11 with IL-6 in a buffer containing 100 μmol / L kynurenine or in a buffer containing no kynurenine. The vertical axis of the graph represents the normalized value, with the amount of A11 binding in the presence of 100 μmol / L kynurenine set to 100, and the horizontal axis represents the time (seconds) elapsed since the start of the interaction. [Figure 9] This is a sensorgram obtained when 800, 400, 200, 100, 50, and 25 nmol / L kynurenine was allowed to interact with IL-6 immobilized on a sensor chip. The vertical axis represents the change in the amount of kynurenine binding to IL-6 (RU) (the response at the start of the interaction experiment was set to 0), and the horizontal axis represents the elapsed time from the start of the interaction experiment. [Figure 10] FIG. 1 shows the structure of 2′-Adenosine-PEG-peptide, an adenosine analog used for immunizing rabbits. [Figure 11] FIG. 1 shows the structure of 5′-Adenosine-PEG-peptide, an adenosine analog used for immunizing rabbits. [Figure 12] FIG. 1 shows the structure of 2′-Adenosine-PEG-biotin, in which the peptide moiety of the adenosine analog used for immunizing rabbits has been substituted with biotin. [Figure 13] FIG. 1 shows the structure of 5′-Adenosine-PEG-biotin, in which the peptide moiety of the adenosine analog used for immunizing rabbits has been substituted with biotin. [Figure 14]The vertical axis shows the amount of binding when each antibody interacts with 2'-Adenosine-PEG-Biotin divided by the amount captured by each antibody (RU) (N_binding_100), and the horizontal axis shows the value 60 seconds after 2'-Adenosine-PEG-Biotin dissociates from each antibody after interaction with 2'-Adenosine-PEG-Biotin divided by the amount captured by each antibody (RU) (N_stability_100). [Figure 15A] These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0002 to adenosine. The sensorgrams show the interaction of SMB0002 with antigen at 7.81, 31.3, 125, and 500 nM, respectively, from bottom to top. [Figure 15B] These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0002 to ATP. The sensorgrams show the interaction of SMB0002 with antigen at 78.1, 313, 1250, and 5000 nM, respectively, from bottom to top. [Figure 15C] These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0089 to adenosine. The sensorgrams show the interaction of SMB0089 with antigen at 7.81, 31.3, 125, and 500 nM, respectively, from bottom to top. [Figure 15D] These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0089 to ATP. The sensorgrams show the interaction of SMB00089 with antigen at 78.1, 313, 1250, and 5000 nM, respectively, from bottom to top. [Figure 15E] These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0104 to adenosine. The sensorgrams show the interaction of SMB0104 with antigen at 7.81, 31.3, and 500 nM, respectively, from bottom to top. [Figure 15F]These are surface plasmon resonance sensorgrams showing the binding (interaction) of clone SMB0104 to ATP. The sensorgrams show the interaction of SMB0104 with antigen at 78.1, 313, 1250, and 5000 nM, respectively, from bottom to top. [Figure 16] These are sensorgrams from surface plasmon resonance analysis showing the binding (interaction) of clone SMB0171 to ATP. The sensorgrams show the interaction of SMB0171 with 5 and 50 μM antigen, from bottom to top. [Figure 17] FIG. 1 shows the results of a competitive ELISA demonstrating that clone SMB0002 binds to adenosine and ATP. [Figure 18] Fig. 10 shows an evaluation of the ability of ATP to inhibit the binding of ATNLSA1-4_D12 to biotin-labeled antigens (a mixture of 5'-Adenosine-PEG-biotin and ATP-PEG-biotin). [Figure 19] This figure shows the concept of a rationally designed antibody library, in which adenosine or ATP is sandwiched between the antibody and antigen, and a library of antibody variable region portions that come into contact with the antigen is created, enabling the acquisition of adenosine / ATP switch antibodies against any antigen. [Figure 20] This figure shows the concept of the adenosine-immunized rabbit antibody library, in which adenosine or ATP is sandwiched between the antibody and antigen, enabling the isolation of adenosine / ATP-switched antibodies against any antigen. [Figure 21] 1 shows the results of ELISA for antibody binding to human IL-6. The vertical axis shows the binding activity of each antibody to human IL-6 in the presence or absence of amino acids and amino acid metabolites (kynurenine, tryptophan, phenylalanine, anthranilic acid, and 3-hydroxykynurenine and kynurenic acid), expressed as absorbance values ​​at a wavelength of 450 nm. [Figure 22]1 shows the results of ELISA for antibody binding to human IL-6. The vertical axis shows the binding activity of the I6NMSC1-3_#03 antibody to human IL-6 in the presence or absence of each small molecule (ATP, adenosine, inosine, PGE2, succinic acid, lactate, kynurenine, and small molecule cocktail), expressed as specific activity values ​​calculated from absorbance at 450 nm. [Figure 23] 1 shows the results of ELISA for antibody binding to human IL-6. The vertical axis shows the binding activity of the I6NMSC1-3_#17 antibody to human IL-6 in the presence or absence of each small molecule (ATP, adenosine, inosine, PGE, succinic acid, lactate, kynurenine, and small molecule cocktail), expressed as specific activity calculated from absorbance at 450 nm. [Figure 24] 1 shows the results of ELISA for antibody binding to HSA. The vertical axis shows the HSA-binding activity of antibody HSNMSC1-4_#22 in the presence or absence of each small molecule (ATP, adenosine, inosine, PGE, succinic acid, lactate, kynurenine, and small molecule cocktail), expressed as absorbance at 450 nm. [Figure 25] This figure shows the results of ELISA of clone I6DL2C5-4_076, obtained from a rationally designed antibody library, against human IL-6 in the presence / absence of 1 mM ATP and / or adenosine. The vertical axis represents absorbance values ​​used to evaluate the antibody's binding activity to human IL-6. The negative control shows the results when M13KO7 Helper Phage was used. [Figure 26] This figure shows the results of ELISA of clone HSDL3C5-4_015, obtained from a rationally designed antibody library, against human serum albumin in the presence / absence of 1 mM ATP and / or adenosine. The vertical axis represents absorbance values ​​used to evaluate the antibody's binding activity to human serum albumin. The negative control shows the results when M13KO7 Helper Phage was used. [Figure 27]This figure shows the results of ELISA of clones 6RAD2C1-4_011 and 6RAD2C1-4_076 obtained from a rationally designed antibody library against human IL-6 receptor in the presence or absence of 1 mM ATP and / or adenosine (referred to as ADO) and in the presence or absence of a small molecule cocktail (SC). The vertical axis represents absorbance values ​​used to evaluate the binding activity of the antibodies to human IL-6 receptor. The negative control shows the results when M13KO7 Helper Phage was used. [Figure 28] Figure 1 shows the results of ELISA for the binding of clone 6RNMSC1-2_F02 to human IL-6R, with the vertical axis representing absorbance values ​​used to evaluate the binding activity of the antibody to human IL-6R in the presence or absence of each small molecule. [Figure 29] 1 shows the results of ELISA for the binding of clone 6RNMSC1-3_G02 to human IL-6R, with the vertical axis representing absorbance values ​​used to evaluate the binding activity of the antibody to human IL-6R in the presence or absence of each small molecule. [Figure 30] Figure 1 shows the results of ELISA for antibody binding to human IL-6R, with the vertical axis representing absorbance values ​​used to evaluate the antibody binding activity to human IL-6R in the presence or absence of each amino acid or amino acid metabolite. [Figure 31] Sensorgrams of the interaction of 6RNMSC1-2_F02 with 1 μmol / L IL-6R in the presence of 100 μmol / L kynurenine, 10 mmol / L ATP, and in the absence of kynurenine or ATP. The solid line indicates the interaction in the presence of kynurenine, the dotted line indicates the interaction in the presence of ATP, and the dashed line indicates the interaction in the absence of these. [Figure 32]This graph shows the interaction of 6RNMSC1-2_F02 with IL-6R immobilized on a CM5 sensor chip in the presence of 100 μmol / L kynurenine, followed by the dissociation of 6RNMSC1-2_F02 from IL-6R in a buffer containing 100 μmol / L kynurenine or in a buffer containing no kynurenine. The vertical axis of the graph represents the normalized value of the amount of 6RNMSC1-2_F02 binding in the presence of 100 μmol / L kynurenine, and the horizontal axis represents the time (seconds) elapsed since 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. [Figure 33] This graph shows the response to IL-6R immobilized on a CM5 sensor chip after 180 seconds of interaction with 5 μg / L of 6RNMSC1-2_F02 as an analyte. 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. [Figure 34] Figure 1 shows antibody binding to membrane-type human IL-6R assessed by FCM. The upper panel shows the results in the presence of Kyunurenine, and the lower panel shows the results in the absence of Kyunurenine. The horizontal axis shows fluorescence intensity, and the vertical axis shows cell number. [Figure 35A]

[0033] Figure 1 shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. Figure 2 shows the ADCC activity of clone 6RNMSC1-2_F02, which binds to hIL-6R in the presence of kynurenine, against BaF cells expressing hIL-6R in the presence (triangles) or absence (circles) of kynurenine. White indicates individual measurements, and black indicates the average. [Figure 35B]Figure 1 shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. Figure 2 shows the ADCC activity of an MRA that binds to hIL-6R regardless of the presence or absence of kynurenine against BaF cells expressing hIL-6R in the presence (triangles) or absence (circles) of kynurenine. White indicates individual measurements, and black indicates the average. [Figure 36] This figure shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. This figure shows the ADCC activity of clone 6RNMSC1-2_F02 against hIL-6R-expressing BaF cells in the presence (triangles) or absence (circles) of clone 6RNMSC1-2_F02, which binds to hIL-6R in the presence of kynurenine. The horizontal axis represents kynurenine concentration, and the vertical axis represents ADCC activity (%). ADCC activity is expressed as the mean and standard deviation. [Figure 37] Figure 1 shows the results of ELISA for the binding of clone 6RNMSC1-2_F02 to human IL-6R in mouse serum. The vertical axis represents absorbance values ​​used to evaluate the binding activity of the antibody to human IL-6R in the presence or absence of Kyunurenine. [Figure 38] This figure shows the results of ELISA of clone I6RLSA1-6_011, obtained from a rationally designed antibody library, against human IL-6 in the presence or absence of ATP and 10 mM adenosine. The vertical axis represents absorbance values ​​used to evaluate the antibody's binding activity to human IL-6. The positive control shows the results obtained using a clone obtained from a rationally designed antibody library that exhibits binding activity to human IL-6, regardless of the presence or absence of a small molecule. The negative control shows the results obtained using M13KO7 Helper Phage. [Figure 39]This figure shows the results of ELISA of clones 6RRLSA1-6_037 and 6RRLSA1-6_045 obtained from a rationally designed antibody library against human IL-6 receptor in the presence or absence of ATP and 10 mM adenosine. The vertical axis represents absorbance values ​​used to evaluate the binding activity of the antibodies to human IL-6 receptor. The negative control shows the results when M13KO7 Helper Phage was used. [Figure 40]

[0039] Figure 1 shows the results of ELISA for 96 clones obtained from a rationally designed antibody library after four rounds of panning using multivalent antibody phage display against human IgA-Fc. The vertical axis shows absorbance values ​​in the absence and presence of ATP and adenosine, respectively, which evaluated the binding activity of the antibodies to human IgA-Fc. [Figure 41]

[0039] Figure 1 shows the results of ELISA for 96 clones obtained from a rationally designed antibody library after four rounds of panning using monovalent antibody phage display against human IgA-Fc. The vertical axis shows absorbance values ​​in the absence and presence of ATP and adenosine, respectively, which evaluated the binding activity of the antibodies to human IgA-Fc. [Figure 42] This figure shows the results of ELISA of clone IADL3C5-4_048 obtained from a rationally designed antibody library against human IgA-Fc in the presence or absence of ATP and 1 mM adenosine. The vertical axis represents absorbance values ​​used to evaluate the antibody's binding activity to human IgA-Fc. The positive control shows the results obtained using a clone obtained from a rationally designed antibody library that exhibits binding activity to human IgA-Fc, regardless of the presence or absence of a small molecule. The negative control shows the results obtained using M13KO7 Helper Phage. [Figure 43]This is a graph showing the amount of binding (binding response (RU)) when 1 μM of each clone was allowed to interact with IL-6R immobilized on a CM5 sensor chip for 120 seconds in the presence or absence of 1 mM of each small molecule. [Figure 44A]

[0033] Figure 1 shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. Figure 2 shows the ADCC activity of clone 6RAD2C1-4_030, which binds to hIL-6R in the presence of ATP, against CHO cells expressing hIL-6R in the presence (triangles) or absence (circles) of ATP. White indicates individual measurements, and black indicates the average. [Figure 44B]

[0033] Figure 1 shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. Figure 2 shows the ADCC activity of clone 6RAD2C1-4_011, which binds to hIL-6R in the presence of ATP, against CHO cells expressing hIL-6R in the presence (triangles) or absence (circles) of ATP. White indicates individual measurements, and black indicates the average. [Figure 44C] Figure 1 shows the ADCC activity of an antibody that binds to an antigen in the presence of a small molecule against cells expressing the antigen. Figure 2 shows the ADCC activity of an MRA that binds to hIL-6R regardless of the presence or absence of ATP against CHO cells expressing hIL-6R in the presence (triangles) or absence (circles) of ATP. White indicates individual measurements, and black indicates the average. [Figure 45] This figure shows the results of ELISA of clone HSADSA1-6_020, obtained from a rationally designed antibody library, against HSA in the presence or absence of ATP and 10 mM adenosine. The vertical axis represents absorbance values ​​used to evaluate the antibody's binding activity to HSA. The positive control shows the results when a clone obtained from a rationally designed antibody library that exhibits binding activity to HSA, regardless of the presence or absence of small molecules, was used. The negative control shows the results when M13KO7 Helper Phage was used. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following definitions and detailed description are provided to facilitate understanding of the invention described herein. amino acid As used herein, amino acids are represented by one-letter or three-letter codes, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V.

[0017] Amino acid modification To modify amino acids in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), a type of stop codon, can also be suitably used.

[0018] As used herein, the meaning of the term "and / or" used to describe the site of amino acid modification includes any combination of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following amino acid modification variations: (a) 33rd place, (b) 55th place, (c) 96th place, (d) 33rd and 55th place, (e) 33rd and 96th place, (f) 55th and 96th place, (g) 33rd, 55th and 96th place.

[0019] Herein, expressions for amino acid modifications may be appropriately expressed by listing the one-letter or three-letter codes for the amino acid before and after the modification before and after a number representing a specific position. For example, the modification N100bL or Asn100bLeu, used when substituting an amino acid contained in an antibody variable region, represents a substitution of Asn at position 100b according to the Kabat numbering system with Leu. That is, the number represents the amino acid position according to the Kabat numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution. Similarly, the modification P238D or Pro238Asp, used when substituting an amino acid in the Fc region contained in an antibody constant region, represents a substitution of Pro at position 238 according to the EU numbering system with Asp. That is, the number indicates the position of the amino acid as expressed in EU numbering, the one-letter or three-letter code of the amino acid written before it indicates the amino acid before substitution, and the one-letter or three-letter code of the amino acid written after it indicates the amino acid after substitution.

[0020] antigen As used herein, the term "antigen" is not limited to a specific structure as long as it contains an epitope to which an antigen-binding domain binds. In another sense, an antigen can be inorganic or organic. Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalcohol In, 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), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 , IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor receptor 1 (IGFR), insulin-like growth factor receptor 2 (IGFR), insulin-like growth factor receptor 3 (IGFR), insulin-like growth factor receptor 4 (IGFR), insulin-like growth factor receptor 5 (IGFR), insulin-like growth factor receptor 6 (IGFR), insulin-like growth factor receptor 7 (IGFR), insulin-like growth factor receptor 8 (IGFR), insulin-like growth factor receptor 9 (IGFR), insulin-like growth factor receptor 1 ... Factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor)receptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, 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 antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, 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, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric 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, factorExamples of antigens include 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 preferably are those expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues or inflammatory tissues.

[0021] Although receptors are also exemplified as antigens above, even when these receptors exist in a soluble form in biological fluids, they can be used as antigens to which antigen-binding molecules containing an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound of the present invention bind. A non-limiting example of such a soluble receptor is a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968).

[0022] Examples of the antigen include membrane-type molecules expressed on the cell membrane and soluble molecules secreted extracellularly from cells. When an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound of the present invention binds to a soluble molecule secreted from cells, the antigen-binding molecule preferably has neutralizing activity, as described below.

[0023] There is no limitation on the solution in which a soluble molecule exists, and the soluble molecule can exist in biological fluids, i.e., all fluids filling the vessels or the spaces between tissues and cells in a living body. In a non-limiting embodiment, the soluble molecule bound by an antigen-binding molecule of the present invention can exist in extracellular fluid. In vertebrates, extracellular fluid refers collectively to components in bone and cartilage, such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, spinal fluid, aspirate, or synovial fluid, as well as transcellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activity of cells, and fluids in the digestive tract and other body cavities), such as alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor).

[0024] When an antigen-binding molecule of the present invention containing an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound binds to a membrane-type molecule expressed on a cell membrane, preferred examples of the antigen-binding molecule include antigen-binding molecules that have cytotoxic activity or that bind or have the ability to bind to a cytotoxic substance, as described below. Furthermore, preferred, non-limiting examples of antigen-binding molecules include antigen-binding molecules that have neutralizing activity instead of or in addition to the property of having cytotoxic activity or the property of binding or having the ability to bind to a cytotoxic substance.

[0025] epitope An epitope, meaning an antigenic determinant present in an antigen, refers to a site on an antigen to which an antigen-binding domain in an antigen-binding molecule disclosed herein binds. Thus, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of an antigen-binding molecule that recognizes the epitope to the antigen. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute the epitope. Furthermore, when the epitope is a sugar chain, the epitope can also be identified by a specific sugar chain structure.

[0026] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope, typically comprising at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0027] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids comprising the epitope is not the single, defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may encompass an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0028] The structure of the antigen-binding domain that binds to the epitope is called the paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between them. The binding strength between this epitope and paratope is called affinity. The sum of the binding strengths when multiple antigens bind to multiple antigen-binding molecules is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding strengths act synergistically, so avidity is higher than affinity.

[0029] Binding activity Methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for IL-6R are exemplified below; however, methods for confirming epitope binding by test antigen-binding molecules containing antigen-binding domains for antigens other than IL-6R can also be appropriately carried out in accordance with the examples below.

[0030] For example, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region of IL-6R cDNA encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to the test antigen-binding molecule containing the IL-6R antigen-binding domain is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide of binding of the antigen-binding molecule to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

[0031] Furthermore, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a conformational epitope can be confirmed as follows. For this purpose, IL-6R-expressing cells are prepared. Examples of such confirmation include when a test antigen-binding molecule containing an IL-6R antigen-binding domain binds strongly to IL-6R-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence forming the extracellular domain of immobilized IL-6R. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity toward human IL-6R-expressing cells.

[0032] Methods for measuring the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) using IL-6R-expressing cells as antigens.

[0033] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody-binding titer toward IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule toward IL-6R-expressing cells to be compared.

[0034] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0035] For example, one suitable method for measuring the antigen-binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain is as follows: First, the test antigen-binding molecule is reacted with cells expressing IL-6R and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the desired concentration. For example, the antigen-binding molecule can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, the Geometric Mean value allows the binding activity of the test antigen-binding molecule, represented by the amount of binding of the test antigen-binding molecule, to be measured.

[0036] Whether a test antigen-binding molecule containing an IL-6R antigen-binding domain shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0037] Specifically, in a cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to wells coated with IL-6R protein.

[0038] The amount of test antigen-binding molecules bound to the wells via the IL-6R protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0039] If a competitor antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of a candidate competitor antigen-binding molecule, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competitor antigen-binding molecule or competes for binding to the same epitope.

[0040] When the structure of the epitope to which a test antigen-binding molecule containing an IL-6R antigen-binding domain binds has been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0041] For example, such binding activity can be measured by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. Alternatively, binding activity to the mutant peptide bound to a column can be measured by flowing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0042] Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with a suitable buffer to the desired concentration and used. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured.

[0043] In this method, "substantially no binding to mutant IL-6R-expressing cells" can be determined, for example, by the following method. First, test and control antigen-binding molecules bound to mutant IL-6R-expressing cells are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the polypeptide complex according to Formula 1 below.

[0044] (Formula 1) ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0045] The Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant IL-6R-expressing cells, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. In this case, it is particularly preferred that the test antigen-binding molecules used to determine the ΔGeo-Mean comparison values ​​for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. An antigen-binding molecule previously confirmed to recognize an epitope in IL-6R is used as a control antigen-binding molecule.

[0046] A test antigen-binding molecule is deemed to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding molecule for the mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding molecule for the IL-6R-expressing cells. The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be determined to be identical.

[0047] target tissue As used herein, the term "target tissue" refers to a tissue containing cells containing an antigen to which an antigen-binding molecule of the present invention binds in a compound-dependent manner, and in which binding of the antigen-binding molecule to a membrane-type molecule expressed in the cells or to a soluble molecule present in the tissue results in a positive pharmacological effect on the living body containing the tissue. In this case, "positive pharmacological effect" refers to an effect that reduces, alleviates, ameliorates, or cures symptoms caused by a pathological site containing the target tissue in the living body containing the tissue. Non-limiting examples of mechanisms that result in such pharmacological effects include, for example, cytotoxic activity and growth inhibition against cancer cells, and immune activation in cancer tissues in the case of symptoms caused by malignant tumors such as cancer. Non-limiting examples of such mechanisms include, for example, blocking activity of inflammatory cytokines in inflammatory tissues and immunosuppression in the case of inflammatory diseases.

[0048] Cancer tissue specific compound As used herein, the term "cancer tissue-specific compound" refers to a compound that is differentially present in cancer tissue compared to non-cancerous tissue. The term "cancer" is used herein generally to refer to malignant neoplasms, which may be metastatic or non-metastatic. Non-limiting examples of carcinomas originating from epithelial tissues such as the digestive tract and skin include brain tumors, skin cancer, head and neck cancer, esophageal cancer, lung cancer, stomach cancer, duodenal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, pancreatic cancer, liver cancer, colorectal cancer, colon cancer, bladder cancer, and ovarian cancer. Non-limiting examples of sarcomas originating from non-epithelial tissues (stroma) such as muscle include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Further, non-limiting examples of hematopoietic cancers include malignant lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; leukemias, including acute or chronic myelocytic leukemia, and acute or chronic lymphatic leukemia; and multiple myeloma. The term "neoplasm," as used broadly herein, refers to any newly formed pathological tissue tumor. In the present invention, a neoplasm results in the formation of a tumor, which is characterized in part by angiogenesis. Neoplasms can be benign, such as hemangiomas, gliomas, and teratomas, or malignant, such as carcinomas, sarcomas, gliomas, astrocytomas, neuroblastomas, and retinoblastomas.

[0049] The term "cancer tissue" refers to tissue containing at least one cancer cell. Thus, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. As used herein, a tumor mass refers to a foci of tumor tissue. The term "tumor" is generally used to refer to benign or malignant neoplasms.

[0050] For example, in some embodiments, a cancer tissue-specific compound can be a compound defined by qualitative cancer tissue specificity, such as being present in cancer tissue but not in non-cancerous tissue, or being absent in cancer tissue but present in non-cancerous tissue. In another embodiment, a cancer tissue-specific compound can be a compound defined by quantitative cancer tissue specificity, such as being present at a different concentration (e.g., higher or lower concentration) in cancer tissue compared to non-cancerous tissue. For example, a cancer tissue-specific compound can be differentially present at a given concentration. However, in general, cancer tissue-specific compounds will exhibit a cytotoxic effect of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 5-fold, at least 10 ... 3 times at least 10 4 times at least 10 5 times at least 10 6The cancer tissue-specific compound may be present at a concentration that is 50% or more times greater than the concentration of the non-cancer tissue, increasing to infinity (i.e., absent in non-cancer tissues), or generally at a concentration that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% (i.e., representing absence). The cancer tissue-specific compound is preferably differentially present at a statistically significant concentration (i.e., as determined using either Welch's t-test or Wilcoxon's rank sum test, p-value is less than 0.05 and / or q-value is less than 0.10). Non-limiting examples of cancer tissue-specific compounds include compounds that are cancer tissue-specific metabolites (cancer tissue-specific metabolites; cancer cell-specific metabolites, immune cell-specific metabolites infiltrating cancer tissue, cancer stromal cell-specific metabolites) produced by metabolic activity specific to cancer cells, immune cells, and stromal cells contained in cancer tissue, such as the following:

[0051] Cancer tissue-specific metabolites The term "metabolism" refers to chemical changes that occur within the tissues of an organism, and includes "anabolism" and "catabolism." Anabolism refers to the biosynthesis or accumulation of molecules, while catabolism refers to the breakdown of molecules. A "metabolite" is an intermediate or product resulting from metabolism. A "primary metabolite" refers to a metabolic product that is directly involved in the growth or reproduction process of a cell or organism, while a "secondary metabolite" refers to a product, such as an antibiotic or pigment, that results from metabolism that biosynthesizes a substance that is not directly involved in the growth or reproduction process or is not directly involved in the life processes common to cells or organisms. A metabolite can be a metabolic product of a "biopolymer" or a "small molecule." A "biopolymer" is a macromolecule composed of one or more types of repeating units. Biopolymers are generally found in biological systems and include molecules with a molecular weight of approximately 5,000 or greater that form structures such as cells that organize organisms and the intercellular and intertissue matrices attached to them, particularly polysaccharides (carbohydrates, etc.), peptides (this term is used to include polypeptides and proteins), polynucleotides, and analogs thereof, such as compounds composed of or containing amino acid analogs or non-amino acid groups. "Small molecules" refer to naturally occurring chemical substances other than "biopolymers" present in living organisms. In one non-limiting embodiment, cancer tissue-specific metabolites described herein preferably include cancer cell-specific small molecule metabolites (Eva Gottfried, Katrin Peter, and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132). Furthermore, metabolites highly produced by immune cells infiltrating cancer tissues and metabolites highly produced by stromal cells (cancer stromal cells or cancer stromal fibroblasts (CAFs)) that support the survival and / or growth of cancer cells are also included. Examples of infiltrating immune cells include dendritic cells, suppressive dendritic cells, suppressive T cells, exhausted T cells, and myeloma-derived suppressor cells (MDSCs).The metabolites of the present invention also include compounds that are released from inside to outside the cells when cells present in cancer tissues (cancer cells, immune cells, stromal cells) die due to apoptosis, necrosis, or the like.

[0052] To identify cancer cell-specific metabolites, analyses at the transcriptome level (e.g., Dhanasekaran et al. (Nature (2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. USA (2004) 101, 811-816, or Perou et al. (Nature (2000) 406, 747-752) are exemplified) or at the proteome level (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15, Hood et al. (Mol. Cell. Proteomics (2005) 4, In addition to the conventional methods (e.g., 1741-1753), metabolic (metabolomic) analysis, centered on metabolic profiling, may be used as appropriate. That is, metabolic profiling may be performed using high-pressure liquid chromatography (HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol. Recognit. (1997) 10, 182-187), mass spectrometry (Gates and Sweeley (Clin. Chem. (1978) 24, 1663-1673) (GC / MS and LC / MS)), ELISA, or the like, alone or in combination, to identify metabolites in a test sample.

[0053] These studies have revealed intratumor heterogeneity, which is structured by altered concentration gradients of metabolites (e.g., glucose or oxygen) and growth factors that enable cancer cells to grow under conditions of low oxygen tension (Dang and Semenza (Trends Biochem. Sci. (1999) 24, 68-72)). These studies also use cell line models to understand changes in energy utilization pathways due to different degrees of tumor malignancy (Vizan et al. (Cancer Res. (2005) 65, 5512-5515)). Non-limiting examples of technical components of the metabolomics platform include sample extraction, separation, detection, spectroscopic analysis, data normalization, delineation of class-specific metabolites, pathway mapping, confirmation, and functional characterization of candidate metabolites, as described in Lawton et al. (Pharmacogenomics (2008) 9, 383). These methods make it possible to identify cancer cell-specific metabolites in desired cancer tissues.

[0054] A non-limiting embodiment of the cancer tissue-specific compound or cancer tissue-specific metabolite used in the present invention is preferably at least one compound selected from the following compounds. "At least one compound" means that the antigen-binding activity of the same antigen-binding domain described below is dependent on one type of cancer tissue-specific compound or cancer tissue-specific metabolite, as well as on multiple types of cancer tissue-specific compounds or cancer tissue-specific metabolites.

[0055] (1) Primary metabolic products of glycolysis or the Krebs cycle, such as lactate, succinate, and citrate A non-limiting embodiment of the cancer tissue-specific compound, particularly the cancer cell-specific metabolite, used in the present invention preferably includes primary metabolites produced as a result of glucose metabolism, such as lactate, succinate, and citrate, which are present at higher concentrations in cancer tissues than in surrounding non-cancerous tissues. A glycolytic phenotype characterized by the upregulation of glycolytic (Embden-Myerhof pathway) enzymes such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH) has long been known as the Warburg effect, a characteristic of solid tumors.

[0056] In other words, it is believed that tumor cells have high expression of the M2 isoform of pyruvate kinase, which is required for glycolysis under anaerobic conditions, rather than the M1 isoform, and this expression is beneficial for tumor cell growth in vivo (Christofk et al. (Nature (2008) 452, 230-233)). Pyruvate produced by pyruvate kinase is feedback inhibited by lactate, which is produced as a result of an equilibrium reaction with lactate dehydrogenase (LDH) under anaerobic conditions. This feedback inhibition promotes mitochondrial respiration (Krebs cycle) and inhibits cell proliferation, so upregulation of LDH, hexokinase, and glucose transporters (GLUT) is said to play an important role in tumor cell proliferation (Fantin et al. (Cancer Cell (2006) 9, 425-434)). Glucose is metabolized via glycolysis, and its final metabolic product, lactic acid, is co-transported with protons around the tumor, resulting in an acidic pH in the tissue surrounding the tumor. Lactic acid, the final product of glycolysis, and succinic acid and citric acid, which are produced by enhanced mitochondrial respiration, are known to accumulate in cancer tissue (Teresa et al. (Mol. Cancer (2009) 8, 41-59)). Non-limiting examples of cancer tissue-specific compounds, particularly cancer cell-specific metabolites, used in the present invention include primary metabolites produced by glycolytic metabolism, such as lactic acid, succinic acid, and citric acid. It is also known that succinic acid, which is present at high concentrations within cells, leaks out of the cells upon cell death (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is thought that succinic acid concentrations are elevated in cancer tissues where cell death occurs frequently.

[0057] (2) Amino acids such as alanine, glutamic acid, and aspartic acid In addition to the glucose metabolism described above, tumor cells, which require a continuous supply of essential and non-essential amino acids necessary for the biosynthesis of biopolymers under anaerobic conditions, are also known to exhibit altered amino acid metabolism. Glutamine, the most widely distributed amino acid in the body, acts as a nitrogen carrier containing two nitrogen atoms in its side chain. Tumor cells with an increased rate of glutamine uptake into cells are said to function as glutamine traps. This increased uptake of glutamine and its conversion to glutamate and lactate is called "glutaminolysis" and is believed to be a characteristic of transformed (tumor) cells (Mazurek and Eigenbrodt, Anticancer Res. (2003) 23, 1149-1154, and Mazurek et al., J. Cell. Physiol. (1999) 181, 136-146). As a result, cancer patients show decreased levels of glutamine in plasma while increasing glutamate concentrations (Droge et al., Immunobiology (1987) 174, 473-479). Furthermore, in lung cancer tissue, 13 by metabolic studies of C radiolabeled glucose 13 C-labeled succinic acid, 13 C-labeled alanine, 13 C-labeled glutamic acid, and 13 A correlation was observed between the concentrations of C-labeled citric acid. Non-limiting examples of cancer tissue-specific compounds used in the present invention include alanine, glutamic acid, aspartic acid, and the like, which accumulate to high concentrations in cancer tissues through glutaminolysis or the like.

[0058] (3) Metabolites of amino acids such as kynurenine Indoleamine 2,3-dioxygenase (IDO) is a tryptophan metabolic enzyme that is highly expressed in many cancers, including melanoma, colon cancer, and kidney cancer (Uyttenhove et al. (Nat. Med. (2003) 9, 1269-127)). Two isoforms are known to exist (Lob et al. (CancerImmunol. Immunother. (2009) 58, 153-157)). IDO catalyzes the conversion of tryptophan to kynurenine (represented by Chemical Formula 1) and is the first enzyme in the de novo pathway of nicotinamide nucleotide (NAD). In gliomas that do not express IDO, kynurenine is produced from tryptophan by hepatic tryptophan 2,3-dioxygenase (TDO) (Opitz et al. (Nature (2011) 478, 7368, 197-203)). IDO is also expressed in dendritic cells infiltrating cancer tissue, and these cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). IDO is also expressed in myeloid-derived suppressor cells (MDSCs) in cancer tissue, and these MDSCs also produce kynurenine (Yu et al. (J. Immunol. (2013) 190, 3783-3797)).

[0059] [ka]

[0060] Kynurenine is known to suppress allogeneic T cell responses (Frumento et al. (J. Exp. Med. (2002) 196, 459-468)). This suppression allows tumor cells to evade anti-tumor immune responses, and it has been proposed that glioma cell proliferation is promoted through an autocrine growth mechanism in which kynurenine acts as an endogenous ligand for the aryl hydrocarbon receptor expressed in gliomas (Opitz et al. (cited above)). Kynurenine is converted to anthranilic acid (represented by [Chemical Formula 2]) by kynurenidase and to 3-hydroxykynurenine (represented by [Chemical Formula 3]) by kynurenine 3-hydroxylase. Both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, a precursor of NAD.

[0061] [ka]

[0062] [ka]

[0063] Kynurenine is converted to kynurenic acid (represented by Chemical Formula 4) by kynurenine aminotransferase. A non-limiting embodiment of the cancer tissue-specific compound, particularly the cancer cell-specific metabolite, used in the present invention preferably includes kynurenine and its metabolites, such as amino acid metabolites such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid.

[0064] [ka]

[0065] (4) Arachidonic acid metabolites such as prostaglandin E2 Prostaglandin E2 (PGE2) ([Chemical Formula 5]) is a metabolite of arachidonic acid called a prostonoid, which includes prostaglandins and thromboxanes, synthesized by cyclooxygenase (COX)-1 / 2 (Warner and Mitchell, FASEB J. (2004) 18, 790-804). PGE2 promotes the proliferation of colon cancer cells and inhibits their apoptosis (Sheng et al., Cancer Res. (1998) 58, 362-366). It is known that the expression of cyclooxygenases is altered in many cancer cells. Specifically, COX-1 is constitutively expressed in almost all tissues, whereas COX-2 is primarily found in tumors, where it is induced by certain inflammatory cytokines and oncogenes (Warner and Mitchell, supra). It has also been reported that overexpression of COX-2 is associated with poor prognosis in breast cancer (Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)) and rapid disease progression in ovarian cancer (Denker et al. (Mod. Pathol. (2006) 19, 1261-1269)). Furthermore, suppressor T cells infiltrating cancer tissue also produce prostaglandin E2 (Curr. Med. Chem. (2011) 18, 5217-5223). Small molecules such as prostaglandins and leukotrienes, which are metabolites of arachidonic acid, are known to act as stimulatory factors that control the autocrine and / or paracrine growth of cancer (Nat. Rev. Cancer (2012) 12 (11)). 782-792). A non-limiting example of the cancer tissue-specific compounds used in the present invention, particularly cancer cell-specific metabolites and immune cell-specific metabolites infiltrating cancer tissue, is preferably an arachidonic acid metabolite such as prostaglandin E2. In addition to prostaglandin E2, thromboxane A2 (TXA2) is also produced at elevated levels in cancer tissues such as colon cancer (J. Lab. Clin. Med. (1993) 122, 518-523), and is a non-limiting example of the arachidonic acid metabolite of the present invention.

[0066]

change

[0067] (5) Nucleosides containing a purine ring structure, such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) It is known that when cancer cells die, a large amount of ATP leaks out of the cell. Therefore, the ATP concentration in cancer tissue is significantly higher than in normal tissue (PLoS One. (2008) 3, e2599). Several types of cells release adenine nucleotides in the form of ATP, ADP, and AMP. Adenosine is metabolized by cell surface extracellular enzymes such as eco-5'-nucleotidase (CD73) (Resta and Thompson (Immunol. Rev. (1998) 161, 95-109) and Sadej et al. (Melanoma Res. (2006) 16, 213-222). Adenosine is a purine nucleoside that is constitutively present in the extracellular environment at low concentrations, but a marked increase in extracellular adenosine concentrations has been reported in hypoxic tissues found in solid tumors (Blay and Hoskin (Cancer Res. (1997) 57, 2602-2605)). CD73 is expressed on the surface of tumor and immune cells (Kobie et al. (J. Immunol. (2006) 177, 6780-6786) and breast cancer (Canbolat et al. (Breast Cancer Res. Treat. Increased adenosine activity has been found in gastric cancer (Durak et al. (Cancer Lett. (1994) 84, 199-202)), pancreatic cancer (Flocke and Mannherz (Biochim. Biophys. Acta (1991) 1076, 273-281)), and glioblastoma (Bardot et al. (Br. J. Cancer (1994) 70, 212-218)). It has been proposed that the accumulation of adenosine in cancer tissues may be due to increased intracellular adenosine production resulting from the dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick and Willis (Biochem. J. (1989) 261, 541-550)).Furthermore, regulatory T cells infiltrating cancer tissues also express ATPase and produce adenosine (Proc. Natl. Acad. Sci. (2006) 103 (35), 13132-13137, Curr. Med. Chem. (2011) 18, 5217-5223). The produced adenosine is thought to create an immunosuppressive environment in cancer tissues via adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011), 18, 5217-23). ​​Non-limiting examples of cancer tissue-specific compounds used in the present invention include ATP, ADP, AMP, adenosine, and the like, which accumulate at high concentrations in cancer tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in the accumulation of high concentrations of inosine.

[0068] (6)Uric acid Uric acid is a product of the metabolic pathway of purine nucleosides in vivo and is released extracellularly, such as into the blood or interstitial space. Recently, it has been revealed that uric acid is released from dead cells present at lesion sites, such as cancer tissues (Nat. Med. (2007) 13, 851-856). A preferred, non-limiting example of a cancer tissue-specific compound used in the present invention is uric acid, which accumulates at high concentrations in cancer tissues due to the metabolism of purine nucleotides such as ATP.

[0069] (7) 1-Methylnicotinamide The enzyme nicotinamide N-methyltransferase is known to be overexpressed in several human cancer tissues. This enzyme converts nicotinamide to 1-methylnicotinamide, a stable metabolite, consuming the methyl group of the methyl donor S-adenosylmethionine (SAM). It has been proposed that overexpression of nicotinamide N-methyltransferase contributes to tumorigenesis through a mechanism that impairs DNA methylation ability due to a decrease in SAM concentration in cancer cells (Ulanovskaya et al. (Nat. Chem. Biol. (2013) 9 (5) 300-306)). 1-methylnicotinamide, a stable metabolic product of this enzyme, is known to be secreted extracellularly by cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). A non-limiting example of the cancer tissue-specific compound used in the present invention is 1-methylnicotinamide, which accumulates at high concentrations in cancer tissues through the metabolism of nicotinamide.

[0070] Inflamed tissue-specific compounds As used herein, the term "inflammatory tissue-specific compound" refers to a compound that is differentially present in inflamed tissue compared to non-inflamed tissue. Joints in rheumatoid arthritis and osteoarthritis Lungs (alveoli) in bronchial asthma and COPD Digestive system in inflammatory bowel disease, Crohn's disease, and ulcerative colitis Fibrotic tissue in liver, kidney, and lung fibrosis Tissues undergoing rejection in organ transplants Blood vessels and heart (myocardium) in arteriosclerosis and heart failure Visceral fat in metabolic syndrome Skin tissue in atopic dermatitis and other dermatitis Spinal nerves in herniated discs and chronic lower back pain Suitable examples include:

[0071] Inflammatory tissue-specific metabolites Inflammatory tissue-specific metabolites are metabolites that are produced at high levels by immune cells infiltrating inflammatory tissues, and metabolites that are produced at high levels specifically by normal cells damaged in inflammatory tissues. Examples of infiltrating immune cells include effector T cells, mature dendritic cells, neutrophils, granulocytes (mast cells), and basophils. Metabolites in the present invention also include compounds that are released from inside to outside the cells when cells (immune cells, normal cells) present in inflammatory tissues die due to apoptosis, necrosis, or the like.

[0072] A non-limiting embodiment of the inflammatory tissue-specific compound or inflammatory tissue-specific metabolite used in the present invention is preferably at least one compound selected from the following compounds. "At least one compound" means that the antigen-binding activity of the same antigen-binding domain described below is dependent on one type of inflammatory tissue-specific compound or inflammatory tissue-specific metabolite, as well as on multiple types of inflammatory tissue-specific compounds or inflammatory tissue-specific metabolites.

[0073] (1) Metabolites of arachidonic acid such as prostaglandin E2 It is known that PGE2 concentrations are high in rheumatoid arthritis and osteoarthritis (Eur. J. Clin. Pharmacol. (1994) 46, 3-7; Clin. Exp. Rheumatol. (1999) 17, 151-160; Am. J. Vet. Res. (2004) 65, 1269-1275). Suitable examples of inflammatory tissue-specific compounds used in the present invention, particularly inflammatory cell-specific metabolites and immune cell-specific metabolites that infiltrate into inflamed tissues, include arachidonic acid metabolites such as prostaglandin E2.

[0074] (2) Nucleosides with a purine ring structure, such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) It is known that ATP concentrations are high in alveoli where inflammation due to bronchial asthma is occurring (Nat. Med. (2007) 13, 913-919). It is also known that ATP concentrations are high in alveoli where inflammation due to COPD is occurring (Am. J. Respir. Crit. Care Med. (2010) 181, 928-934). Furthermore, it has been observed that adenosine concentrations are high in the synovial fluid of patients with rheumatoid arthritis (Journal of Pharmaceutical and Biomedical Analysis (2004) 36 877-882). Furthermore, it is known that ATP concentrations are high in tissues where rejection is occurring due to GVHD (Nat. Med. (2010) 16, 1434-1438). It is also known that adenosine concentrations are elevated in fibrotic tissues in the lungs, liver, and kidneys (FASEB J. (2008) 22, 2263-2272, J. Immunol. (2006) 176, 4449-4458, J. Am. Soc. Nephrol. (2011) 22 (5), 890-901, PLoS ONE J. (2010) 5 (2), e9242). It has also been observed that ATP concentrations are elevated in fibrotic tissues of patients with pulmonary fibrosis (Am. J. Respir. Crit. Care Med. (2010) 182, 774-783). Non-limiting examples of inflammatory tissue-specific compounds used in the present invention include ATP, ADP, AMP, adenosine, and the like, which accumulate at high concentrations in inflamed tissues through the metabolism of purine nucleotides such as ATP. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in the accumulation of high concentrations of inosine.

[0075] (3)Uric acid Uric acid is a product of the metabolic pathway of purine nucleosides in vivo and is released extracellularly, such as into the blood or interstitial space. Recently, it has been revealed that uric acid released from cells undergoing necrosis promotes inflammatory responses (J. Clin. Invest. (2010) 120 (6), 1939-1949). A preferred, non-limiting example of the inflamed tissue-specific compound used in the present invention is uric acid, which accumulates at high concentrations in inflamed tissues due to the metabolism of purine nucleotides such as ATP.

[0076] antigen-binding domain As used herein, the "antigen-binding domain" may be any domain with any structure as long as it binds to the target antigen. Examples of such domains include the variable regions of the heavy and light chains of antibodies, a module called an A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectin (International Publication No. WO2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes, Affibody (International Publication No. WO1995 / 001937) using an IgG binding domain consisting of a 58-amino acid three-helix bundle of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeat (AR)) which are regions exposed on the molecular surface of ankyrin repeats (AR) with a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Preferred examples of the antigen-binding domain of the present invention include an anticalin molecule, which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center, found in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish, are repeatedly stacked (International Publication WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present invention include antigen-binding domains comprising the variable regions of the heavy and light chains of antibodies.Suitable examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0077] The antigen-binding domains in the antigen-binding molecules of the present invention can bind to the same epitope. Here, the same epitope can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, the antigen-binding domains in the antigen-binding molecules of the present invention can bind to different epitopes. Here, the different epitopes can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0078] specific "Specific" refers to a state in which one of the specifically binding molecules does not substantially bind to any molecules other than the one or more other molecules to which it binds. This term is also used when the antigen-binding domain is specific for a specific epitope among multiple epitopes contained in a certain antigen. Furthermore, when the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitopes. Here, "not substantially binding" is determined according to the method described in the above section on binding activity, and refers to the binding activity of the specific binding molecule for molecules other than the other molecule being 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity for the other molecule.

[0079] Cytotoxic activity In one non-limiting aspect, the present invention provides an antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a cancer tissue-specific compound and has cytotoxic activity against cells that express a membrane-type molecule on their cell membrane, as well as a pharmaceutical composition comprising the antigen-binding molecule as an active ingredient. In the present invention, cytotoxic activity includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T cell-mediated cytotoxicity. In the present invention, CDC activity refers to cytotoxic activity mediated by the complement system. Meanwhile, ADCC activity refers to the activity of immune cells, etc., binding to the Fc region of an antigen-binding molecule that contains an antigen-binding domain that binds to a membrane-type molecule expressed on the cell membrane of target cells via Fcγ receptors expressed on the immune cells, causing the immune cells to inflict damage on the target cells. Whether or not 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, edited by Coligan et al. (1993)).

[0080] Specifically, first, effector cells, a complement solution, and target cells are prepared. (1) Preparation of effector cells Spleen cells are isolated from spleens removed from CBA / N mice or other mice in RPMI1640 medium (Invitrogen). The spleen cells are washed with the same medium containing 10% fetal bovine serum (FBS, HyClone) and then diluted to a concentration of 5 × 10 6 Effector cells can be prepared by adjusting the volume to 1 / mL. (2) Preparation of complement solution A complement solution can be prepared by diluting Baby Rabbit Complement (CEDARLANE) 10-fold with 10% FBS-containing medium (Invitrogen). (3) Preparation of target cells

[0081] 0.2 mCi of antigen-expressing cells 51 The target cells can be radiolabeled by culturing them with Cr-sodium chromate (GE Healthcare Biosciences) in 10% FBS-containing DMEM medium at 37°C for 1 hour. After radiolabeling, the cells were washed three times with 10% FBS-containing RPMI 1640 medium and diluted to a concentration of 2 x 10 5 The target cells can be prepared by adjusting the concentration to 1 / mL.

[0082] ADCC activity or CDC activity can be measured by the method described below. For measuring ADCC activity, 50 μl of target cells and antigen-binding molecules are added to a 96-well U-bottom plate (Becton Dickinson) and allowed to react for 15 minutes at room temperature. Then, 100 μl of effector cells are added to the plate, which is then left to stand in a carbon dioxide incubator for 4 hours. The final concentration of the antigen-binding molecule can be set to, for example, 0 or 10 μg / ml. After standing, the radioactivity of 100 μl of supernatant collected from each well is measured using a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). Cytotoxicity (%) can be calculated using the measured values ​​according to the formula: (AC) / (BC) x 100. A represents the radioactivity (cpm) in each sample, B represents the radioactivity (cpm) in the sample to which 1% NP-40 (nacalai tesque) was added, and C represents the radioactivity (cpm) in the sample containing only target cells.

[0083] On the other hand, for measuring CDC activity, 50 μl of target cells and antigen-binding molecules are added to a 96-well flat-bottom plate (Becton Dickinson) and allowed to react on ice for 15 minutes. Then, 100 μl of complement solution is added to the plate, which is then left to stand in a carbon dioxide incubator for 4 hours. The final concentration of the antigen-binding molecule can be set to, for example, 0 or 3 μg / mL. After standing, the radioactivity of 100 μl of supernatant collected from each well is measured using a gamma counter. Cytotoxic activity can be calculated in the same manner as for measuring ADCC activity.

[0084] Modified antigen-binding molecules conjugated with cytotoxic substances such as chemotherapeutic agents, toxic peptides, or radioactive chemicals, as described below, can also be suitably used as antigen-binding molecules with cytotoxic activity of the present invention. Such modified antigen-binding molecules (hereinafter referred to as "antigen-binding molecule-drug conjugates") can be obtained by chemically modifying the obtained antigen-binding molecules. Methods already established in the field of antibody-drug conjugates, etc., can be used as appropriate to modify antigen-binding molecules. Modified antigen-binding molecules conjugated with a toxic peptide can also be obtained by expressing a fusion gene in which a gene encoding the toxic peptide and a gene encoding the antigen-binding molecule of the present invention are linked in-frame in suitable host cells, followed by isolation from the culture medium of the cells.

[0085] Neutralizing activity In one non-limiting aspect, the present invention provides a pharmaceutical composition for inducing an immune response, comprising as an active ingredient an antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a cancer tissue-specific compound and has neutralizing activity against the membrane-type molecule. In another non-limiting aspect, the present invention provides a pharmaceutical composition for inducing an immune response, comprising as an active ingredient an antigen-binding molecule that contains an antigen-binding domain whose antigen-binding activity changes depending on the concentration of a cancer tissue-specific compound and has cytotoxic activity against cells expressing the membrane-type molecule on their cell membrane as well as neutralizing activity against the membrane-type molecule. Generally, neutralizing activity refers to the activity of inhibiting the biological activity of a ligand that has biological activity against cells, such as a virus or toxin. In other words, a substance with neutralizing activity refers to a substance that binds to the ligand or a receptor to which the ligand binds, and inhibits the binding of the ligand to the receptor. A receptor whose binding to the ligand is blocked by neutralizing activity can no longer exert biological activity mediated by the receptor. When the antigen-binding molecule is an antibody, an antibody with such neutralizing activity is generally called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand with that in the presence or absence of the test substance.

[0086] For example, IL-6, represented by SEQ ID NO: 27, is considered to be the primary ligand for the IL-6 receptor. The IL-6 receptor, a type I membrane protein whose amino terminus forms the extracellular domain, forms a heterotetramer with the gp130 receptor, whose dimerization is induced by IL-6 (Heinrich et al. (Biochem. J. (1998) 334, 297-314)). The formation of this heterotetramer activates Jaks associated with the gp130 receptor. Jaks autophosphorylate and phosphorylate the receptor. The phosphorylation sites on the receptor and Jaks serve as binding sites for SH2-containing Stat family molecules, such as Stat3, as well as MAP kinases, PI3 / Akt, and other SH2-containing proteins and adaptors. Stats bound to the gp130 receptor are then phosphorylated by Jaks. The phosphorylated Stats then form dimers, translocate into the nucleus, and regulate the transcription of target genes. Jak or Stat receptors can also participate in signaling cascades via other classes of receptors. Dysregulated IL-6 signaling cascades are observed in the pathology of autoimmune diseases, inflammation, and cancers such as multiple myeloma and prostate cancer. Stat3, which can act as an oncogene, is constitutively activated in many cancers. In prostate cancer and multiple myeloma, there is crosstalk between the signaling cascade from the IL-6 receptor and the signaling cascade from members of the epidermal growth factor receptor (EGFR) family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

[0087] Because intracellular signal cascades vary depending on the cell type, target molecules can be appropriately selected for each target cell type and are not limited to the factors listed above. Neutralizing activity can be evaluated by measuring the activation of in vivo signals. In addition, activation of in vivo signals can be detected using the transcriptional induction effect on target genes downstream of the in vivo signal cascade as an indicator. Changes in the transcriptional activity of target genes can be detected using the reporter assay principle. Specifically, a reporter gene such as GFP (Green Fluorescence Protein) or luciferase can be placed downstream of the transcription factor or promoter region of the target gene, and the reporter activity can be measured to determine changes in transcriptional activity as reporter activity. Commercially available kits for measuring in vivo signal activation can be used as appropriate (e.g., Mercury Pathway Profiling Luciferase System (Clontech)).

[0088] Furthermore, the neutralizing activity of antigen-binding molecules can be assessed by measuring the proliferation activity of target cells as a method for measuring the neutralizing activity of receptor ligands such as those of the EGF receptor family, which act on signal cascades that normally promote cell proliferation. For example, the following method is preferably used to assess or measure the inhibitory effect, based on 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 cell proliferation inhibitory activity can be assessed or measured in vitro by measuring the neutralizing activity of an anti-HB-EGF antibody added to the culture medium. 3A common method is to measure the uptake of [H]-labeled thymidine by viable cells as an indicator of DNA replication capacity. More convenient methods include the dye exclusion method, which measures the cell's ability to exclude dyes such as trypan blue, under a microscope, and the MTT assay. The latter method utilizes the ability of viable cells to convert the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue formazan product. Specifically, a test antibody and a ligand are added to the culture medium of test cells. 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, allowing MTT to be taken up by the cells. As a result, the yellow compound MTT is converted to a blue compound by succinate dehydrogenase in the mitochondria within the cells. This blue product is dissolved and colored, and its absorbance is measured to provide an indicator of viable cell count. In addition to MTT, commercially available reagents such as MTS, XTT, WST-1, and WST-8 (e.g., Nacalai Tesque) can be used favorably. When measuring activity, a control antibody having the same isotype as the anti-HB-EGF antibody but lacking the cell growth inhibitory activity can be used in the same manner as the anti-HB-EGF antibody, and activity can be determined by determining whether the anti-HB-EGF antibody exhibits stronger cell growth inhibitory activity than the control antibody.

[0089] Suitable cells for evaluating activity include, for example, the RMG-1 cell line, an ovarian cancer cell line whose proliferation is promoted by HB-EGF, and mouse Ba / F3 cells transformed with a vector ligated to express a gene encoding hEGFR / mG-CSFR, a fusion protein in which the extracellular domain of human EGFR and the intracellular domain of mouse G-CSF receptor are fused in-frame. Thus, those skilled in the art can appropriately select cells for evaluating activity and use them to measure the cell proliferation activity.

[0090] antibody As used herein, the term "antibody" refers to a natural or partially or completely synthetically produced immunoglobulin. Antibodies can be isolated from natural sources, such as plasma or serum, or from the culture supernatant of antibody-producing hybridoma cells. Alternatively, antibodies can be partially or completely synthesized using techniques such as genetic recombination. Examples of antibodies include immunoglobulin isotypes and their isotypic subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, the antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4. Multiple allotype sequences due to genetic polymorphisms for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these may be used in the present invention. In particular, for human IgG1 sequences, the amino acid sequence at positions 356-358 (EU numbering) may be DEL or EEM. Furthermore, for the human Igκ (Kappa) constant region and the human Igλ (Lambda) constant region, multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and either of these sequences may be used in the present invention.

[0091] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0092] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. The monoclonal antibodies of the present invention also include "humanized antibodies" and "chimeric antibodies."

[0093] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0094] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody production, can be obtained by expressing the IL-6R gene, whose nucleotide sequence is disclosed in SEQ ID NO: 2. Specifically, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), is expressed in place of the IL-6R protein represented by SEQ ID NO: 1. Purified native IL-6R protein can also be used as a sensitizing antigen.

[0095] The purified IL-6R protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also be obtained by degrading the IL-6R protein using a protease. However, the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be any sequence selected from the amino acid sequence corresponding to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids constituting the peptide used as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0096] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in International Publication Nos. WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0097] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0098] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0099] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages: -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0100] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0101] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R is confirmed in the serum, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0102] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0103] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

[0104] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

[0105] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0106] More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0107] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0108] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0109] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0110] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0111] Screening and monocloning of the desired antibody can be suitably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

[0112] To screen for hybridomas producing the monoclonal antibodies of the present invention by FACS, first, cells expressing IL-6R are prepared. Preferred cells for screening are mammalian cells overexpressing IL-6R. By using non-transformed mammalian cells as a control host cell, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, hybridomas producing IL-6R monoclonal antibodies can be obtained by selecting hybridomas producing antibodies that do not bind to host cells but bind to cells overexpressing IL-6R.

[0113] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody capable of binding to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

[0114] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

[0115] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0116] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0117] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells that produce the anti-IL-6R antibody. To do this, total RNA is usually first extracted from the hybridoma. The following methods can be used to extract mRNA from cells. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0118] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or similar. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be isolated from hybridomas. cDNA encoding antibody V regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, the SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002; Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, which will be described later, can be introduced at both ends of the cDNA.

[0119] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether or not the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0120] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0121] The resulting 5'-RACE cDNA library is used as a template for PCR amplification of antibody genes. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0122] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

[0123] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibodies can be screened using the binding activity of the reconstituted immunoglobulins to IL-6R as an indicator. For example, when the goal is to obtain antibodies against IL-6R, it is more preferable that the antibodies bind to IL-6R specifically. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0124] Methods for detecting the binding of an antibody to IL-6R-expressing cells are known. Specifically, the binding of an antibody to IL-6R-expressing cells can be detected by techniques such as the above-mentioned FACS. Fixed preparations of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of an antibody.

[0125] Panning methods using phage vectors are also suitable for screening antibodies using binding activity as an index. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be enriched.

[0126] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region. The two genes are digested with the same combination of restriction enzymes and fused in frame to construct a chimeric antibody expression vector.

[0127] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the Examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells are transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0128] For antibody gene expression, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. By co-transfecting the same host cells with vectors incorporating the H chain and L chain, antibody molecules comprising both H and L chains can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (see International Publication WO 1994 / 011523).

[0129] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. All of these expression systems can be applied to isolating the antigen-binding domains of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / 0, NS0, etc.), BHK (baby hamster kidney cell line), Hela, Vero, HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May 2001, Unit 5.9, Table 5.9.1)) (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0130] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0131] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris -Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0132] Expression systems for antibody genes using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the antibody gene of interest is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0133] In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing a fusion gene with an antibody gene inserted therein is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the goat that received the embryo produces milk from which the desired antibody can be obtained as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0134] When the antigen-binding molecules described herein are administered to humans, the antigen-binding domain of the antigen-binding molecule may be an antigen-binding domain derived from a recombinant antibody that has been artificially modified for purposes such as reducing heterologous antigenicity to humans. Examples of recombinant antibodies include humanized antibodies. These modified antibodies are produced appropriately using known methods.

[0135] The antibody variable region used to prepare the antigen-binding domain of the antigen-binding molecule described herein is typically composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are essentially the regions that determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences that make up FRs often show high identity even among antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by CDR grafting.

[0136] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which CDRs from non-human animals, such as mouse antibodies, are grafted onto human antibodies are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting mouse antibody CDRs onto human FRs. In overlap extension PCR, a nucleotide sequence encoding the mouse antibody CDR to be grafted is added to a primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting human FRs that are highly identical to the mouse FRs is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0137] The nucleotide sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform complementary strand synthesis. This reaction links the human FRs via the mouse CDR sequences.

[0138] The V region gene, in which three CDRs and four FRs are finally linked, is amplified in its entirety using primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A humanized antibody expression vector can be constructed by inserting the DNA obtained as described above and DNA encoding a human antibody C region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996 / 002576).

[0139] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select FRs of a human antibody that form a good antigen-binding site with the CDRs when linked via the CDRs. If necessary, amino acid residues in the FRs can be substituted so that the CDRs of a reshaped human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. By measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Cancer Res., (1993) 53, 851-856).

[0140] Alternatively, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0141] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) by phage display. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0142] In addition to the above, methods for obtaining antibody genes may also be used, as appropriate, including B cell cloning techniques such as those described in Bernasconi et al. (Science (2002) 298, 2199-2202) or International Publication WO2008 / 081008 (identification and cloning of the coding sequence for each antibody, isolation thereof, and use to construct expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4)).

[0143] EU numbering and Kabat numbering According to the method used in the present invention, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Herein, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids in the variable regions are represented according to the Kabat numbering, and the amino acids in the constant regions are represented according to the EU numbering based on the Kabat amino acid positions.

[0144] Target tissue-specific compound-dependent antigen-binding domain To obtain an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity changes depending on the concentration of a target tissue-specific compound, i.e., a target tissue-specific compound-dependent antigen-binding domain (or an antigen-binding molecule comprising said domain), the techniques described above in the section on binding activity can be applied as appropriate. Specific examples are provided below as a non-limiting example. For example, to confirm that the antigen-binding activity of an antigen-binding domain (or an antigen-binding molecule comprising said domain) in the presence of a target tissue-specific compound is higher than the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising said domain) in the absence of the target tissue-specific compound, the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising said domain) is compared in the absence and presence, or in the presence of low and high concentrations, of the target tissue-specific compound. 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 said domain) in the presence of a high concentration of a target tissue-specific compound is higher than the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising said domain) in the presence of a low concentration of the target tissue-specific compound, the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule comprising said domain) in the presence of a low concentration and a high concentration of the target tissue-specific compound is compared.

[0145] Furthermore, in the present invention, the expression "the antigen-binding activity is higher in the presence of a target tissue-specific compound than in the absence of the compound" can also be expressed as "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) is lower in the absence of a target tissue-specific compound than in the presence of the compound." Note that in the present invention, "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) is lower in the absence of a target tissue-specific compound than in the presence of the compound" can also be expressed as "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) is weaker in the absence of a target tissue-specific compound than in the presence of the compound."

[0146] Furthermore, in the present invention, the expression "the antigen-binding activity 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" can also be expressed as "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) in the presence of a low concentration of a target tissue-specific compound is lower than the antigen-binding activity in the presence of a high concentration of the compound." Note that in the present invention, "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) in the presence of a low concentration of a target tissue-specific compound is lower than the antigen-binding activity in the presence of a high concentration of the compound" can also be expressed as "the antigen-binding activity of the antigen-binding domain (or an antigen-binding molecule containing the domain) in the presence of a low concentration of a target tissue-specific compound is weaker than the antigen-binding activity in the presence of a high concentration of the compound."

[0147] Conditions for measuring antigen-binding activity, other than the concentration of the target tissue-specific compound, can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurements can be performed in HEPES buffer at 37°C. Measurements can be performed using, for example, Biacore (GE Healthcare). When measuring the binding activity between an antigen-binding domain (or an antigen-binding molecule containing the domain) and an antigen, if the antigen is a soluble molecule, the binding activity to soluble molecules can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding domain (or an antigen-binding molecule containing the domain) is immobilized. If the antigen is a membrane-type molecule, the binding activity to membrane-type molecules can be evaluated by passing the antigen-binding domain (or an antigen-binding molecule containing the domain) as an analyte through a chip on which the antigen is immobilized.

[0148] As long as the antigen-binding activity of an antigen-binding domain contained in an antigen-binding molecule of the present invention (or an antigen-binding molecule comprising said 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, but preferably the ratio of the KD (dissociation constant) for the antigen in the absence of the target tissue-specific compound to the KD in the presence of the compound, KD (in the absence of compound) / KD (in the presence of compound), is 2 or more, more preferably the KD (in the absence of compound) / KD (in the presence of compound) is 10 or more, and even more preferably the KD (in the absence of compound) / KD (in the presence of compound) is 40 or more. The upper limit of the KD (in the absence of compound) / KD (in the presence of compound) is not particularly limited, and may be any value, such as 400, 1000, or 10,000, as long as it can be produced within the skills of a person skilled in the art. If no binding activity to the antigen is observed in the absence of a target tissue-specific compound, this upper limit becomes an infinite value.

[0149] As long as the antigen-binding activity of an antigen-binding domain contained in an antigen-binding molecule of the present invention (or an antigen-binding molecule containing the domain) in the presence of a low concentration of a target tissue-specific compound 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 in the presence of a low concentration of the compound to the antigen-binding activity in the presence of a high concentration of the compound is not particularly limited. Preferably, the ratio of the KD (Dissociation constant) for the antigen in the presence of a low concentration of the target tissue-specific compound to the KD in the presence of a high concentration, KD (in the presence of a low concentration of the compound) / KD (in the presence of a high concentration of the compound), is 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the KD (in the presence of a low concentration of compound) / KD (in the presence of a high concentration of compound) value is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be achieved by a person skilled in the art. If no antigen-binding activity is observed in the presence of a low concentration of a target tissue-specific compound, this upper limit becomes an infinite numerical value.

[0150] As a value of antigen-binding activity, KD (dissociation constant) can be used when the antigen is a soluble molecule, but apparent KD (apparent dissociation constant) can be used when the antigen is a membrane-type molecule. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0151] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio of antigen-binding activity of an antigen-binding domain of the present invention (or an antigen-binding molecule comprising said domain) in the absence of a target tissue-specific compound to that in the presence of said compound. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of binding activity ratio, the ratio of kd (dissociation rate constant) for an antigen in the absence of a target tissue-specific compound to kd (dissociation rate constant) in the presence of said compound, kd (in the absence of compound) / kd (in the presence of compound), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There are no particular upper limits to the value of Kd (in the absence of compound) / kd (in the presence of compound), and any value, such as 50, 100, or 200, may be used as long as it is within the common technical knowledge of a person skilled in the art. In the absence of a target tissue-specific compound, if no binding activity to the antigen is observed, no dissociation occurs, and therefore this upper limit becomes an infinite numerical value.

[0152] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio of antigen-binding activity of an antigen-binding domain of the present invention (or an antigen-binding molecule comprising said domain) in the presence of a low concentration of a target tissue-specific compound to that in the presence of a high concentration. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of binding activity ratio, the ratio of kd (dissociation rate constant) for an antigen in the presence of a low concentration of a target tissue-specific compound to the kd (dissociation rate constant) in the presence of a high concentration of said compound, kd (in the presence of a low concentration of compound) / kd (in the presence of a high concentration of compound), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There are no particular upper limits to the value of Kd (in the presence of a low concentration of compound) / kd (in the presence of a high concentration of compound), and any value, such as 50, 100, or 200, may be used as long as it is within the common technical knowledge of a person skilled in the art. In the presence of a low concentration of a target tissue-specific compound, if no binding activity to the antigen is observed, no dissociation occurs, and therefore this upper limit becomes an infinite numerical value.

[0153] When the antigen is a soluble molecule, kd (dissociation rate constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type molecule, apparent kd (apparent dissociation rate constant) can be used. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare) or a flow cytometer. In the present invention, when measuring the antigen-binding activity of an antigen-binding domain (or an antigen-binding molecule containing the domain) at a certain concentration of a target tissue-specific compound, it is preferable to keep all conditions other than the concentration of the compound the same.

[0154] For example, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by screening antigen-binding domains (or antigen-binding molecules) comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding domain (or antigen-binding molecule) in the absence of a target tissue-specific compound; (b) measuring the antigen-binding activity of the antigen-binding domain (or antigen-binding molecule) in the presence of a target tissue-specific compound; (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.

[0155] For example, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by screening for antigen-binding domains (or antigen-binding molecules) comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding domain (or antigen-binding molecule) in the presence of a low concentration of a target tissue-specific compound; (b) determining 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; (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 the antigen-binding activity in the presence of a high concentration of the compound.

[0156] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by screening antigen-binding domains (or antigen-binding molecules) or a library thereof, comprising the following steps (a) to (c): (a) contacting an antigen-binding domain (or antigen-binding molecule) or a library thereof with an antigen in the presence of a target tissue-specific compound; (b) placing the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a) in the absence of the compound; (c) isolating the antigen-binding domain (or antigen-binding molecule) dissociated in step (b).

[0157] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by screening antigen-binding domains (or antigen-binding molecules) or a library thereof, which comprises the following steps (a) to (c): (a) contacting an antigen with an antigen-binding domain (or antigen-binding molecule) or a library thereof in the presence of a high concentration of a target tissue-specific compound; (b) placing the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a) in the presence of a low concentration of the compound; (c) isolating the antigen-binding domain (or antigen-binding molecule) dissociated in step (b).

[0158] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by screening antigen-binding domains (or antigen-binding molecules) or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains (or antigen-binding molecules) with an antigen in the absence of a target tissue-specific compound; (b) selecting antigen-binding domains (or antigen-binding molecules) that do not bind to the antigen in step (a); (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; (d) isolating the antigen-binding domain (or antigen-binding molecule) that bound to the antigen in step (c).

[0159] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by screening antigen-binding domains (or antigen-binding molecules) or a library thereof, which comprises the following steps (a) to (d): (a) contacting a 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; (b) selecting antigen-binding domains (or antigen-binding molecules) that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain (or antigen-binding molecule) selected in step (b) to bind to an antigen in the presence of a high concentration of the compound; (d) isolating the antigen-binding domain (or antigen-binding molecule) that bound to the antigen in step (c).

[0160] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains (or antigen-binding molecules) with a column onto which an antigen has been immobilized in the presence of a target tissue-specific compound; (b) eluting the antigen-binding domain (or antigen-binding molecule) bound to the column in step (a) from the column in the absence of the compound; (c) isolating the antigen-binding domain (or antigen-binding molecule) eluted in step (b).

[0161] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains (or antigen-binding molecules) with a column onto which an antigen has been immobilized in the presence of a high concentration of a target tissue-specific compound; (b) eluting the antigen-binding domain (or antigen-binding molecule) bound to the column in step (a) from the column in the presence of a low concentration of the compound; (c) isolating the antigen-binding domain (or antigen-binding molecule) eluted in step (b).

[0162] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains (or antigen-binding molecules) through a column onto which an antigen has been immobilized in the absence of a target tissue-specific compound; (b) recovering the antigen-binding domain (or antigen-binding molecule) that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain (or antigen-binding molecule) recovered in step (b) to bind to an antigen in the presence of the compound; (d) isolating the antigen-binding domain (or antigen-binding molecule) that bound to the antigen in step (c).

[0163] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains (or antigen-binding molecules) through a column onto which an antigen has been immobilized in the presence of a low concentration of a target tissue-specific compound; (b) recovering the antigen-binding domain (or antigen-binding molecule) that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain (or antigen-binding molecule) recovered in step (b) to bind to an antigen in the presence of a high concentration of the compound; (d) isolating the antigen-binding domain (or antigen-binding molecule) that bound to the antigen in step (c).

[0164] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains (or antigen-binding molecules) with an antigen in the presence of a target tissue-specific compound; (b) obtaining the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a); (c) placing the antigen-binding domain (or antigen-binding molecule) obtained in step (b) in the absence of a compound; (d) isolating antigen-binding domains (or antigen-binding molecules) whose antigen-binding activity is weaker than the criterion selected in step (b) in step (c).

[0165] Furthermore, in one embodiment provided by the present invention, an antigen-binding domain (or an antigen-binding molecule comprising said 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 said compound can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a 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; (b) obtaining the antigen-binding domain (or antigen-binding molecule) bound to the antigen in step (a); (c) placing the antigen-binding domain (or antigen-binding molecule) obtained in step (b) in the presence of a low concentration of a compound; (d) isolating antigen-binding domains (or antigen-binding molecules) whose antigen-binding activity is weaker than the criterion selected in step (b) in step (c).

[0166] The above steps may be repeated two or more times. Thus, the present invention provides an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the absence of a target tissue-specific compound is lower than its antigen-binding activity in the presence of the compound, or an antigen-binding domain (or an antigen-binding molecule comprising said domain) whose antigen-binding activity in the presence of a low concentration of a target tissue-specific compound is lower than its antigen-binding activity in the presence of a high concentration of the compound, obtained by the above-mentioned screening method, which further comprises repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is typically no more than 10 times.

[0167] In the screening methods of the present invention, a target tissue-specific compound can be a compound defined by quantitative target tissue specificity, such as being present at a different concentration (e.g., higher or lower) in target tissue compared to non-target tissue. For example, a target tissue-specific compound can be differentially present at any concentration. However, generally, a target tissue-specific compound will exhibit a differential expression of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 10 ... 3 times at least 10 4 times at least 10 5 times at least 10 6 It is possible for the ATP to be present in increasing concentrations, up to infinity (ie, absent in non-target tissues), by 2-fold or more.

[0168] The threshold for distinguishing between low and high concentrations can be set appropriately depending on the compound. For example, in a non-limiting embodiment of the threshold for ATP or adenosine, the low concentration condition can be set appropriately from the threshold values ​​of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, or 0 M. Depending on the set threshold, the high concentration condition can be set to at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 10 ... 3 times at least 10 4 times at least 10 5 times at least 10 6 In a non-limiting embodiment of PGE2, the threshold value for the low concentration condition can be appropriately set from 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, or 0 M. Depending on 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 3 times at least 10 4 times at least 10 5 times at least 10 6 Furthermore, in one non-limiting embodiment of Kyunurenine, the threshold value for the low concentration condition can be appropriately set from 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, or 0 M. Depending on the set threshold value, the high concentration condition can be set to 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 value. 3 times at least 10 4 times at least 10 5 times at least 10 6 The value can be appropriately set from 1 / 2 times.

[0169] The antigen-binding activity of an antigen-binding domain (or antigen-binding molecule) can be measured by methods known to those skilled in the art, and conditions other than the concentration of the target tissue-specific compound can be appropriately determined by those skilled in the art. 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 constant), apparent kd (Apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, FACS, etc.

[0170] 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 means the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound.

[0171] 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 means the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity in the absence of a target tissue-specific compound is lower than that in the presence of the compound.

[0172] As long as the antigen-binding activity in the absence of a target tissue-specific compound is lower than the antigen-binding activity in the presence of the compound, the difference in antigen-binding activity between the presence and absence of the compound is not particularly limited, but preferably the antigen-binding activity in the presence of the compound is at least 2-fold, more preferably at least 10-fold, and even more preferably at least 40-fold greater than the antigen-binding activity in the absence of the compound. The upper limit of the difference in antigen-binding activity is not particularly limited, and may be any value, such as 400-fold, 1000-fold, or 10,000-fold, as long as it can be achieved by those skilled in the art. If no antigen-binding activity is observed in the absence of a target tissue-specific compound, this upper limit becomes infinite.

[0173] The antigen-binding domains (or antigen-binding molecules containing said domains) of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains (or antigen-binding molecules), and for example, the above-mentioned antigen-binding domains (or antigen-binding molecules) can be screened. For example, antigen-binding domains (or antigen-binding molecules) having native sequences may be screened, or antigen-binding domains (or antigen-binding molecules) with substituted amino acid sequences may be screened.

[0174] Library According to one embodiment, the antigen-binding domains of the present invention (or antigen-binding molecules containing the domains) can be obtained from a library primarily consisting of multiple antigen-binding molecules with different sequences, each of which contains at least one amino acid residue that alters the binding activity of the antigen-binding molecule to an antigen dependent on a target tissue-specific compound. Examples of such compounds include (1) primary metabolites of the glycolytic pathway or the Krebs cycle, such as lactate, succinate, and citrate; (2) amino acids, such as alanine, glutamate, and aspartate; (3) kynurenine and its metabolites, such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid; (4) arachidonic acid metabolites, 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 provides an example of such a target tissue-specific compound, a library mainly consisting of multiple antigen-binding molecules with different sequences, each of whose antigen-binding domains contains at least one amino acid residue that alters the binding activity of the antigen-binding molecule toward an adenosine- and / or ATP-dependent antigen.

[0175] As used herein, the term "library" refers to multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. 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 are antigen-binding molecules or fusion polypeptides containing antigen-binding molecules with sequences that differ from one another.

[0176] As used herein, the term "different sequences" in the description of multiple antigen-binding molecules with different sequences means that the sequences of the individual antigen-binding molecules in the library are different from each other. In other words, the number of different sequences in the library reflects the number of independent clones with different sequences in the library, and is sometimes referred to as the "library size." In a typical phage display library, 10 6From 10 12 By applying known techniques such as ribosome display, the library size can be increased to 10 14 However, 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, also called the "library equivalent number," indicates that there may be 10 to 10,000 individual clones with the same amino acid sequence. Therefore, the term "different in sequence from each other" in the present invention means that the sequences of the individual antigen-binding molecules in the library, excluding the library equivalent number, are different from each other, more specifically, there may be 10 or more antigen-binding molecules with different sequences from each other. 6 From 10 14 molecules, preferably 10 7 From 10 12 molecules, more preferably 10 8 From 10 11 , particularly preferably 10 8 From 10 10 It means to exist.

[0177] Furthermore, the term "plurality" in the description of a library of the present invention consisting primarily of a plurality of antigen-binding molecules generally refers to a collection of two or more types of the substance, for example, the antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors, or viruses of the present invention. For example, if two or more substances differ from each other in a specific trait, this indicates that there are two or more types of the substance. An example would be variant amino acids observed at specific amino acid positions in an amino acid sequence. For example, if there are two or more antigen-binding molecules of the present invention that have substantially the same, preferably identical, sequences other than flexible residues or specific variant amino acids at highly diverse, surface-exposed amino acid positions, then there are plural antigen-binding molecules of the present invention. In another example, if there are two or more polynucleotide molecules of the present invention that have substantially the same, preferably identical sequences other than bases encoding flexible residues or bases encoding specific variant amino acids at highly diverse, surface-exposed amino acid positions, then there are plural polynucleotide molecules of the present invention.

[0178] Furthermore, the term "mainly consisting of" in the description of a library of the present invention consisting mainly of a plurality of antigen-binding molecules reflects the number of antigen-binding molecules whose antigen-binding activity varies depending on the concentration of a target tissue-specific compound among the number of independent clones with different sequences in the library. Specifically, the term "mainly consisting of" refers to the number of antigen-binding molecules whose antigen-binding activity varies depending on the concentration of a target tissue-specific compound in the library. 4 It is preferable that the antigen-binding domain of the present invention has at least 10 antigen-binding molecules that exhibit such binding activity. 5 More preferably, the antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 6 The antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 7 Preferably, the antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 8 The antigen-binding domains of the present invention can be obtained from libraries containing antigen-binding molecules present in the library. Alternatively, they can be suitably expressed as the proportion of antigen-binding molecules whose antigen-binding activity 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 domains of the present invention can be obtained from libraries in which antigen-binding molecules exhibiting such binding activity account for 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, even more preferably 2% to 20%, and particularly preferably 4% to 10% of the number of independent clones with different sequences in the library. Fusion polypeptides, polynucleotide molecules, and vectors can also be expressed as the number of molecules or the proportion of all molecules, as described above. Viruses can also be expressed as the number of virus individuals or the proportion of all individuals, as described above.

[0179] Adenosine and / or amino acids that change the binding activity of the antigen-binding domain to an antigen depending on the presence or absence of ATP The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be prepared in any manner, and may include pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, and antibodies or libraries prepared from immune cells such as B cells of animals immunized with a conjugate of an adjuvant agent such as a highly immunogenic T cell epitope peptide appropriately linked to adenosine or ATP. A suitable, non-limiting example of such a T cell epitope peptide is the p30 helper peptide derived from Tetanus toxin (represented by SEQ ID NO: 4, also referred to as Fragment C (FrC)).

[0180] As described above, examples of amino acids that change the antigen-binding activity of an antigen-binding molecule depending on the presence or absence of adenosine and / or ATP include amino acids that form an adenosine and / or ATP-binding motif. The position of the amino acid in the antigen-binding domain containing the amino acid is not limited to a specific position, and can be any position in the heavy chain variable region or light chain variable region that forms the antigen-binding domain, as long as the antigen-binding activity of the antigen-binding domain changes depending on the presence or absence of adenosine and / or ATP. That is, the antigen-binding domains of the present invention can be obtained from a library primarily composed of antigen-binding molecules with different sequences, in which the heavy chain antigen-binding domain contains an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP. In a non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily composed of antigen-binding molecules with different sequences, in which the heavy chain CDR1, CDR2, and / or CDR3 contains an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP. In another non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences that contain amino acids in FR1, FR2, FR3, and / or FR4 of the heavy chain that change the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP.

[0181] Furthermore, in one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library primarily composed of antigen-binding molecules with different sequences, in which the heavy and / or light chain antigen-binding domains contain amino acids that change the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP. In a non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library primarily composed of antigen-binding molecules with different sequences, in which the heavy and / or light chain CDR1, CDR2, and / or CDR3 contain amino acids that change the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP. In another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library primarily composed of antigen-binding molecules with different sequences, in which the heavy and / or light chain FR1, FR2, FR3, and / or FR4 contain amino acids that change the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP.

[0182] Non-limiting examples of such amino acids include any one or more of the amino acids at positions 52, 52a, 53, 96, 100a, and 100c in the heavy chain variable region. Non-limiting examples of such amino acids include any one or more of the amino acids at positions 52, 52a, 53, Arg at position 53, Gly at position 96, Leu at position 100a, and Trp at position 100c in the heavy chain variable region.

[0183] Any framework sequence can be used for the light and / or heavy chain variable region of an antigen-binding molecule, as long as the heavy and / or light chain antigen-binding domain contains amino acids whose antigen-binding activity changes depending on the presence or absence of adenosine and / or ATP. The origin of the framework sequence can be obtained from any non-human animal organism or human. Preferably, the any organism includes an organism selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In a particularly preferred embodiment, the framework sequence of the light and / or heavy chain variable region of the antigen-binding molecule preferably has a human germline framework sequence. Therefore, in one aspect of the present invention, if the framework sequence is completely human, it is expected that the antigen-binding molecule of the present invention will evoke little or no immunogenic response when administered to humans (e.g., for the treatment of a disease). In the above sense, "comprising a germline sequence" of the present invention means that a portion of the framework sequence of the present invention is identical to a portion of any human germline framework sequence. For example, an antigen-binding molecule of the present invention "comprising a germline sequence" also includes an antigen-binding molecule of the present invention in which the heavy chain FR2 sequence is a combination of heavy chain FR2 sequences from multiple different human germline framework sequences. Furthermore, an antigen-binding molecule of the present invention "comprising a germline sequence" also includes an antigen-binding molecule of the present invention in which the framework sequence has been substituted. Examples of such substituted sequences include, in particular, sequences in which some amino acids in a human germline framework sequence have been substituted with amino acids that alter the antigen-binding activity of the antigen-binding molecule depending on the presence or absence of adenosine and / or ATP.

[0184] Suitable examples of frameworks include currently known fully human framework region sequences included on websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ). These framework region sequences can be appropriately used as germline sequences 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 appropriately selected from Vκ, which are classified into seven subgroups, Vλ, which are classified into ten subgroups, and VH, which are classified into seven subgroups.

[0185] Fully human VH sequences include, but are not limited to, sequences from the VH1 subgroup (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), the VH2 subgroup (e.g., VH2-5, VH2-26, VH2-70), the VH3 subgroup (e.g., VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-3), and the VH4 subgroup (e.g., VH4-5, VH4-6, VH4-70, VH4-8, VH4-9, VH4-10, VH4-11, VH4-13, VH4-15, VH4-16, VH4-20, VH4-21, VH4-23, VH4-3). Suitable examples of VH sequences include those of the VH4 subgroup (VH4-0, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74), the VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61), the VH5 subgroup (VH5-51), the VH6 subgroup (VH6-1), and the VH7 subgroup (VH7-4, VH7-81). 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 antigen-binding molecules of the present invention based on this sequence information. Completely human frameworks or framework subregions other than these can also be suitably used.

[0186] Completely human Vκ sequences include, but are not limited to, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18, which are classified into the Vk1 subgroup, and A1, A2, A3, A5, A7, A17, and A19, which are classified into the Vk2 subgroup. Preferred examples thereof include 18, A19, A23, O1, O11, A11, A27, L2, L6, L10, L16, L20, and L25, which are classified into the Vk3 subgroup, B3, which is classified into the Vk4 subgroup, B2, which is classified into the Vk5 subgroup (also referred to as Vk5-2 in this specification), and A10, A14, and A26, which are classified into the Vk6 subgroup (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)).

[0187] Fully human Vλ sequences include, but are not limited to, 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, which are classified into the VL1 subgroup; V2-1, V2-6, V2-7, and V2-8, which are classified into the VL1 subgroup; Preferred examples thereof include V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19; ​​V3-2, V3-3, and V3-4, which are classified into the VL3 subgroup; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classified into the VL4 subgroup; and V5-1, V5-2, V5-4, and V5-6, which are classified into the VL5 subgroup (Kawasaki et al., Genome Res. (1997) 7, 250-261).

[0188] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used together with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding domain depending on the presence or absence of adenosine and / or ATP" of the present invention. Other examples of fully human frameworks that can be used together with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding domain depending on 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)).

[0189] Although the present invention is not bound by any particular theory, it is believed that one reason the use of germline sequences is expected to eliminate adverse immune responses in most individuals is that somatic mutations frequently occur in the variable regions of immunoglobulins as a result of the affinity maturation step that occurs during a normal immune response. These mutations occur primarily around the CDRs, whose sequences are hypervariable, but also affect residues in the framework regions. These framework mutations are absent from germline genes and are unlikely to be immunogenic in patients. On the other hand, the normal human population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are expected to be less immunogenic or non-immunogenic in patients. To maximize the likelihood of immune tolerance, genes encoding the variable regions can be selected from a commonly present set of functional germline genes.

[0190] To prepare antigen-binding molecules of the present invention in which amino acids that change the antigen-binding activity of the antigen-binding domain depending on the presence or absence of adenosine and / or ATP are contained in the variable region sequence, heavy chain variable region sequence, light chain variable region sequence, CDR sequence, or framework sequence, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used.

[0191] For example, a library comprising a plurality of antigen-binding molecules of the present invention with different sequences can be prepared by combining a light chain variable region selected as a CDR sequence and / or framework sequence that already contains at least one amino acid residue that alters 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 prepared as a randomized variable region sequence library.

[0192] Furthermore, the sequences of the heavy and / or light chain variable regions selected as CDR and / or framework sequences already contain at least one amino acid residue that alters the antigen-binding activity of the antigen-binding domain depending on the presence or absence of adenosine or ATP. It is also possible to design these sequences so that they contain various amino acids as residues other than the amino acid residue. In the present invention, such residues are referred to as "flexible residues." As long as the antigen-binding activity of the antigen-binding molecules of the present invention changes depending on the concentration of the tissue-specific compound, the number and location of the flexible residues are not limited to specific embodiments. That is, one or more flexible residues may be contained in the CDR and / or FR sequences of the heavy and / or light chains. Flexible residues and other amino acids that can be substituted for them to create libraries can be identified by crystal structure analysis or mutagenesis of the antibody complex with adenosine and / or ATP. For example, antibody residues not involved in adenosine and / or ATP binding can be identified by crystal structure analysis of the antibody complex with adenosine and / or ATP. Amino acids that can maintain an appropriate level of compound binding can be selected even when residues identified as not involved in adenosine and / or ATP binding are substituted with other amino acids. This allows for the design of a library in which selected amino acids appear at selected residues. In this case, it is possible to design a library primarily consisting of multiple antigen-binding molecules so that the antigen-binding molecules are a collection of antigen-binding molecules in which residues identified as not involved in adenosine and / or ATP binding have been substituted with different amino acids. In other words, combining individual flexible residues substituted with different amino acids results in sequence diversity for antigen-binding molecules containing the flexible residues.

[0193] Furthermore, antigen-binding molecules containing these residues can be designed so that at least one of the residues identified to be involved in adenosine and / or ATP binding is any residue selected from those residues and residues different from those residues. Non-limiting examples of amino acids identified to be involved in adenosine and / or ATP binding include any one or more of the amino acids at positions 52, 52a, 53, 96, 100a, and 100c contained in the heavy chain variable region. Non-limiting examples of such amino acids include any one or more of the amino acids at positions 52, 52a, 53, 96, 100a, and 100c contained in the heavy chain variable region. For example, if Leu at position 100a is identified as being involved in adenosine and / or ATP binding, the amino acid residue at position 100a of the antigen-binding molecules contained in the library may be an amino acid residue selected from Leu and any of the flexible residues His, Met, Leu, Arg, Trp, and Tyr.

[0194] Non-limiting examples of flexible residues include the amino acids at positions 31, 32, 33, 35, 50, 55, 56, 57, 58, 59, 95, 96, 97, 98, 99, 100, 100a, and 100b in the heavy chain variable region. Non-limiting examples of such amino acids include 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 in the light chain variable region.

[0195] In one non-limiting embodiment, the flexible residue is an amino acid contained in the heavy chain variable region: the amino acid at position 31 is Asp, Gly, Asn, Ser, Arg, or Thr; the amino acid at position 32 is Ala, Phe, His, Asn, Ser, or Tyr; the amino acid at position 33 is Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg, Trp, Val, Tyr, or Thr; The amino acid at position 35 is His, Ser, Thr, Tyr, or Asn; The amino acid at position 50 is Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Arg, Thr, Trp, Val, Tyr, or Ser; the amino acid at position 55 is Ala, Glu, Asp, Gly, Leu, Thr, Ser, Arg, or Asn; the amino acid at position 56 is any of Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Thr, Trp, Val, and Tyr; the amino acid at position 57 is Ala, Lys, Arg, Thr, or Ile; the amino acid at position 58 is Asp, Gly, Phe, His, Ser, Thr, Tyr, or Asn; The amino acid at position 59 is either Leu or Tyr, the amino acid at position 95 is Ala, Ile, Lys, Met, Leu, Arg, Trp, Val, Tyr, or Phe; the amino acid at position 96 is Ala, Asp, Asn, or Ser; the amino acid at position 97 is Ala, Asp, Gly, Ile, His, Lys, Met, Leu, Asn, Ser, Val, Tyr, or Arg; the amino acid at position 98 is Ala, Glu, Asp, Gly, Phe, Ile, His, Met, Leu, Asn, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Lys; the amino acid at position 99 is Ala, Glu, Asp, Phe, His, Lys, Asn, Gln, Ser, Arg, Trp, Val, Tyr, or Gly; The amino acid at position 100 is Ala, Glu, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asp; The amino acid at position 100a is Ala, Phe, Ile, His, Lys, Met, Arg, Trp, Val, or Tyr, or The amino acid at position 100b is Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asn; Examples of amino acids include:

[0196] In one non-limiting embodiment, the flexible residue is an amino acid contained in the light chain variable region: the amino acid at position 26 is Ala, Ser, or Thr; The amino acid at position 27 is either Thr or Ser, the amino acid at position 27a is Gly, Asn, Thr, or Ser; The amino acid at position 27b is either Asn or Asp, The amino acid at position 27c is either Ile or Val, The amino acid at position 28 is either Asp or Gly, the amino acid at position 29 is Ala, Asp, Phe, Ser, Arg, Thr, Tyr, or Gly; The amino acid at position 31 is Glu, Asp, Lys, or Asn; the amino acid at position 32 is Ala, Asp, Ser, Thr, or Tyr; The amino acid at position 50 is Asp, Gly, Lys, Asn, Gln, Ser, Arg, Tyr, or Glu; the amino acid at position 51 is Asp, Gly, Lys, Asn, Thr, or Val; the amino acid at position 52 is Ala, Asp, Asn, Thr, or Ser; the amino acid at position 53 is Glu, Asp, His, Asn, Gln, Ser, Tyr, or Lys; The amino acid at position 54 is either Lys or Arg, The amino acid at position 55 is either Leu or Pro, the amino acid at position 89 is Ala, Gly, Phe, Leu, Asn, Gln, Thr, Val, Tyr, or Ser; The amino acid at position 90 is Ala, Leu, Thr, Val, or Ser; the amino acid at position 91 is Ala, Asp, Phe, His, Lys, Asn, Ser, Arg, Thr, Trp, Val, or Tyr; the amino acid at position 92 is Glu, Asp, Ser, Arg, Thr, Val, Tyr, or Ala; the amino acid at position 93 is Ala, Asp, Ile, Asn, Ser, Arg, Thr, Val, Tyr, or Gly; the amino acid at position 94 is Ala, Asp, Gly, Ile, Asn, Arg, Thr, or Ser; the amino acid at position 95 is Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asn; the amino acid at position 95a is Ala, Glu, Asp, Gly, Ile, His, Lys, Leu, Gln, Pro, Ser, Arg, Thr, Tyr, or Asn; The amino acid at position 96 is Ala, Asp, Gly, Phe, His, Lys, Leu, Asn, Gln, Pro, Ser, Thr, Trp, Tyr, or Val; or The amino acid at position 97 is Ala, Gly, Ile, Met, Leu, Ser, or Val Examples of amino acids include:

[0197] As used herein, "flexible residues" refers to amino acid residue variations present at positions in the light and heavy chain variable regions where amino acids are highly diverse when comparing the amino acid sequences of known and / or natural antibodies or antigen-binding domains, with several different amino acids present at that position. Highly diverse positions are typically present in the CDR regions. In one embodiment, data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.) (1987 and 1991) are useful for determining highly diverse positions in known and / or natural antibodies. Additionally, several databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html) provide collected sequences and their arrangements of numerous human light and heavy chains. Information on these sequences and their arrangements is useful for determining highly diverse positions in the present invention. According to the present invention, an amino acid position is said to be highly diverse if it has a diversity of preferably about 2 to about 20, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 possible different amino acid residues at that position. In some embodiments, an amino acid position may have a diversity of preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, preferably about 12 possible different amino acid residues.

[0198] Furthermore, a library of the present invention comprising a plurality of antigen-binding molecules with different sequences can be prepared by combining a light chain variable region into which 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 has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library. Similarly, a library of the present invention comprising a plurality of antigen-binding molecules with different sequences can be prepared by combining a light chain variable region into which 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 has been introduced with other amino acid residues designed as flexible residues.

[0199] Even when combining a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of the target tissue-specific compound has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library, it is possible to design the light chain variable region sequence to contain flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the presence or absence of adenosine and / or ATP, the number and position of the flexible residues are not limited to any particular embodiment. That is, one or more flexible residues may be contained in the CDR sequences and / or FR sequences of the heavy and / or light chains.

[0200] A suitable example of a heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases or humans with increased blood antibody titers after vaccination, cancer patients, or patients with autoimmune diseases can be suitably used as a randomized variable region library.

[0201] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length can also be used as a randomized variable region library. In this case, since diversity in the gene sequences of heavy chain CDR3 is observed, it is also possible to replace only the CDR3 sequence. The criterion for generating amino acid diversity in the variable regions of antigen-binding molecules is to provide diversity to amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is determined to be surface-exposed and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Suitable software available for this purpose includes the SYBYL Biopolymer Module software (Tripos Associates). Generally, and preferably, when an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Furthermore, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0202] In a non-limiting embodiment of the present invention, amino acids in the variable regions, including the CDR regions and / or framework regions, can be appropriately modified to improve antibody stability. Non-limiting examples of such amino acids include those at positions 1, 5, 10, 30, 48, and 58. More specifically, Gln at position 1, Gln at position 5, Asp at position 10, Asn at position 30, Leu at position 48, and Asn at position 58 can be substituted. To improve antibody stability, these amino acids can be substituted with the corresponding amino acids contained in the germline sequence. A non-limiting example of such a germline sequence is the sequence of VH3-21. In this case, Gln at position 1 can be substituted with Glu, Gln at position 5 with Val, Asp at position 10 with Gly, Asn at position 30 with Ser, Leu at position 48 with Val, and Asn at position 58 with Tyr.

[0203] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy individuals and whose repertoire is unbiased, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344). The amino acid sequence comprising a naive sequence described in the present invention refers to an amino acid sequence obtained from such a naive library.

[0204] Fc area The Fc region comprises an amino acid sequence derived from the constant region of an antibody heavy chain. The Fc region is a portion of the antibody heavy chain constant region, spanning from the N-terminus of the hinge region of the papain cleavage site at approximately amino acid position 216 (EU numbering) to the hinge, CH2, and CH3 domains. The Fc region can be obtained from human IgG1, but is not limited to a specific IgG subclass. Preferred examples of the Fc region include Fc regions that have FcRn-binding activity in the acidic pH range, as described below. Preferred examples of the Fc region also include Fc regions that have Fcγ receptor-binding activity, as described below. Non-limiting examples of such Fc regions include the Fc regions of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), and IgG4 (SEQ ID NO: 8).

[0205] Fcγ receptor (FcγR) Fcγ receptors (also referred to as FcγR) refer to receptors capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and refer to any member of a family of proteins substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131, i.e., FcγRIIa (H) and FcγRIIa (R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIIIa (including allotypes V158 and F158, i.e., FcγRIIIa (V) and FcγRIIIa (V)). These include, but are not limited to, FcγRIII (CD16), including FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms or allotypes. Suitable examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI are shown in SEQ ID NOs: 9 (NM_000566.3) and 10 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of human FcγRIIa (allotype H131) are shown in SEQ ID NOs: 11 (BC020823.1) and 12 (AAH20823.1), respectively (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 12 is substituted with Arg). The polynucleotide sequence and amino acid sequence of FcγRIIb are shown in SEQ ID NOs: 12 (BC020823.1) and 12 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIa are shown in SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIa are shown in SEQ ID NOs: 15 (BC033678.1) and 16 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are shown in SEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (database accession numbers such as RefSeq are shown in parentheses). Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the above-described FACS or ELISA formats, as well as by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0206] FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), are composed of an α chain that binds to the Fc region of IgG and a common γ chain with ITAMs that transduce activation signals intracellularly. On the other hand, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its cytoplasmic domain. These receptors are expressed on many immune cells, including macrophages, mast cells, and antigen-presenting cells. Binding of these receptors to the Fc region of IgG transmits activation signals that promote the phagocytic activity of macrophages, the production of inflammatory cytokines, mast cell degranulation, and enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are referred to herein as activating Fcγ receptors.

[0207] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses activation signals from the BCR, resulting in the suppression of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic activity and the ability to produce inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are referred to herein as inhibitory Fcγ receptors.

[0208] Binding activity of the Fc region to FcγR As described above, examples of Fc regions contained in the antigen-binding molecules of the present invention include Fc regions that have binding activity to Fcγ receptors. Non-limiting examples of such Fc regions include the Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), and IgG4 (SEQ ID NO: 8). Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the FACS and ELISA formats described above, as well as ALPHA screens (Amplified Luminescent Proximity Homogeneous Assays) and BIACORE methods that utilize surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0209] The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. If the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

[0210] For example, donor beads are bound to antigen-binding molecules containing biotin-labeled Fc regions, and acceptor beads are bound to Fcγ receptors tagged with glutathione S-transferase (GST). In the absence of competing antigen-binding molecules containing Fc region variants, antigen-binding molecules with native Fc regions interact with Fcγ receptors, generating a signal at 520-620 nm. Antigen-binding molecules containing untagged Fc region variants compete with the interaction between antigen-binding molecules with native Fc regions and Fcγ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar is known. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in frame in an operably linked vector in cells, and purifying the gene using a glutathione column. The resulting signals are suitably analyzed by fitting them to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0211] One of the substances (ligand) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is passed over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of shift (i.e., the change in mass on the sensor chip surface) on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and affinity (KD) can be calculated from the ratio of these constants. Inhibition assays are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0212] Fcγ receptor (FcγR) binding engineered Fc region In addition to the Fc regions represented by human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8), Fc regions that are modified for FcγR binding and have higher Fcγ receptor-binding activity than that of the Fc region of native human IgG can also be used as appropriate for the present invention. As used herein, "Fc region of native human IgG" refers to an Fc region in which the sugar chain attached to position 297 (EU numbering) of the Fc region of human IgG1, IgG2, IgG3, or IgG4, exemplified by SEQ ID NO: 5, 6, 7, or 8, is a fucose-containing sugar chain. Such Fc regions with modified FcγR binding can be prepared by modifying the amino acids of the Fc region of native human IgG. Whether the FcγR-binding activity of an Fc region with modified FcγR binding is higher than that of the Fc region of native human IgG can be determined appropriately using the method described above in the section on binding activity.

[0213] In the present invention, "amino acid modification" or "amino acid modification" of an Fc region includes modifying the amino acid sequence to a different amino acid sequence from that of the starting Fc region. Any Fc region can be used as the starting Fc region as long as the modified variant of the starting Fc region can bind to human Fcγ receptors in the neutral pH range. Furthermore, Fc regions that have been further modified using an already modified Fc region can also be suitably used as the Fc region of the present invention. The starting Fc region can refer to the polypeptide itself, a composition containing the starting Fc region, or the amino acid sequence encoding the starting Fc region. The starting Fc region can include known Fc regions produced by recombinant means, as outlined in the antibody section. The source of the starting Fc region can be obtained from any non-human animal organism or human. Preferably, the organism is selected from the group consisting of mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cows, horses, camels, and non-human primates. In another embodiment, the starting Fc region can also be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Preferably, the starting Fc region can be obtained from human IgG1, but is not limited to a particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting Fc region. Similarly, as used herein, it is meant that the Fc region of any class or subclass of IgG from any of the above-mentioned organisms can preferably be used as the starting Fc region. Examples of naturally occurring IgG variants or engineered forms are described in known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, International Publication Nos. WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0214] Examples of modifications include one or more mutations, such as substitution of amino acid residues different from those of the starting Fc region, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the starting Fc region. Preferably, the amino acid sequence of the modified Fc region comprises at least a portion of a non-naturally occurring Fc region. Such variants necessarily have less than 100% sequence identity or similarity with the starting Fc region. In a preferred embodiment, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100%, and most preferably about 95% to less than 100%, to the amino acid sequence of the starting Fc region. In one non-limiting embodiment of the present invention, there is at least one amino acid difference between the starting Fc region and the FcγR-binding modified Fc region of the present invention. The amino acid differences between the starting Fc region and the altered FcγR binding Fc regions of the present invention can be preferably identified by the differences in specific amino acids at the amino acid residue positions specified by the EU numbering system described above. Methods for creating such variants are exemplified in the section "Amino acid modifications."

[0215] The FcγR-binding modified Fc regions (FcγR-binding modified Fc regions) included in the antigen-binding molecules of the present invention, which have higher Fcγ receptor-binding activity than that of the Fc region of native human IgG, can be obtained by any method, but specifically, the FcγR-binding modified Fc regions can be obtained by modifying the amino acids of a human IgG-type immunoglobulin used as the starting Fc region. Preferred IgG-type immunoglobulin Fc regions for modification include, for example, the Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and variants thereof) exemplified by SEQ ID NOs: 5, 6, 7, or 8.

[0216] Amino acids at any position can be modified to other amino acids, as long as the Fcγ receptor-binding activity is higher than that of the Fc region of native human IgG. When the antigen-binding molecule contains the Fc region of human IgG1 as the human Fc region, it is preferable that the molecule contains a modification that results in higher Fcγ receptor-binding activity than that of the Fc region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain. Such amino acid modifications have been reported, for example, in International Publications WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO2006 / 020114, WO2006 / 116260 and WO2006 / 023403.

[0217] Examples of amino acids that can be modified in this way include those at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, and 247 (EU numbering). 249th, 250th, 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 28th 6th, 288th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313rd, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th , 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440. By modifying these amino acids, it is possible to obtain an Fc region (FcγR binding-altered Fc region) that has higher Fcγ receptor-binding activity than that of the Fc region of native human IgG.

[0218] Particularly preferred modifications for use in the present invention include, for example, those represented by EU numbering in the Fc region: The amino acid at position 221 is either Lys or Tyr; the amino acid at position 222 is Phe, Trp, Glu, or Tyr; the amino acid at position 223 is Phe, Trp, Glu, or Lys; the amino acid at position 224 is Phe, Trp, Glu, or Tyr; The amino acid at position 225 is either Glu, Lys, or Trp; the amino acid at position 227 is Glu, Gly, Lys, or Tyr; the amino acid at position 228 is Glu, Gly, Lys, or Tyr; The amino acid at position 230 is Ala, Glu, Gly, or Tyr; the amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 232 is Glu, Gly, Lys, or Tyr; the amino acid at position 233 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 234 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 235 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 236 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 238 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 239 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 240 is Ala, Ile, Met, or Thr; the amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or Tyr; the amino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The amino acid at position 244 is His; The amino acid at position 245 is Ala, the amino acid at position 246 is Asp, Glu, His, or Tyr; the amino acid at position 247 is Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The amino acid at position 249 is Glu, His, Gln, or Tyr; The amino acid at position 250 is either Glu or Gln, The amino acid at position 251 is Phe, the amino acid at position 254 is either Phe, Met, or Tyr; The amino acid at position 255 is either Glu, Leu, or Tyr; The amino acid at position 256 is either Ala, Met, or Pro; the amino acid at position 258 is Asp, Glu, His, Ser, or Tyr; The amino acid at position 260 is either Asp, Glu, His, or Tyr; the amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; the amino acid at position 263 is Ala, Ile, Met, or Thr; the amino acid at position 264 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 265 is Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 266 is Ala, Ile, Met, or Thr; the amino acid at position 267 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 268 is Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp; the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 270 is Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 271 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 272 is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 273 is either Phe or Ile; the amino acid at position 274 is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 275 is either Leu or Trp, the amino acid at position 276 is any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 279 is Ala, the amino acid at position 280 is Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr; the amino acid at position 281 is Asp, Lys, Pro, or Tyr; The amino acid at position 282 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 283 is Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; the amino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or Tyr; the amino acid at position 285 is Asp, Glu, Lys, Gln, Trp, or Tyr; The amino acid at position 286 is Glu, Gly, Pro, or Tyr; the amino acid at position 288 is Asn, Asp, Glu, or Tyr; the amino acid at position 290 is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr; the amino acid at position 291 is Asp, Glu, Gly, His, Ile, Gln, or Thr; the amino acid at position 292 is Ala, Asp, Glu, Pro, Thr, or Tyr; the amino acid at position 293 is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 294 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 296 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the amino acid at position 297 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 298 is Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 300 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; the amino acid at position 301 is Asp, Glu, His, or Tyr; The amino acid at position 302 is Ile; the amino acid at position 303 is Asp, Gly, or Tyr; the amino acid at position 304 is Asp, His, Leu, Asn, or Thr; The amino acid at position 305 is Glu, Ile, Thr, or Tyr; the amino acid at position 311 is Ala, Asp, Asn, Thr, Val, or Tyr; The amino acid at position 313 is Phe; The amino acid at position 315 is Leu; The amino acid at position 317 is Glu or Gln, the amino acid at position 318 is any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 322 is any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 323 is Ile; the amino acid at position 324 is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 325 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 326 is Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 327 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 328 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 330 is any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 332 is Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 333 is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr; the amino acid at position 334 is Ala, Glu, Phe, Ile, Leu, Pro, or Thr; the amino acid at position 335 is Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 336 is either Glu, Lys, or Tyr; The amino acid at position 337 is either Glu, His, or Asn; the amino acid at position 339 is Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr; The amino acid at position 376 is either Ala or Val; The amino acid at position 377 is either Gly or Lys; The amino acid at position 378 is Asp, The amino acid at position 379 is Asn, The amino acid at position 380 is either Ala, Asn, or Ser; The amino acid at position 382 is either Ala or Ile; The amino acid at position 385 is Glu; The amino acid at position 392 is Thr; The amino acid at position 396 is Leu, The amino acid at position 421 is Lys; The amino acid at position 427 is Asn, The amino acid at position 428 is either Phe or Leu; The amino acid at position 429 is Met; The amino acid at position 434 is Trp, The amino acid at position 436 is Ile, or the amino acid at position 440 is Gly, His, Ile, Leu, or Tyr; The modification may involve modification of at least one amino acid selected from the group consisting of: In addition, the number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of modification of two or more amino acids include those shown in Table 1 (Table 1-1 to Table 1-3).

[0219] [Table 1-1]

[0220] Table 1-2 is a continuation of Table 1-1. [Table 1-2]

[0221] Table 1-3 is a continuation of Table 1-2. [Table 1-3]

[0222] The pH conditions for measuring the binding activity of an Fcγ receptor-binding domain contained in an antigen-binding molecule of the present invention to an Fcγ receptor can be appropriately set within the acidic to neutral pH range. The acidic to neutral pH range used for measuring the binding activity of an Fcγ receptor-binding domain contained in an antigen-binding molecule of the present invention to an Fcγ receptor typically refers to a pH range of 5.8 to 8.0. The range is preferably any pH value between 6.0 and 7.4, and is preferably selected from pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4, with pH 6.15 to 7.4, which is close to the pH of cancer tissue, being particularly preferred (Vaupel et al., Cancer Res. (1989) 49, 6449-6665). Regarding the temperature used as a measurement condition, the binding affinity between an Fcγ receptor-binding domain and a human Fcγ receptor can be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between a human Fcγ receptor-binding domain and an Fcγ receptor. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, can also be used to determine the binding affinity between an Fcγ receptor-binding domain and an Fcγ receptor. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0223] As used herein, "the binding activity of an Fc region with altered FcγR binding to an Fcγ receptor is higher than the binding activity of a native Fc region to an Fcγ receptor" means that the binding activity of an Fc region with altered FcγR binding to any one of the human Fcγ receptors, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb, is higher than the binding activity of a native Fc region to these human Fcγ receptors. For example, based on the above-mentioned analytical method, the binding activity of an antigen-binding molecule comprising an Fc region with altered FcγR binding is 105% or higher, preferably 110% or higher, 115% or higher, 120% or higher, 125% or higher, particularly preferably 130% or higher, 135% or higher, 140% or higher, 145% or higher, 150% or higher, 155% or higher, 160% or higher, compared to the binding activity of an antigen-binding molecule comprising a native Fc region of human IgG as a control. % or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more binding activity. As the native Fc region, a starting Fc region or a native Fc region of an antibody of the same subclass can be used.

[0224] In the present invention, the native Fc region of native human IgG used as a control is preferably an Fc region of native human IgG in which the sugar chain attached to the amino acid at position 297 (EU numbering) is a fucose-containing sugar chain. Whether or not the sugar chain attached to the amino acid at position 297 (EU numbering) is a fucose-containing sugar chain can be determined by the method described in Non-Patent Document 6. For example, it is possible to determine whether or not the sugar chain attached to the Fc region of native human IgG is a fucose-containing sugar chain by the method described below. The test native human IgG is reacted with N-Glycosidase F (Roche diagnostics) to release glycans from the test native human IgG (Weitzhandler et al. (J. Pharma. Sciences (1994) 83, 12, 1670-1675)). Next, the reaction mixture is reacted with ethanol to remove proteins (Schenk et al. (J. Clin. Investigation (2001) 108 (11) 1687-1695)). The resulting concentrate is then fluorescently labeled with 2-aminopyridine (Bigge et al. (Anal. Biochem. (1995) 230 (2) 229-238)). The fluorescently labeled 2-AB-glycans are removed by solid-phase extraction using a cellulose cartridge and analyzed by normal-phase chromatography. By observing the peaks in the chromatogram, it is possible to determine whether the glycans bound to the native Fc region of human IgG are fucose-containing glycans.

[0225] As a control antigen-binding molecule containing a native Fc region of an antibody of the same subclass, an antigen-binding molecule containing the Fc region of an IgG monoclonal antibody can be used as appropriate. The structures of the Fc regions are set forth in SEQ ID NOs: 5 (Database Accession No. AAC82527.1 with an A added to the N-terminus), 6 (Database Accession No. AAB59393.1 with an A added to the N-terminus), 7 (Database Accession No. CAA27268.1), and 8 (Database Accession No. AAB59394.1 with an A added to the N-terminus). Furthermore, when an antigen-binding molecule containing an Fc region of an antibody of a certain isotype is used as a test substance, the effect of the Fcγ receptor-binding activity of the antigen-binding molecule containing the test Fc region is verified by using an antigen-binding molecule containing an Fc region of an IgG monoclonal antibody of that specific isotype as a control. As described above, an antigen-binding molecule containing an Fc region verified to have high Fcγ receptor-binding activity is appropriately selected.

[0226] Fc region with selective Fcγ receptor binding activity Other preferred examples of Fcγ receptor-binding domains for use in the present invention include Fcγ receptor-binding domains that have higher binding activity for a specific Fcγ receptor than for other Fcγ receptors (Fcγ receptor-binding domains that have selective Fcγ receptor-binding activity). When an antibody is used as the antigen-binding molecule (an Fc region is used as the Fcγ receptor-binding domain), a single antibody molecule can only bind to a single Fcγ receptor, and therefore a single antigen-binding molecule cannot bind to other activating FcγRs when bound to an inhibitory Fcγ receptor, and cannot bind to other activating Fcγ receptors or inhibitory Fcγ receptors when bound to an activating Fcγ receptor.

[0227] Fc region with higher binding activity to activating Fcγ receptors than to inhibitory Fcγ receptors As described above, preferred examples of activating Fcγ receptors include FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc; FcγRIII (CD16), which includes FcγRIIa, FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Preferred examples of inhibitory Fcγ receptors include FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

[0228] Herein, an example of a region having a higher binding activity to a specific Fcγ receptor than to other Fcγ receptors is one in which the binding activity to an activating Fcγ receptor is higher than the binding activity to an inhibitory Fcγ receptor, which means that the binding activity of the Fc region to any one of the human Fcγ receptors, FcγRIa, FcγRIIa, FcγRIIIa, and / or FcγRIIIb, is higher than the binding activity to FcγRIIb. For example, based on the above-mentioned analytical methods, the binding activity of an antigen-binding molecule comprising an Fc region towards any one of human Fcγ receptors FcγRIa, FcγRIIa, FcγRIIIa, and / or FcγRIIIb is 105% or more, preferably 110% or more, 120% or more, 130% or more, 140% or more, particularly preferably 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 450% or more, 500% or more, 750% or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times or more that of the binding activity towards FcγRIIb. An Fc region whose binding activity to activating Fcγ receptors is higher than that to inhibitory Fcγ receptors can be suitably contained in an antigen-binding molecule of the present invention in which the antigen-binding domain binds to a membrane-type molecule. IgG1 antibodies comprising such an Fc region are known to have enhanced ADCC activity, as described below, and therefore, antigen-binding molecules comprising such Fc regions are also useful as antigen-binding molecules to be contained in the pharmaceutical compositions of the present invention.

[0229] In one non-limiting embodiment of the present invention, examples of Fc regions whose binding activity to activating Fcγ receptors is higher than that to inhibitory Fcγ receptors (having selective binding activity to inhibitory Fcγ receptors) include Fc regions at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, and 236 according to the EU numbering system described above. , 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 247th, 249th, 250th, 251st, 254th, 255th, 256th, 258th, 260th, 2 62nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 33 Preferred examples of such Fc regions include those in which at least one amino acid selected from the group consisting of positions 0, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 has been altered to an amino acid different from that of a native Fc region.

[0230] In one non-limiting embodiment of the present invention, preferred examples of Fc regions whose binding activity to activating Fcγ receptors is higher than that to inhibitory Fcγ receptors (which have selective binding activity to inhibitory Fcγ receptors) include Fc regions in which multiple amino acids listed in Tables 1-1 to 1-3 have been altered to amino acids different from those in native Fc regions.

[0231] Fc region with higher binding activity to inhibitory Fcγ receptors than to activating Fcγ receptors Herein, an example of a region having higher binding activity to a specific Fcγ receptor than to other Fcγ receptors is one in which the binding activity to an inhibitory Fcγ receptor is higher than the binding activity to an activating Fcγ receptor, which means that the binding activity of the Fc region to FcγRIIb is higher than the binding activity to any of the human Fcγ receptors FcγRIa, FcγRIIa, FcγRIIIa, and / or FcγRIIIb. For example, based on the above-mentioned analytical methods, the binding activity of an antigen-binding molecule comprising an Fc region to FcγRIIb means that the binding activity to any one of the human Fcγ receptors FcγRIa, FcγRIIa, FcγRIIIa, and / or FcγRIIIb is 105% or more, preferably 110% or more, 120% or more, 130% or more, 140% or more, particularly preferably 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 450% or more, 500% or more, 750% or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times or more. An Fc region whose binding activity to inhibitory Fcγ receptors is higher than that to activating Fcγ receptors can be suitably contained in an antigen-binding molecule of the present invention, in which the antigen-binding domain binds to a soluble molecule.

[0232] In one non-limiting embodiment of the present invention, preferred examples of Fc regions whose binding activity to inhibitory Fcγ receptors is higher than that to activating Fcγ receptors (which have selective binding activity to inhibitory Fcγ receptors) include Fc regions in which the amino acid at position 238 or 328 (EU numbering) of the Fc region has been altered to an amino acid different from that in a native Fc region.

[0233] In a non-limiting embodiment of the present invention, preferred examples of Fc regions whose binding activity to inhibitory Fcγ receptors is higher than that to activating Fcγ receptors (which have selective binding activity to inhibitory Fcγ receptors) include Fc regions in which one or more amino acids according to EU numbering have been altered to Asp at position 238 or Glu at position 328. Furthermore, Fc regions or alterations described in US2009 / 0136485 can also be appropriately selected as Fc regions with selective binding activity to inhibitory Fcγ receptors.

[0234] In a non-limiting embodiment of the present invention, preferred examples include Fc regions in which the amino acids at position 238 (EU numbering) have been altered to Asp or the amino acid at position 328 (EU numbering) have been altered to Glu.

[0235] Furthermore, in a non-limiting embodiment of the present invention, as exemplified in PCT / JP2012 / 054624, a substitution of Pro at position 238 (EU numbering) with Asp, and a substitution of Trp at position 237 (EU numbering), Phe at position 237 (EU numbering), Val at position 267 (EU numbering), Gln at position 267 (EU numbering), Asn at position 268 (EU numbering), Gly at position 271 (EU numbering), and a substitution of Glu at position 272 (EU numbering) with Glu. The amino acid at position 326 in the EU numbering system is Leu, the amino acid at position 326 in the EU numbering system is Gln, the amino acid at position 326 in the EU numbering system is Glu, the amino acid at position 326 in the EU numbering system is Met, the amino acid at position 239 in the EU numbering system is Asp, the amino acid at position 267 in the EU numbering system is Ala, the amino acid at position 234 in the EU numbering system is Trp, the amino acid at position 234 in the EU numbering system is Tyr, the amino acid at position 237 in the EU numbering system is Ala, the amino acid at position 237 in the EU numbering system is The amino acid at position 237 in EU numbering is Asp, the amino acid at position 237 in EU numbering is Glu, the amino acid at position 237 in EU numbering is Leu, the amino acid at position 237 in EU numbering is Met, the amino acid at position 237 in EU numbering is Tyr, the amino acid at position 330 in EU numbering is Lys, the amino acid at position 330 in EU numbering is Arg, the amino acid at position 233 in EU numbering is Asp, the amino acid at position 268 in EU numbering is Asp, the amino acid at position 268 in EU numbering is Glu, the amino acid at position 326 of the EU numbering is Asp, the amino acid at position 326 of the EU numbering is Ser, the amino acid at position 326 of the EU numbering is Thr, the amino acid at position 323 of the EU numbering is Ile, the amino acid at position 323 of the EU numbering is Leu, the amino acid at position 323 of the EU numbering is Met, the amino acid at position 296 of the EU numbering is Asp, the amino acid at position 326 of the EU numbering is Ala, the amino acid at position 326 of the EU numbering is Asn,and Fc regions in which the amino acid at position 330 (EU numbering) has been altered to Met.

[0236] Glycosylated Fc region The Fc region contained in the antigen-binding molecules provided by the present invention may also include an Fc region in which the composition of the sugar chains bound to the Fc region has been modified so that the proportion of Fc regions bound to fucose-deficient sugar chains is increased, or so that the proportion of Fc regions to which bisecting N-acetylglucosamine has been added is increased. It is known that removing a fucose residue from the N-acetylglucosamine at the reducing end of an N-glycoside-linked complex sugar chain bound to an antibody Fc region enhances affinity for FcγRIIIa (Non-Patent Document 6). IgG1 antibodies containing such an Fc region are known to have enhanced ADCC activity, as described below, and therefore antigen-binding molecules containing such Fc regions are also useful as antigen-binding molecules contained in the pharmaceutical compositions of the present invention. Examples of antibodies in which a fucose residue has been removed from the N-acetylglucosamine at the reducing end of an N-glycoside-linked complex sugar chain bound to the antibody Fc region include the following antibodies: Antibodies with modified glycosylation (International Publication WO1999 / 054342, etc.), Antibodies lacking fucose that is added to sugar chains (International Publication Nos. WO2000 / 061739, WO2002 / 031140, WO2006 / 067913, etc.),

[0237] More specifically, as a non-limiting example of an antibody in which the fucose residue has been removed from the N-acetylglucosamine at the reducing end of the complex N-glycoside-linked sugar chain attached to the antibody Fc region, an antibody lacking fucose added to its sugar chain (International Publication Nos. WO2000 / 061739, WO2002 / 031140, WO2006 / 067913, etc.) is produced by modifying the activity of forming a sugar chain structure in a polypeptide to be glycosylated, thereby producing a host cell with a reduced ability to add fucose to its sugar chain. By expressing a desired antibody gene in the host cell, the antibody lacking fucose in its sugar chain can be recovered from the culture medium of the host cell. Preferred, but non-limiting examples of the activity of an enzyme or transporter that forms a glycosylation structure on a polypeptide include fucosyltransferase (EC 2.4.1.152), fucose transporter (SLC35C1), GMD (GDP-mannose 4,6-dehydratase) (EC 4.2.1.47), Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase) (EC 1.1.1.271), and GFPP (GDP-β-L-fucose pyrophosphorylase) (EC 2.7.7.30). The structure of these enzymes or transporters is not necessarily specified as long as they can exert their activity. Herein, proteins capable of exerting these activities are referred to as functional proteins. A non-limiting example of a method for modifying these activities is deletion of these activities. To prepare host cells lacking these activities, known methods, such as methods for disrupting the genes encoding these functional proteins to render them nonfunctional, can be appropriately employed (International Publication Nos. WO2000 / 061739, WO2002 / 031140, WO2006 / 067913, etc.). Host cells lacking such activities can be prepared by methods, such as disrupting the genes encoding these functional proteins endogenous to CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridoma cells, or the like, to render them nonfunctional.

[0238] Antibodies having sugar chains with bisecting GlcNAc are known (International Publication No. WO 2002 / 079255, etc.). In a non-limiting embodiment, to produce antibodies having sugar chains with bisecting GlcNAc, host cells are produced that express a gene encoding a functional protein having GnTIII (β-1,4-mannosyl-glycoprotein, 4-β-N-acetylglucosaminyltransferase) (EC 2.4.1.144) activity or GalT (β-1,4-galactosyltransferase) (EC 2.4.1.38) activity. In another non-limiting preferred embodiment, a host cell is produced that co-expresses, in addition to the above-mentioned functional protein, a gene encoding a functional protein having human ManII (mannosidase II) (3.2.1.114) activity, a gene encoding a functional protein having GnTI (β-1,2-acetylglucosaminyltransferase I) (EC 2.4.1.94) activity, a gene encoding a functional protein having GnTII (β-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143) activity, a gene encoding a functional protein having ManI (mannosidase) (EC 3.2.1.113) activity, and α-1,6-fucosyltransferase (EC 2.4.1.68) (International Publication WO2004 / 065540).

[0239] By transducing an expression vector containing an antibody gene into host cells with a low ability to add fucose to sugar chains as described above, and into host cells with the activity to form sugar chains containing bisecting GlcNAc structures, antibodies in which the fucose residue has been removed from the N-acetylglucosamine at the reducing end of the complex-type N-glycoside-linked sugar chain attached to the antibody Fc region, and antibodies having sugar chains with a bisecting GlcNAc, can be produced, respectively. The methods for producing these antibodies can also be applied to producing antigen-binding molecules of the present invention containing modified Fc regions in which the composition of sugar chains attached to the Fc region has been modified so that the proportion of Fc regions with fucose-deficient sugar chains or the proportion of Fc regions with bisecting N-acetylglucosamine added is increased. The composition of sugar chains attached to the Fc region contained in the antigen-binding molecules of the present invention produced by such production methods can be confirmed by the method described above under "Fc Regions with Modified Fcγ Receptor (FcγR) Binding."

[0240] Multispecific or multiparatopic antigen-binding molecules An antigen-binding molecule comprising at least two antigen-binding domains, wherein at least one antigen-binding domain binds to a first epitope in an antigen molecule and at least one other antigen-binding domain binds to a second epitope in the antigen molecule, is called a multispecific antigen-binding molecule from the viewpoint of the specificity of its reaction. When an antigen-binding molecule binds to two different epitopes via two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a bispecific antigen-binding molecule. When an antigen-binding molecule binds to three different epitopes via three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a trispecific antigen-binding molecule.

[0241] The paratope in the antigen-binding domain that binds to a first epitope in an antigen molecule and the paratope in the antigen-binding domain that binds to a second epitope structurally different from the first epitope are structurally different from each other. Therefore, an antigen-binding molecule that contains at least two antigen-binding domains, in which at least one antigen-binding domain binds to a first epitope in an antigen molecule and at least one other antigen-binding domain binds to a second epitope in the antigen molecule, is called a multiparatopic antigen-binding molecule from the perspective of its structural specificity. When an antigen-binding molecule binds to two different epitopes via two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a biparatopic antigen-binding molecule. Furthermore, when an antigen-binding molecule binds to three different epitopes via three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a tripleparatopic antigen-binding molecule.

[0242] Multivalent, multispecific or multiparatopic antigen-binding molecules comprising one or more antigen-binding domains and methods for their preparation are also described in non-patent literature such as Conrath et al. (J. Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302), and Kontermann RE (2011) Bispecific Antibodies (Springer-Verlag), as well as in patent literature such as International Publication No. WO1996 / 034103 or WO1999 / 023221. The antigen-binding molecules of the present invention can be produced using the multispecific or multiparatopic antigen-binding molecules and methods for their preparation described therein.

[0243] Bispecific antibodies and methods for producing them As one embodiment of the above-mentioned multispecific or multiparatopic antigen-binding molecules and methods for preparing them, bispecific antibodies and methods for producing them are exemplified below. Bispecific antibodies are antibodies that contain two types of variable regions that specifically bind to different epitopes. IgG-type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein et al., Nature (1983) 305, 537-540).

[0244] When producing bispecific antibodies using recombinant techniques such as those described above in the antibody section, a method can be used in which genes encoding heavy chains containing two desired variable regions are introduced into cells and co-expressed. However, even considering the heavy chain combinations in such co-expression methods, the resulting mixture contains (i) a heavy chain combination in which one heavy chain containing a variable region that binds to a first epitope is paired with another heavy chain containing a variable region that binds to a second epitope, (ii) a heavy chain combination in which only heavy chains containing a variable region that binds to the first epitope are paired, and (iii) a heavy chain combination in which only heavy chains containing a variable region that binds to the second epitope are paired, in a molecular ratio of 2:1:1. It is difficult to purify antigen-binding molecules containing the desired heavy chain combination from a mixture of these three types of heavy chain combinations.

[0245] When producing bispecific antibodies using such recombinant techniques, bispecific antibodies containing heterogeneous heavy chain combinations can be preferentially secreted by modifying the CH3 domains of the heavy chains with appropriate amino acid substitutions. Specifically, the amino acid side chains in the CH3 domain of one heavy chain are replaced with larger side chains (knobs) and the amino acid side chains in the CH3 domain of the other heavy chain are replaced with smaller side chains (holes) so that the knobs can be positioned in the holes, promoting heterologous heavy chain formation and inhibiting homologous heavy chain formation (International Publication No. WO1996027011; Ridgway et al., Protein Engineering (1996) 9, 617-621; Merchant et al., Nat. Biotech. (1998) 16, 677-681).

[0246] Furthermore, techniques for producing bispecific antibodies are known that utilize methods for controlling the association of polypeptides or heteromultimers composed of polypeptides in the association of heavy chains. Specifically, a method can be employed to produce bispecific antibodies by modifying amino acid residues that form an interface within the heavy chain, thereby inhibiting the association of heavy chains with identical sequences and controlling the formation of two heavy chains with different sequences (International Publication No. WO 2006 / 106905). Such methods can also be employed when producing bispecific antibodies.

[0247] In one non-limiting embodiment of the present invention, the Fc region comprised in the antigen-binding molecule may suitably be two polypeptides that form an Fc region derived from the bispecific antibody described above. More specifically, two polypeptides that form an Fc region, in which the amino acid at position 349 (EU numbering) in the amino acid sequence of one polypeptide is Cys and the amino acid at position 366 (Trp), and the amino acid at position 356 (EU numbering) in the amino acid sequence of the other polypeptide is Cys, Ser, Ala, and Val, as indicated by EU numbering, are preferably used.

[0248] In another non-limiting embodiment of the present invention, an Fc region preferably comprises two polypeptides that form an Fc region, wherein the amino acid at position 409 (EU numbering) in the amino acid sequence of one polypeptide is Asp, and the amino acid at position 399 (EU numbering) in the amino acid sequence of the other polypeptide is Lys. In this embodiment, the Asp at position 409 may be replaced by Glu, and the Lys at position 399 may be replaced by Arg. Furthermore, in addition to the Lys at position 399, Asp may also be preferably added as the amino acid at position 360 or Asp as the amino acid at position 392.

[0249] In another non-limiting embodiment of the present invention, an Fc region preferably includes two polypeptides that form an Fc region, wherein the amino acid at position 370 (EU numbering) in the amino acid sequence of one polypeptide is Glu, and the amino acid at position 357 (EU numbering) in the amino acid sequence of the other polypeptide is Lys.

[0250] In yet another non-limiting embodiment of the present invention, the Fc region preferably includes two polypeptides that form an Fc region, wherein the amino acid at position 439 (EU numbering) in the amino acid sequence of one polypeptide is Glu, and the amino acid at position 356 (EU numbering) in the amino acid sequence of the other polypeptide is Lys.

[0251] In another non-limiting embodiment of the present invention, the Fc region may be any of the following embodiments in combination: (i) two polypeptides forming an Fc region, wherein in the amino acid sequence of one polypeptide, the amino acid at position 409 is Asp and the amino acid at position 370 is Glu according to EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys and the amino acid at position 357 is Lys according to EU numbering (in this embodiment, Asp may be substituted for Glu at amino acid position 370 according to EU numbering, and Asp may be substituted for Glu at amino acid position 370 according to EU numbering), (ii) two polypeptides forming an Fc region, wherein in the amino acid sequence of one polypeptide, the amino acid at position 409 is Asp and the amino acid at position 439 is Glu according to EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys and the amino acid at position 356 is Lys according to EU numbering (in this embodiment, the Glu at position 439 according to EU numbering may be replaced by Asp at position 360, Asp at position 392, or Asp at position 439); (iii) two polypeptides forming an Fc region, wherein the amino acid at position 370 and the amino acid at position 439 are Glu and Glu, respectively, as indicated by EU numbering in the amino acid sequence of one polypeptide, and the amino acid at position 357 and the amino acid at position 356 are Lys and Lys, respectively, as indicated by EU numbering in the amino acid sequence of the other polypeptide; or two polypeptides forming an Fc region, wherein in the amino acid sequence of one polypeptide, the amino acid at position 409 is Asp, the amino acid at position 370 is Glu, and the amino acid at position 439 is Glu, as indicated by EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys, the amino acid at position 357 is Lys, and the amino acid at position 356 is Lys, as indicated by EU numbering (in this embodiment, the amino acid at position 370 (EU numbering) does not need to be substituted with Glu, and further, in addition to not substituting the amino acid at position 370 with Glu, the Glu at position 439 may be replaced with Asp, or the Glu at position 439 may be replaced with Asp, or the Glu at position 392 may be replaced with Asp); is preferably used.

[0252] Furthermore, in another non-limiting embodiment of the present invention, two polypeptides that form an Fc domain, in which the amino acid at position 356 (EU numbering) in the amino acid sequence of one polypeptide is Lys, and the amino acid at position 435 (EU numbering) and the amino acid at position 439 (Glu) in the amino acid sequence of the other polypeptide are also preferably used.

[0253] Furthermore, in another non-limiting embodiment of the present invention, two polypeptides that form an Fc domain, in which the amino acids at positions 356 and 357, as indicated by EU numbering, in the amino acid sequence of one polypeptide, are Lys and Lys, respectively, and the amino acids at positions 370, 435, and 439, as indicated by EU numbering, in the amino acid sequence of the other polypeptide, are also preferably used.

[0254] In addition to the above-described heterogeneous heavy chain assembly techniques, the CrossMab technique (Scaefer et al. (Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192)), known as a heterogeneous light chain assembly technique, in which a light chain forming a variable region that binds to a first epitope and a light chain forming a variable region that binds to a second epitope are assembled with a heavy chain forming a variable region that binds to the first epitope and a heavy chain forming a variable region that binds to the second epitope, respectively, can also be used to produce the multispecific or multiparatopic antigen-binding molecules provided by the present invention. Fab-Arm Exchange (Labrijn et al. (Proc. Natl. Acad. Sci. USA (2013) 110, 5145-5150), WO2008119353), a technique for associating heterologous heavy chains, takes advantage of the exchange that occurs between heavy chains of different IgG4s to associate a heavy chain forming a variable region that binds to a first epitope with a heavy chain forming a variable region that binds to a second epitope, can also be used to create the multispecific or multiparatopic antigen-binding molecules provided by the present invention.

[0255] Effector cells In the present invention, "effector cells" refer to T cells (CD4 + (Helper lymphocytes) T cells and / or CD8 + Although the term "effector cell" can be used in the broadest sense to include leukocytes such as (cytotoxic) T cells, polymorphonuclear leukocytes (neutrophils, eosinophils, basophils, mast cells), monocytes, macrophages, histiocytes, or natural killer cells (NK cells), NK-like T cells, Kupffer cells, Langerhans cells, or lymphokine-activated killer cells (LAK cells), B lymphocytes, or antigen-presenting cells such as dendritic cells or macrophages, examples of suitable effector cells include CD8 +(Cytotoxic) T cells, NK cells, or macrophages. Any membrane-type molecule expressed on the cell membrane of effector cells can be used as an antigen to which at least one antigen-binding domain contained in the antigen-binding molecule of the present invention binds. Suitable membrane-type molecules include, but are not limited to, polypeptides constituting TCR, CD3, CD2, CD28, CD44, CD16, CD32, CD64, or NKG2D or NK cell-activating ligands.

[0256] cytotoxic substances A cytotoxic substance may be conjugated to the antigen-binding molecule of the present invention so that the antigen-binding molecule can bind to cancer cells and exert cytotoxic activity. The cytotoxic substance may be a chemotherapeutic agent exemplified below, or a compound disclosed in Curr Opin Chem Biol (2010) 14, 529-37 or WO 2009 / 140242, and these compounds are conjugated to the antigen-binding molecule via an appropriate linker or the like. When the antigen-binding molecule of the present invention is used as a pharmaceutical composition, the cytotoxic substance can be conjugated to the antigen-binding molecule before administration to a subject (e.g., a human subject, a patient), or can be administered before, after, or simultaneously with administration.

[0257] Modified antigen-binding molecules conjugated with cytotoxic substances such as chemotherapeutic agents, toxic peptides, or radioactive chemicals, as described below, can also be suitably used as antigen-binding molecules with cytotoxic activity of the present invention. Such modified antigen-binding molecules (hereinafter referr...

Claims

1. An antigen-binding molecule whose binding activity to an antigen changes depending on the concentration of a target tissue-specific compound, wherein the antigen-binding molecule comprises an antibody heavy chain variable region and a light chain variable region, the compound is at least one compound selected from adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate, and the antigen-binding molecule binds to CEACAM5.

2. The antigen-binding molecule according to claim 1, wherein the target tissue is cancer tissue.

3. An antigen-binding molecule according to any one of claims 1 to 2, having neutralizing activity.

4. An antigen-binding molecule according to any one of claims 1 to 3, which has cytotoxic activity.

5. The antigen-binding molecule according to any one of claims 1 to 4, comprising an Fc region.

6. The antigen-binding molecule according to claim 5, wherein the Fc region is an Fc region included in the constant region described in SEQ ID NO: 5, 6, 7, or 8.

7. The antigen-binding molecule according to claim 5, wherein the Fc region includes an FcγR-binding modified Fc region having a higher binding activity to the Fcγ receptor than the binding activity to the Fcγ receptor of the Fc region of natural human IgG.

8. Among the amino acid sequences of the modified Fc region of the FcγR bond mentioned above, positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, and 250, as represented by EU numbering. , 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 27th 2nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 2 92nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th , 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th The antigen-binding molecule according to claim 7, wherein at least one amino acid selected from the group consisting of amino acids at positions 1, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, or 440 is different from the amino acids in the Fc region of natural human IgG.

9. The amino acid sequence of the modified Fc region of the aforementioned FcγR binding is represented by EU numbering: The amino acid at position 221 is either Lys or Tyr. The amino acid at position 222 is one of Phe, Trp, Glu, or Tyr. The amino acid at position 223 is one of Phe, Trp, Glu, or Lys. The amino acid at position 224 is one of Phe, Trp, Glu, or Tyr. The amino acid at position 225 is either Glu, Lys, or Trp. The amino acid at position 227 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 228 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 230 is one of Ala, Glu, Gly, or Tyr. The amino acid at position 231 is one of Glu, Gly, Lys, Pro, or Tyr. The amino acid at position 232 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 233 is one of the following: Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 234 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 235 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 236 is one of the following: Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 237 is one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 238 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 239 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 240 is one of Ala, Ile, Met, or Thr. The amino acid at position 241 is one of Asp, Glu, Leu, Arg, Trp, or Tyr. The amino acid at position 243 is one of Leu, Glu, Leu, Gln, Arg, Trp, or Tyr. The amino acid at position 244 is His. The amino acid at position 245 is Ala. The amino acid at position 246 is one of Asp, Glu, His, or Tyr. The amino acid at position 247 is one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr. The amino acid at position 249 is one of Glu, His, Gln, or Tyr. The amino acid at position 250 is either Glu or Gln. The amino acid at position 251 is Phe, The amino acid at position 254 is either Phe, Met, or Tyr. The amino acid at position 255 is either Glu, Leu, or Tyr. The amino acid at position 256 is either Ala, Met, or Pro. The amino acid at position 258 is one of Asp, Glu, His, Ser, or Tyr. The amino acid at position 260 is one of Asp, Glu, His, or Tyr. The amino acid at position 262 is one of Ala, Glu, Phe, Ile, or Thr. The amino acid at position 263 is one of Ala, Ile, Met, or Thr. The amino acid at position 264 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 265 is one of the following: Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 266 is one of Ala, Ile, Met, or Thr. The amino acid at position 267 is one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 268 is one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp. The amino acid at position 269 is one of the following: Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 270 is one of the following: Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 271 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 272 is one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 273 is either Phe or Ile. The amino acid at position 274 is one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 275 is either Leu or Trp. The amino acid at position 276 is one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 278 is one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. The amino acid ranked 279th is Ala. The amino acid at position 280 is one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr. The amino acid at position 281 is one of Asp, Lys, Pro, or Tyr. The amino acid at position 282 is one of Glu, Gly, Lys, Pro, or Tyr. The amino acid at position 283 is one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr. The amino acid at position 284 is one of Asp, Glu, Leu, Asn, Thr, or Tyr. The amino acid at position 285 is one of Asp, Glu, Lys, Gln, Trp, or Tyr. The amino acid at position 286 is one of Glu, Gly, Pro, or Tyr. The amino acid at position 288 is one of Asn, Asp, Glu, or Tyr. The amino acid at position 290 is one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr. The amino acid at position 291 is one of Asp, Glu, Gly, His, Ile, Gln, or Thr. The amino acid at position 292 is one of Ala, Asp, Glu, Pro, Thr, or Tyr. The amino acid at position 293 is one of the following: Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 294 is one of the following: Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 295 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 296 is one of the following: Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val. The amino acid at position 297 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 298 is one of the following: Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 299 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. The amino acid at position 300 is one of the following: Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. The amino acid at position 301 is one of Asp, Glu, His, or Tyr. The amino acid at position 302 is Ile. The amino acid at position 303 is either Asp, Gly, or Tyr. The amino acid at position 304 is one of Asp, His, Leu, Asn, or Thr. The amino acid at position 305 is one of Glu, Ile, Thr, or Tyr. The amino acid at position 311 is one of Ala, Asp, Asn, Thr, Val, or Tyr. The amino acid at position 313 is Phe, The amino acid at position 315 is Leu. The amino acid at position 317 is Glu or Gln. The amino acid at position 318 is one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr. The amino acid at position 320 is one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 322 is one of the following: Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 323 is Ile. The amino acid at position 324 is one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 325 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 326 is one of the following: Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 327 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 328 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 329 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 330 is one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 331 is one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 332 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 333 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr. The amino acid at position 334 is one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr. The amino acid at position 335 is one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr. The amino acid at position 336 is either Glu, Lys, or Tyr. The amino acid at position 337 is either Glu, His, or Asn. The amino acid at position 339 is one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr. The amino acid at position 376 is either Ala or Val. The amino acid at position 377 is either Gly or Lys. The amino acid at position 378 is Asp. The amino acid at position 379 is Asn. The amino acid at position 380 is either Ala, Asn, or Ser. The amino acid at position 382 is either Ala or Ile. The amino acid at position 385 is Glu, The amino acid at position 392 is Thr. The amino acid at position 396 is Leu. The amino acid ranked 421st is Lys. The amino acid at position 427 is Asn. The amino acid at position 428 is either Phe or Leu. The amino acid at position 429 is Met. The amino acid at position 434 is Trp. The amino acid at position 436 is Ile, or The amino acid at position 440 is one of Gly, His, Ile, Leu, or Tyr. The antigen-binding molecule according to claim 8, comprising at least one amino acid selected from the group consisting of the following.

10. The antigen-binding molecule according to claim 5, wherein the Fc region is modified such that the composition of the glycan attached to EU numbering position 297 of the Fc region has a higher proportion of the Fc region to which a fucose-deficient glycan is attached, or a higher proportion of the Fc region to which bisecting N-acetylglucosamine is attached.

11. The antigen-binding molecule according to any one of claims 5 and 7 to 10, wherein the binding activity of the Fc region to FcRn under acidic pH conditions is enhanced compared to the binding activity of the Fc region to FcRn represented by any one of SEQ ID NOs: 5, 6, 7, or 8.

12. The aforementioned Fc region is the amino acid sequence of the Fc region included in the constant region described in SEQ ID NO: 5, 6, 7, or 8, specifically the positions represented by EU numbering at positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, and 332. The antigen-binding molecule according to claim 11, wherein the Fc region is substituted with at least one amino acid selected from the group consisting of amino acids at positions 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, or 447.

13. The aforementioned Fc region is an amino acid sequence of an Fc region included in the constant region described in Sequence ID No. 5, 6, 7, or 8, represented by EU numbering: The amino acid at position 238 is Leu. The amino acid at position 244 is Leu. The amino acid at position 245 is Arg. The amino acid ranked 249th is Pro. The amino acid at position 250 is either Gln or Glu. The amino acid at position 251 is one of Arg, Asp, Glu, or Leu. The amino acid at position 252 is one of Phe, Ser, Thr, or Tyr. The amino acid at position 254 is either Ser or Thr. The amino acid at position 255 is one of Arg, Gly, Ile, or Leu. The amino acid at position 256 is one of the following: Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr. The amino acid at position 257 is one of Ala, Ile, Met, Asn, Ser, or Val. The amino acid at position 258 is Asp. The amino acid at position 260 is Ser. The amino acid at position 262 is Leu. The amino acid at rank 270 is Lys. The amino acid at position 272 is either Leu or Arg. The amino acid at position 279 is one of the following: Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 283 is one of the following: Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 285 is Asn. The amino acid at position 286 is Phe, The amino acid at position 288 is either Asn or Pro. The amino acid ranked 293rd is Val. The amino acid at position 307 is one of Ala, Glu, Gln, or Met. The amino acid at position 311 is one of the following: Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp. The amino acid ranked 309th is Pro. The amino acid at position 312 is either Ala, Asp, or Pro. The amino acid at position 314 is either Ala or Leu. The amino acid at position 316 is Lys. The amino acid ranked 317th is Pro. The amino acid at position 318 is either Asn or Thr. The amino acid at position 332 is one of the following: Phe, His, Lys, Leu, Met, Arg, Ser, or Trp. The amino acid at position 339 is either Asn, Thr, or Trp. The amino acid ranked 341st is Pro. The amino acid at position 343 is one of the following: Glu, His, Lys, Gln, Arg, Thr, or Tyr. The amino acid ranked 375th is Arg. The amino acid at position 376 is one of the following: Gly, Ile, Met, Pro, Thr, or Val. The amino acid ranked 377th is Lys. The amino acid at position 378 is either Asp, Asn, or Val. The amino acid at position 380 is either Ala, Asn, Ser, or Thr. The amino acid at position 382 is one of the following: Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 385 is one of the following: Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr. The amino acid at position 386 is one of the following: Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr. The amino acid at position 387 is one of Ala, Arg, His, Pro, Ser, or Thr. The amino acid at position 389 is either Asn, Pro, or Ser. The amino acid at position 423 is Asn. The amino acid at position 427 is Asn. The amino acid at position 428 is either Leu, Met, Phe, Ser, or Thr. The amino acid at position 430 is one of the following: Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr. The amino acid at position 431 is either His or Asn. The amino acid at position 433 is one of Arg, Gln, His, Ile, Lys, Pro, or Ser. The amino acid at position 434 is one of Ala, Gly, His, Phe, Ser, Trp, or Tyr. The amino acid at position 436 is one of Arg, Asn, His, Ile, Leu, Lys, Met, or Thr. The amino acid at position 438 is one of Lys, Leu, Thr, or Trp. The amino acid at position 440 is Lys. The amino acid at position 442 is Lys, or The amino acid at position 308 is either Ile, Pro, or Thr. The antigen-binding molecule according to claim 12, comprising at least one amino acid selected from the group consisting of the following.

14. The antigen-binding molecule according to any one of claims 1 to 13, which is a multispecific or multiparatopic antigen-binding molecule containing multiple antigen-binding domains.

15. The antigen-binding molecule according to claim 14, wherein the antigen to which at least one antigen-binding domain binds is CEACAM5, and the antigen to which at least one antigen-binding domain binds is a membrane-type molecule expressed on the cell membrane of effector cells.

16. The antigen-binding molecule according to claim 15, wherein the effector cell is an NK cell, a macrophage, or a T cell.

17. The antigen-binding molecule according to claim 15 or 16, wherein the membrane molecule expressed on the cell membrane of effector cells is a polypeptide constituting the TCR, CD2, CD3, CD28, CD44, CD16, CD32, CD64, or NKG2D.

18. The antigen-binding molecule according to claim 14, wherein the antigen to which at least one antigen-binding domain binds is a membrane-type molecule expressed on the cell membrane of cancer cells, and the antigen to which at least one antigen-binding domain binds is a cytotoxic substance.

19. An antigen-binding molecule according to any one of claims 14 to 18, which is an antibody fragment.

20. An antigen-binding molecule according to any one of claims 1 to 18, which is an antibody.

21. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 20.

22. The pharmaceutical composition according to claim 21, which is administered in combination with further therapeutic agents.

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