Antigen-binding molecule for promoting elimination of antigen having plural bioactivities

Abstract

To provide an antigen-binding molecule for reducing an antigen concentration in plasma.SOLUTION: The present invention provides an antigen-binding molecule that promotes the elimination of, from blood, an antigen having two or more bioactivities, which are difficult to inhibit only with a single antigen-binding molecule, thus reducing the bioactivities. The present invention further provides a method for producing the antigen-binding molecule, and a pharmaceutical composition containing the molecule as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen-binding molecules that can reduce the in vivo physiological activity of antigens that have multiple physiological activities and therefore are difficult to inhibit in vitro by promoting their elimination from the blood (serum or plasma), and to pharmaceutical compositions containing such antigen-binding molecules as active ingredients. [Background technology]

[0002] Many known diseases are caused by an excessive increase in the amount of physiologically active substances (e.g., cytokines) present in plasma compared to healthy individuals, which disrupts the balance of physiological activities maintained in healthy individuals and leads to the onset of these diseases. One effective means of treating such diseases is to inhibit the physiological activity of the excess physiologically active substances. For example, antibodies that neutralize physiological activity by binding to a physiologically active antigen could be an effective therapeutic agent.

[0003] However, when an antigen has two or more physiological activities, one type of neutralizing antibody can usually inhibit only one of the physiological activities, and it is expected that it will be difficult to treat a disease caused by the physiologically active substance with such an antibody.

[0004] High Mobility Group Box 1 (HMGB1) is an example of a physiologically active substance with two or more physiological activities. HMGB1 was identified as a member of the HMG family, a nuclear protein that contributes to the stability of DNA conformation by binding to DNA. HMGB1 consists of 215 amino acids and is structurally broadly divided into three domains: the HMG A box, the HMG B box, and the acidic carboxyl terminus. Normally present intracellularly as a DNA-binding protein, it is released extracellularly from inflammatory cells and necrotic cells via active or passive mechanisms. Released HMGB1 is known to bind to various substances, such as DNA, lipopolysaccharide (LPS), and interleukin (IL)-1β, and activate various cell surface receptors, such as RAGE (Receptor for Advanced Glycation Endproducts), TLR4 (Toll-Like Receptor 4), and IL-1 receptor, thereby transducing signals into cells and inducing various inflammatory responses (Non-Patent Document 1). Furthermore, in sepsis model mice administered with LPS, elevated blood levels of HMGB1 were observed, and the administration of a polyclonal antibody against HMGB1 reduced the mortality rate of the mice (Non-Patent Document 2), suggesting that HMGB1 plays an important role in the onset of sepsis. Patent Document 1 discloses that several high-affinity monoclonal antibodies against HMGB1 were produced, which inhibited the binding of HMGB1 to RAGE or TLR4 and reduced the mortality rate in sepsis model mice. However, there is no description of the acquisition of an antibody that inhibits both the action of HMGB1 on RAGE and TLR4, and it is expected that it will be difficult to inhibit the multiple activities of HMGB1 with a single antibody.

[0005] Antibodies (IgG) maintain long plasma retention by binding to the neonatal Fc receptor (FcRn). Binding of IgG to FcRn is only observed under acidic conditions (pH 6.0) and is almost nonexistent under neutral conditions (pH 7.4). Normally, IgG is nonspecifically taken up by cells via endocytosis, but returns to the cell surface by binding to FcRn in endosomes under acidic conditions. Under neutral plasma conditions, it dissociates from FcRn. IgG that does not bind to FcRn proceeds to lysosomes and is degraded there. Introducing a mutation into the Fc region of IgG that abolishes FcRn binding under acidic conditions prevents IgG from recycling from endosomes to plasma, significantly impairing IgG plasma retention. A method for improving IgG plasma retention by enhancing FcRn binding under acidic conditions has been reported. By introducing amino acid substitutions into the Fc region of IgG to improve its binding to FcRn under acidic conditions, the recycling efficiency from endosomes to plasma increases, resulting in improved plasma retention. Conversely, it has been reported that when binding to FcRn under neutral conditions increases, even if IgG returns to the cell surface by binding to FcRn under the acidic conditions of the endosome, it does not dissociate from FcRn in plasma under neutral conditions, resulting in no change in plasma retention or even worsening (Non-Patent Documents 3-5).

[0006] Recently, an antibody that binds to an antigen in a pH-dependent manner has been reported (Patent Document 2). This antibody binds strongly to an antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in endosomes. After dissociating from the antigen, the antibody is recycled into plasma by FcRn and can re-bind to the antigen, allowing a single antibody to repeatedly bind to multiple antigens. Because the plasma retention of an antigen is significantly shorter than that of antibodies that have an FcRn-mediated recycling mechanism, binding of an antigen to an antibody typically prolongs its plasma retention and increases the antigen concentration in plasma. On the other hand, it has been reported that the above-mentioned pH-dependent antibody dissociates from the antigen in endosomes during the FcRn-mediated recycling process, thereby promoting the elimination of antigens from plasma compared to conventional antibodies (Patent Document 2). However, no antibody engineering techniques are known that can further improve the effect of promoting the elimination of antigens from plasma. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO1995 / 002187 [Patent Document 2] WO2009 / 125825 [Non-patent literature]

[0008] [Non-Patent Document 1] Sims GP et al., Annu. Rev. Immunol. (2010) 28, 367-388. [Non-patent document 2] Wang H et al., Science (1999) 285, 248-251. [Non-patent document 3] Yeung YA et al., J. Immunol. (2009) 182, 7663-71. [Non-patent document 4] Datta-Mannan A et al., J. Biol. Chem. (2007) 282, 1709-17. [Non-patent document 5] Dall'Acqua WF et al., J. Immunol. (2002) 169, 5171-80. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention was made in light of these circumstances, and aims to provide antigen-binding molecules that can reduce the physiological activity of antigens that have two or more physiological activities and therefore whose physiological activities are difficult to inhibit in vitro with a single antigen-binding molecule by promoting their elimination from blood (serum or plasma) with a single antigen-binding molecule in vivo. The present invention also aims to provide methods for producing the antigen-binding molecules and pharmaceutical compositions containing the antigen-binding molecules as active ingredients. [Means for solving the problem]

[0010] As a result of intensive research, the present inventors have found that even for an antigen-binding molecule that inhibits some physiological activities in vitro but not the remaining physiological activities of an antigen having two or more physiological activities, (i) binds to human FcRn (Neonatal Fc Receptor) under acidic pH conditions; (ii) binds to human FcRn and / or human Fcγ receptors more strongly than native human IgG under a neutral pH range condition; and (iii) The antigen-binding activity changes depending on the ion concentration. We have newly discovered that by imparting this property, it is possible to promote the elimination of antigens from the blood (serum or plasma), thereby reducing their physiological activity in vivo.

[0011] The present invention is based on such findings and specifically relates to the following inventions. [1] An antigen-binding molecule that reduces an antigen concentration in plasma, the antigen-binding molecule having the following characteristics (1) to (6): (1) The antigen-binding molecule comprises an antigen-binding domain and at least one receptor-binding domain; (2) The receptor-binding domain has the activity of binding to human FcRn (Neonatal Fc Receptor) under acidic pH conditions; (3) the binding activity of the receptor-binding domain to a human Fc receptor is higher than that of native human IgG under a neutral pH range condition; (4) The antigen-binding activity of the antigen-binding domain changes depending on the ion concentration conditions. (5) The antigen has two or more physiological activities; (6) Upon binding of the antigen-binding molecule, one or more physiological activities of the antigen are inhibited, while at least one physiological activity is maintained. [2] The antigen-binding molecule of [1], which, upon binding to an antigen, inhibits one or more of the antigen's binding activities with target molecules while maintaining its binding activity with at least one target molecule. [3] the antigen-binding molecule of [1] or [2], wherein the reduction in plasma antigen concentration is due to enhanced uptake of the antigen into cells; [4] The antigen-binding molecule of any one of [1] to [3], wherein the antigen concentration in plasma is reduced, thereby reducing the physiological activity of the antigen in the body. [5] The antigen-binding molecule of any one of [1] to [4], wherein the antigen is HMGB1 (High Mobility Group Box 1). [6] The antigen-binding molecule of [5], which inhibits the binding of HMGB1 to RAGE (Receptor for Advanced Glycation Endproducts). [7] The antigen-binding molecule of [5] or [6], which inhibits the binding of HMGB1 to TLR4 (Toll-Like Receptor 4). [8] The antigen-binding molecule of any one of [1] to [4], wherein the antigen is CTGF (Connective Tissue Growth Factor). [9] the antigen-binding molecule of any one of [1] to [8], wherein the human Fc receptor is human FcRn;

[10] the antigen-binding molecule of [9], wherein the receptor-binding domain comprises an IgG Fc region in which at least one amino acid has been modified;

[11] The amino acid modifications in the Fc region of IgG are: 234, 235, 236, 237, 238, 239, 244, 245, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 267, 270, 272, 274, 279, 280, 282, 283, 284, 285, 286, 288, 289, 293, 295, 297, 298, 303, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 325, 326, 327, 328, 329, 330, 332, 334, 338, 339, 340, 341, 343, 345, 360, 361, 362, 375, 376, 377, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 391, 413, 422, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, The antigen-binding molecule of

[10] , wherein the modification is at least one amino acid selected from positions 437, 438, 440, and 442.

[12] The amino acid modification in the Fc region of IgG is based on EU numbering; The 234th amino acid is Arg, The 235th amino acid is Gly, Lys, or Arg; The 236th amino acid is Ala, Asp, Lys, or Arg; The 237th amino acid is Lys, Met, or Arg; The amino acid at position 238 is Ala, Asp, Lys, Leu, or Arg; The 239th amino acid is Asp or Lys, The 244th amino acid is Leu, The 245th amino acid is Arg, The 248th amino acid is Ile or Tyr, The 249th amino acid is Pro, The 250th amino acid is Ala, Glu, Phe, Ile, Met, Gln, Ser, Val, Trp, Gly, His, Leu, Asn, or Tyr; The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, Trp, or Tyr; The 253rd amino acid is Val, The 254th amino acid is Ala, Gly, His, Ile, Gln, Ser, Val, or Thr; The 255th amino acid is Ala, Asp, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Gly, Ser, Trp, Tyr, or Glu; The 256th amino acid is Ala, Asp, Glu, Arg, Asn, Pro, Thr, Ser, or Gln; The 257th amino acid is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The 258th amino acid is Asp or His, The 260th amino acid is Ser, The 262nd amino acid is Leu, The 265th amino acid is Ala, The 267th amino acid is Met or Leu, The 270th amino acid is Lys or Phe, The 272nd amino acid is Ala, Leu, or Arg; The 274th amino acid is Ala, The 279th amino acid is Leu, Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 280th amino acid is Ala, Gly, His, Lys, Asn, Gln, Arg, Ser, Thr, or Glu; The 282nd amino acid is Ala or Asp, The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 284th amino acid is Lys, The 285th amino acid is Asn, The 286th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr or Glu; The 288th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Val, Trp, Tyr or Ser; The 289th amino acid is His, The 293rd amino acid is Val, The 295th amino acid is Met, The 297th amino acid is Ala, The 298th amino acid is Gly. The 303rd amino acid is Ala, The 305th amino acid is Ala or Thr, The 307th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The 308th amino acid is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The 309th amino acid is Ala, Asp, Glu, Pro, His, or Arg; The 311th amino acid is Ala, His, Glu, Lys, Leu, Met, Ser, Val, Trp, or Ile; The 312th amino acid is Ala, Asp, Pro, or His; The 313th amino acid is Tyr or Phe, The 314th amino acid is Ala, Leu, Lys, or Arg; The 315th amino acid is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, Tyr or His; The 316th amino acid is Ala, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Asp; The 317th amino acid is Ala or Pro, The 318th amino acid is Asn or Thr, The 325th amino acid is Ala, Gly, Met, Leu, Ile, or Ser; The 326th amino acid is Asp, The 327th amino acid is Gly, The amino acid at position 328 is Arg, Asp, Glu, or Tyr; The 329th amino acid is Lys or Arg, The 330th amino acid is Leu, The 332nd amino acid is Glu, Phe, His, Lys, Leu, Met, Arg, Ser, Trp, or Val; The 334th amino acid is Leu, The 338th amino acid is Ala, The 339th amino acid is Asn, Thr, or Trp; The 340th amino acid is Ala, The 341st amino acid is Pro, The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 345th amino acid is Ala, The 360th amino acid is His, The 361st amino acid is Ala, The 362nd amino acid is Ala, The 375th amino acid is Ala or Arg, The 376th amino acid is Ala, Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys, The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Thr, or Ser; The 382nd amino acid is Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Trp, Tyr, or Val; The 384th amino acid is Ala, The 385th amino acid is Ala, Gly, Lys, Ser, Thr, Asp, His, or Arg; The amino acid at position 386 is Arg, Asp, Ile, Met, Ser, Thr, Lys, or Pro; The 387th amino acid is Ala, Arg, His, Pro, Ser, Thr, or Glu; The 389th amino acid is Ala, Asn, Pro, or Ser; The 390th amino acid is Ala, The 391st amino acid is Ala, The 413th amino acid is Ala, The 423rd amino acid is Asn, The 424th amino acid is Ala or Glu, The 427th amino acid is Asn, The 428th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The 431st amino acid is His or Asn, The 433rd amino acid is Arg, Gln, His, Ile, Pro, Ser, or Lys; The 434th amino acid is Ala, Phe, Gly, Met, His, Ser, Trp, or Tyr; The 435th amino acid is Lys, Arg, or Asn; The 436th amino acid is Ala, His, Ile, Leu, Glu, Phe, Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp, or Val; The 437th amino acid is Arg, Amino acid 438 is Lys, Leu, Thr, or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys, The antigen-binding molecule of

[11] , wherein the modification is at least one amino acid selected from the group consisting of:

[13] the antigen-binding molecule of any one of

[10] to

[12] , wherein the IgG Fc region is an IgG Fc region derived from a non-human animal;

[14] the antigen-binding molecule of any one of

[10] to

[12] , wherein the IgG Fc region is an IgG Fc region of human origin;

[15] the antigen-binding molecule of any one of [1] to [8], wherein the human Fc receptor is a human Fcγ receptor;

[16] the antigen-binding molecule of

[15] , wherein the receptor-binding domain comprises an IgG Fc region in which at least one amino acid has been modified;

[17] The amino acid modifications in the Fc region of IgG are: 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, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, The antigen-binding molecule of

[16] , wherein the modification is at least one amino acid selected from the amino acids 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 341, 343, 375, 376, 377, 378, 379, 380, 382, ​​385, 386, 387, 389, 392, 396, 421, 423, 427, 428, 429, 430, 431, 433, 434, 436, 438, 440, and 442.

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

[17] , wherein the modification is at least one amino acid selected from the following:

[19] the antigen-binding molecule of any one of

[16] to

[18] , wherein the human Fcγ receptor is FcγRIa, FcγRIIa, FcγRIIb, or FcγRIIIa;

[20] the antigen-binding molecule of

[17] , wherein the amino acid modifications in the IgG Fc region are Asp at amino acid position 238 and Gly at amino acid position 271 (EU numbering);

[21] In the Fc region of IgG, the following residues (EU numbering) are also present: 233, 234, 237, 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 264, 265, 266, 267, 268, 269, 270, 272, 279, 283, 285, 286, 288, 293, 296, 307, 308, 309, 311, 312, 314, 316, 317, 318, 326, 327, 330, 331, 332, The antigen-binding molecule of

[20] , wherein at least one amino acid selected from the amino acids 333, 339, 341, 343, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 389, 396, 423, 427, 428, 430, 431, 433, 434, 436, 438, 440, and 442 has been modified.

[22] The amino acid modification in the Fc region of IgG is based on EU numbering; The 233rd amino acid is Asp, The 234th amino acid is Tyr, The 237th amino acid is Asp, The 264th amino acid is Ile, The 265th amino acid is Glu, Amino acid 266 is Phe, Met, or Leu; The 267th amino acid is Ala, Glu, Gly, or Gln; The 268th amino acid is Asp or Glu, The 269th amino acid is Asp, The amino acid at position 272 is Asp, Phe, Ile, Met, Asn, or Gln; The 296th amino acid is Asp, The 326th amino acid is Ala or Asp, The 327th amino acid is Gly, The 330th amino acid is Lys or Arg, The 331st amino acid is Ser, The 332nd amino acid is Thr, The 333rd amino acid is Thr, Lys, or Arg; The 396th amino acid is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr; The antigen-binding molecule of

[21] , wherein the modification is at least one amino acid selected from the group consisting of:

[23] In the Fc region of IgG, the following residues (EU numbering) are also found: 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 389, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, The antigen-binding molecule of any one of

[20] to

[22] , wherein at least one amino acid selected from positions 427, 428, 430, 431, 433, 434, 436, 438, 440, and 442 has been modified.

[24] The amino acid modification in the Fc region of IgG is based on EU numbering; The 244th amino acid is Leu, The 245th amino acid is Arg, The 249th amino acid is Pro, The 250th amino acid is Gln or Glu, The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, or Tyr; The 254th amino acid is Ser or Thr, The 255th amino acid is Arg, Gly, Ile, or Leu; The 256th amino acid is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr; The 257th amino acid is Ala, Ile, Met, Asn, Ser, or Val; The 258th amino acid is Asp, The 260th amino acid is Ser, The 262nd amino acid is Leu, The 270th amino acid is Lys, The 272nd amino acid is Leu or Arg, The 279th amino acid is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 285th amino acid is Asn, The 286th amino acid is Phe, The 288th amino acid is Asn or Pro, The 293rd amino acid is Val, The 307th amino acid is Ala, Glu, Gln, or Met; The 308th amino acid is Ile, Pro, or Thr; The 309th amino acid is Pro, The 311th amino acid is Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp, The 312th amino acid is Ala, Asp, or Pro; The 314th amino acid is Ala or Leu, The 316th amino acid is Lys, The 317th amino acid is Pro, The 318th amino acid is Asn or Thr, Amino acid 332 is Phe, His, Lys, Leu, Met, Arg, Ser, or Trp; The 339th amino acid is Asn, Thr, or Trp; The 341st amino acid is Pro, The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 375th amino acid is Arg, The 376th amino acid is Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys, The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Ser, or Thr; Amino acid 382 is Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 385th amino acid is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; The 386th amino acid is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; The 387th amino acid is Ala, Arg, His, Pro, Ser, or Thr; The 389th amino acid is Asn, Pro, or Ser; The 423rd amino acid is Asn, The 427th amino acid 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 431st amino acid is His or Asn, The 433rd amino acid is Arg, Gln, His, Ile, Lys, Pro, or Ser; The 434th amino acid is Ala, Gly, His, Phe, Ser, Trp, or Tyr; The 436th amino acid is Arg, Asn, His, Ile, Leu, Lys, Met, or Thr; Amino acid 438 is Lys, Leu, Thr, or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys, The antigen-binding molecule of

[23] , wherein the modification is at least one amino acid selected from the group consisting of:

[25] the antigen-binding molecule of any one of

[16] to

[24] , wherein the IgG Fc region is an IgG Fc region derived from a non-human animal;

[26] the antigen-binding molecule of any one of

[16] to

[24] , wherein the IgG Fc region is an IgG Fc region derived from a human;

[27] the antigen-binding molecule of any one of [1] to

[26] , wherein the ion concentration is hydrogen ion concentration (pH), and the antigen-binding activity is lower under an acidic pH range than under a neutral pH range.

[28] the antigen-binding molecule of

[27] , wherein the acidic pH is the pH within an endosome;

[29] the antigen-binding molecule of

[27] or

[28] , wherein the neutral pH is the pH in plasma;

[30] The antigen-binding molecule of any one of

[27] to

[29] , wherein the acidic pH is pH 5.5 to 6.5, and the neutral pH is pH 7.0 to 8.0.

[31] the antigen-binding molecule of any one of

[27] to

[30] , wherein the ratio of antigen-binding activity under an acidic pH range condition to that under a neutral pH range condition, expressed as KD(acidic pH) / KD(neutral pH), is 2 or greater;

[32] the antigen-binding molecule of any one of

[27] to

[31] , wherein at least one amino acid is substituted with histidine and / or at least one histidine is inserted in the antigen-binding domain;

[33] the antigen-binding molecule of any one of

[27] to

[32] , which further has lower antigen-binding activity under a low calcium ion concentration condition than under a high calcium ion concentration condition;

[34] the antigen-binding molecule of any one of [1] to

[26] , wherein the ion concentration is a calcium ion concentration, and the antigen-binding activity is lower under a low calcium ion concentration condition than under a high calcium ion concentration condition;

[35] the antigen-binding molecule of

[33] or

[34] , wherein the low calcium ion concentration is the calcium ion concentration in an endosome;

[36] the antigen-binding molecule of any one of

[33] to

[35] , wherein the high calcium ion concentration is the calcium ion concentration in plasma;

[37] The antigen-binding molecule of any one of

[33] to

[36] , wherein the low calcium ion concentration is a calcium ion concentration of 0.1 μM to 30 μM, and the high calcium ion concentration is a calcium ion concentration of 100 μM to 10 mM.

[38] the antigen-binding molecule of any one of

[33] to

[37] , wherein the ratio of antigen-binding activity under a low calcium ion concentration condition to that under a high calcium ion concentration condition, KD (low calcium ion concentration) / KD (high calcium ion concentration), is 2 or greater;

[39] the antigen-binding molecule of any one of [1] to

[38] , wherein the antigen-binding domain is obtained from a library.

[40] the antigen-binding molecule of any one of [1] to

[39] , wherein the antigen-binding molecule is an antibody;

[41] The antigen-binding molecule of

[40] , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[42] A pharmaceutical composition comprising, as an active ingredient, the antigen-binding molecule of any one of [1] to

[41] .

[43] The pharmaceutical composition according to

[42] , for treating a disease thought to be caused in part by the antigen.

[44] The pharmaceutical composition of

[43] , wherein the antigen is HMGB1.

[45] The pharmaceutical composition according to

[43] or

[44] , wherein the disease is sepsis.

[46] The pharmaceutical composition of

[43] , wherein the antigen is CTGF.

[47] The pharmaceutical composition according to

[43] or

[46] , wherein the disease is fibrosis.

[48] ​​A method for producing the antigen-binding molecule of [1], comprising the steps of: (a) selecting antigens having two or more types of physiological activity; (b) obtaining an antigen-binding domain; (c) obtaining at least one receptor-binding domain; (d) selecting, from the antigen-binding domains obtained in step (b), domains whose antigen-binding activity changes depending on ion concentration conditions; (e) selecting, from the receptor-binding domains obtained in step (c), domains that have human FcRn-binding activity under acidic pH conditions and whose human Fc receptor-binding activity under neutral pH conditions is higher than the binding activity of native human IgG to human Fc receptors; (f) preparing an antigen-binding molecule in which the antigen-binding domain selected in step (d) and the receptor-binding domain selected in step (e) are linked; and (g) selecting, from the antigen-binding molecules prepared in step (f), antigen-binding molecules that, upon binding to an antigen, inhibit one or more physiological activities of the antigen while maintaining at least one physiological activity.

[49] The method according to

[48] , wherein step (b) is a step of obtaining an antigen-binding domain and further modifying at least one amino acid in the domain.

[50] The method of

[48] or

[49] , wherein step (c) is a step of obtaining a domain that binds to human FcRn and further modifying at least one amino acid in the domain.

[51] Step (f) (f) preparing a polynucleotide in which a polynucleotide encoding the antigen-binding domain selected in step (d) and a polynucleotide encoding the receptor-binding domain selected in step (e) are linked, and using the linked polynucleotide to prepare an antigen-binding molecule in which the antigen-binding domain selected in step (d) and the receptor-binding domain selected in step (e) are linked; The method according to any one of

[48] to

[50] ,

[52] Step (g) (g) selecting, from the antigen-binding molecules prepared in step (f), antigen-binding molecules that, upon binding to the antigen, inhibit one or more of the antigen's binding activities to target molecules while maintaining binding activity to at least one target molecule; The method according to any one of

[48] to

[51] ,

[53] The method of any one of

[48] to

[52] , wherein the human Fc receptor is human FcRn or human Fcγ receptor.

[0012] The present invention further relates to the following:

[54] A method for reducing the plasma antigen concentration by administering the antigen-binding molecule of any one of [1] to

[41] or the antigen-binding molecule produced by the production method of any one of

[48] to

[53] .

[55] A method for promoting intracellular uptake of an antigen, comprising administering the antigen-binding molecule of any one of [1] to

[41] or the antigen-binding molecule produced by the production method of any one of

[48] to

[53] .

[56] A method for reducing the physiological activity of an antigen in a living body, comprising administering the antigen-binding molecule of any one of [1] to

[41] or the antigen-binding molecule produced by the production method of any one of

[48] to

[53] .

[57] the method of any one of

[54] to

[56] , wherein the antigen-binding molecule is an antibody.

[58] The method according to

[57] , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[59] A therapeutic agent for a disease, comprising, as an active ingredient, the antigen-binding molecule of any one of [1] to

[41] or the antigen-binding molecule produced by the production method of any one of

[48] to

[53] .

[60] The therapeutic agent according to

[59] , wherein the antigen is HMGB1.

[61] The therapeutic agent according to

[59] or

[60] , wherein the disease is sepsis.

[62] The therapeutic agent according to

[59] , wherein the antigen is CTGF.

[63] The therapeutic agent according to

[59] or

[62] , wherein the disease is fibrosis.

[64] A kit for use in the method of any one of

[54] to

[58] , comprising the antigen-binding molecule of any one of [1] to

[41] or the antigen-binding molecule produced by the production method of any one of

[48] to

[53] .

[0013] The present invention further relates to the following:

[101] A method for screening for an antibody whose antigen-binding activity changes depending on conditions, comprising the steps of: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) selecting cells that bind to a certain amount or more of the antigen from the cells of step (b); (d) subjecting the cells of step (c) to second conditions; and (e) A step of selecting cells from the cells of step (d) that have a reduced amount of antigen binding compared to step (c).

[102] The method according to

[101] , comprising the steps of: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) contacting the cells of step (b) with an anti-IgG antibody; (d) selecting cells from the cells of step (c) that bind to a certain amount or more of antigen and a certain amount or more of anti-IgG antibody; (e) subjecting the cells of step (d) to second conditions; and (f) A step of selecting cells from the cells of step (e) that have a reduced amount of antigen binding compared to step (d).

[103] The method according to

[102] , comprising the steps of: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) concentrating antigen-bound cells from among the cells of step (b); (d) contacting the cells of step (c) with an anti-IgG antibody; (e) selecting cells from the cells of step (d) that have bound to a certain amount or more of antigen and a certain amount or more of anti-IgG antibody; (f) subjecting the cells of step (e) to second conditions; and (g) A step of selecting cells from the cells of step (f) that have a reduced amount of antigen binding compared to step (e).

[104] The method according to any one of

[101] to

[103] , wherein the first condition and the second condition are ion concentration conditions.

[105] The method according to

[104] , wherein the first condition is a neutral pH and the second condition is an acidic pH.

[106] The method according to

[104] , wherein the first condition is a high calcium ion concentration and the second condition is a low calcium ion concentration.

[107] The method according to

[104] , wherein the first condition is a neutral pH and a high calcium ion concentration, and the second condition is an acidic pH and a low calcium ion concentration.

[108] The method according to

[105] or

[107] , wherein the neutral pH is pH 7.0 to 8.0, and the acidic pH is pH 5.5 to 6.5.

[109] The method according to any one of

[106] to

[108] , wherein the high calcium ion concentration is a calcium ion concentration of 100 μM to 10 mM, and the low calcium ion concentration is a calcium ion concentration lower than that.

[110] The method of any one of

[101] to

[109] , wherein the antibody-producing cells are cells collected from blood, spleen, and / or lymph nodes.

[111] The method of any one of

[101] to

[110] , wherein the antibody-producing cells are B cells.

[112] The method according to

[111] , wherein the antibody-producing cells are rabbit B cells.

[113] The method of any one of

[101] to

[112] , wherein the antigen and / or anti-IgG antibody is fluorescently labeled.

[114] The method of any one of

[101] to

[113] , wherein the cell sorting step is performed using FACS.

[115] The method according to any one of

[101] to

[114] , wherein the step of concentrating cells is carried out using MACS.

[0014] Furthermore, the present invention relates to the following inventions. [a] A method for treating a disease thought to be caused in part by a physiologically active antigen, a method for reducing plasma antigen concentration, a method for promoting antigen uptake into cells, or a method for reducing the physiological activity of an antigen in a living body, comprising the step of administering an antigen-binding molecule of the present invention. [b] a therapeutic agent for a disease thought to be caused in part by a physiologically active antigen, an agent for reducing plasma antigen concentration, an agent for promoting antigen uptake into cells, or an agent for reducing the physiological activity of an antigen in the body, which comprises an antigen-binding molecule of the present invention as an active ingredient. [c] an antigen-binding molecule of the present invention for use in a method for treating a disease thought to be caused in part by a physiologically active antigen, a method for reducing the plasma antigen concentration, a method for promoting antigen uptake into cells, or a method for reducing the physiological activity of an antigen in a living body. [d] use of an antigen-binding molecule of the present invention in the production of a therapeutic agent for a disease thought to be caused in part by a physiologically active antigen, an agent for reducing plasma antigen concentration, an agent for promoting antigen uptake into cells, or an agent for reducing the physiological activity of an antigen in the body. [e] A process for producing a therapeutic agent for a disease thought to be caused in part by a physiologically active antigen, an agent for reducing plasma antigen concentration, an agent for promoting antigen uptake into cells, or an agent for reducing the physiological activity of an antigen in a living body, the process comprising a step of using an antigen-binding molecule of the present invention. Examples of the disease include diseases thought to be caused in part by HMGB1, diseases thought to be caused in part by CTGF, and diseases thought to be caused in part by IgE. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the results of FACS sorting of anti-HMGB1 antibody-producing B cells. (A) shows the results of the first sorting run under neutral pH and high calcium ion concentration conditions, and (B) shows the results of the second sorting run under acidic pH and low calcium ion concentration conditions. The numbers (1, 2, 3) in the figure indicate the gate numbers. [Figure 2]1 is a graph plotting the HMGB1-binding activity of anti-HMGB1 antibodies, showing the results plotted for antibodies produced by B cells derived from (A) gate 1, (B) gate 2, and (C) gate 3 in FACS sorting. [Figure 3] This graph shows the number of antibodies whose antigen-binding ability is independent of pH and / or calcium ion concentration (gray area) and the number of antibodies whose antigen-binding ability is dependent on pH and / or calcium ion concentration (black area) among the antibodies produced by B cells derived from gates 1, 2, and 3, respectively. [Figure 4-1] FIG. 1 shows sensorgrams of anti-HMGB1 antibodies (HMG233-IgG1 and HMG236-IgG1) and human HMGB1 in Biacore. [Figure 4-2] This is a continuation of Figure 4-1 and shows sensorgrams of anti-HMGB1 antibodies (HMG481-IgG1 and HMG487-IgG1) and human HMG1. [Figure 5] 1 is a graph showing the results of RAGE ELISA for anti-HMGB1 antibodies (HMG233, HMG236, HMG481, HMG487, HMG446). The absorbance of the control without antibody was set at 100, and the relative value for each antibody is shown. [Figure 6] 1 is a graph showing the results of TLR4 / MD-2 ELISA for anti-HMGB1 antibodies (HMG233, HMG236, HMG481, HMG487, HMG446). The absorbance of the control without antibody was set at 100, and the relative value for each antibody is shown. [Figure 7] Fig. 1 shows the time course of human HMGB1 serum concentrations in normal mice. [Figure 8] Fig. 10 shows the time course of serum anti-human HMGB1 antibody concentrations in normal mice. [Figure 9] FIG. 1 shows that antigens can be removed from plasma by using antibodies that bind to target antigens in a pH-dependent manner and have FcRn-binding activity in the neutral pH range. [Figure 10]Fig. 1 shows sensorgrams of 6RKE02-IgG1 against hsIL-6R at pH 7.4 and pH 6.0. [Figure 11] FIG. 1 shows the results of biological activity evaluation using human gp130-expressing BaF3 cells (BaF / gp130). [Figure 12] FIG. 1 shows the time course of plasma hsIL-6R concentration after antibody administration in an infusion test using normal mice. [Figure 13] FIG. 1 shows the protocol for an in vivo efficacy test (Test 1) using normal mice, and the SAA suppression effect (mean±SE) of antibody administration. [Figure 14] FIG. 1 shows the protocol for an in vivo efficacy test (Test 2) using normal mice, and the SAA suppression effect (mean±SE) of antibody administration. [Figure 15] FIG. 1 shows a non-limiting mechanism of action by which soluble antigens are eliminated from plasma by administration of antibodies that bind to antigens in an ion concentration-dependent manner and have enhanced Fcγ receptor binding at neutral pH compared to existing neutralizing antibodies. [Figure 16] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when H54 / L28-IgG1 or Fv4-IgG1 that binds to human IL-6 receptor in a pH-dependent manner was administered to the mice. [Figure 17] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered with Fv4-IgG1, which binds to human IL-6 receptor in a pH-dependent manner, Fv4-IgG1-F760, a modified Fv4-IgG1 that lacks mouse FcγR binding, Fv4-IgG1-F1022, a modified Fv4-IgG1 with enhanced mouse FcγR binding, or Fv4-IgG1-Fuc, a low-fucose Fv4-IgG1 antibody. [Figure 18]FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when the mice were administered antigen-binding molecules comprising as a heavy chain Fv4-IgG1, VH3-IgG1-F1022, or VH3-IgG1-F1093, a modified form of VH3-IgG1-F1022 that has improved FcRn binding in the acidic pH range. [Figure 19] FIG. 1 shows the concentration time courses of the antigen-binding molecules in the plasma of human FcRn transgenic mice when the antigen-binding molecules comprise, as a heavy chain, Fv4-IgG1, VH3-IgG1-F1022, or VH3-IgG1-F1093, a modified form of VH3-IgG1-F1022 that has improved FcRn binding in the acidic pH range. [Figure 20] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered with Fv4-IgG1, Fv4-IgG1-F1087, a variant of Fv4-IgG1 with enhanced mouse FcγR binding (particularly enhanced binding to mouse FcγRIIb and mouse FcγRIII), or Fv4-IgG1-F1182, a variant of Fv4-IgG1 with enhanced mouse FcγR binding (particularly enhanced binding to mouse FcγRI and mouse FcγRIV). [Figure 21] FIG. 1 shows the concentration time courses of the antigen-binding molecules in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified forms of Fv4-IgG1-F1087 that have improved FcRn binding in the acidic pH range, were administered to the mice. [Figure 22] FIG. 1 shows the concentration time courses of the antigen-binding molecules in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1182, or Fv4-IgG1-F1181, a modified form of Fv4-IgG1-F1182 with improved FcRn binding in the acidic pH range, was administered to the mice. [Figure 23] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when the mice were administered Fv4-IgG1, Fv4-IgG1-F1087, or Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified forms of Fv4-IgG1-F1087 with improved FcRn binding in the acidic pH range. [Figure 24] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when the mice were administered Fv4-IgG1, Fv4-IgG1-F1182, or Fv4-IgG1-F1181, a modified form of Fv4-IgG1-F1182 with improved FcRn binding in the acidic pH range. [Figure 25] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of normal mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 26] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIII-deficient mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 27]FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of Fc receptor γ chain-deficient mice when the mice were administered Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 28] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIIb-deficient mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 29] FIG. 10 shows the results of evaluating the platelet aggregation activity of omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets derived from donors with the FcγRIIa polymorphism (R / H). [Figure 30] FIG. 10 shows the results of evaluating the platelet aggregation activity of omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets derived from a donor with the FcγRIIa polymorphism (H / H). [Figure 31] This figure shows the results of evaluating CD62p expression on the membrane surface of washed platelets. The solid black bars show the results when platelets were reacted with PBS and then stimulated with ADP, while the open bars show the results when platelets were reacted with immune complexes and then stimulated with ADP. [Figure 32] This figure shows the results of evaluating the expression of activated integrins on the membrane surface of washed platelets. The solid black bars show the results when platelets were reacted with PBS and then stimulated with ADP, while the open bars show the results when platelets were reacted with immune complexes and then stimulated with ADP. [Figure 33] The horizontal axis represents the relative binding activity of each PD variant to FcγRIIb, and the vertical axis represents the relative binding activity of each PD variant to FcγRIIa type R. The value for the amount of binding of each PD variant to each FcγR is divided by the value for the amount of binding of the control antibody IL6R-F652 (SEQ ID NO: 162) / IL6R-L (IL6R-F652 is an antibody heavy chain containing an altered Fc in which Pro at position 238 (EU numbering) is replaced with Asp) to each FcγR, and the resulting value is multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The plot labeled F652 in the figure represents the value for IL6R-F652 / IL6R-L. [Figure 34] The vertical axis shows the relative FcγRIIb-binding activity of variants obtained by introducing each alteration into GpH7-B3 (SEQ ID NO: 168) / GpL16-k0 (SEQ ID NO: 169) which does not have the P238D alteration, and the horizontal axis shows the relative FcγRIIb-binding activity of variants obtained by introducing each alteration into IL6R-F652 (SEQ ID NO: 162) / IL6R-L which has the P238D alteration. The value for the amount of FcγRIIb binding of each variant was divided by the value for the amount of FcγRIIb binding of the antibody before the alteration was introduced, and then multiplied by 100 to obtain the relative binding activity. Region A includes alterations that exhibit an FcγRIIb-binding-enhancing effect when introduced into GpH7-B3 / GpL16-k0 that does not have P238D and when introduced into IL6R-F652 / IL6R-L that has P238D, while Region B includes alterations that exhibit an FcγRIIb-binding-enhancing effect when introduced into GpH7-B3 / GpL16-k0 that does not have P238D but do not exhibit an FcγRIIb-binding-enhancing effect when introduced into IL6R-F652 / IL6R-L that has P238D. [Figure 35] 1 shows the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Figure 36]Figure 1 shows a diagram of the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex and the model structure of the Fc (WT) / FcγRIIb extracellular region complex superimposed by the least-squares method based on the Cα interatomic distances for the FcγRIIb extracellular region and Fc CH2 domain A. [Figure 37] The crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex and the model structure of the Fc (WT) / FcγRIIb extracellular region complex were superimposed using the least-squares method based on the Cα interatomic distances for the Fc CH2 domain A and the Fc CH2 domain B alone, and the detailed structures around P238D were compared. [Figure 38]

[0046] FIG. 10 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, a hydrogen bond is observed between the main chain of Gly at position 237 (EU numbering) in Fc CH2 domain A and Tyr at position 160 in FcγRIIb. [Figure 39] FIG. 10 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, electrostatic interaction is observed between Asp at position 270 (EU numbering) in Fc CH2 domain B and Arg at position 131 in FcγRIIb. [Figure 40] The horizontal axis shows the relative binding activity of each 2B variant to FcγRIIb, and the vertical axis shows the relative binding activity of each 2B variant to FcγRIIa type R. The binding amount of each 2B variant to each FcγR was divided by the binding amount of the control antibody before alteration (altered Fc in which Pro at position 238 (EU numbering) was substituted with Asp) to each FcγR, and the resulting value was multiplied by 100 to obtain the relative binding activity of each 2B variant to each FcγR. [Figure 41] FIG. 10 shows Glu at position 233 (EU numbering) of Fc Chain A in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, and surrounding residues in the extracellular region of FcγRIIb. [Figure 42]FIG. 10 shows the Ala at position 330 (EU numbering) of Fc Chain A in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, and surrounding residues in the extracellular region of FcγRIIb. [Figure 43] Figure 1 shows the structure of Pro at position 271 (EU numbering) of Fc Chain B, obtained by superimposing the crystal structures of the Fc (P238D) / FcγRIIb extracellular region complex and the Fc (WT) / FcγRIIIa extracellular region complex using the least-squares method based on the Cα interatomic distances relative to Fc Chain B. [Figure 44] This is a diagram of the Fc (P208) / FcγRIIb extracellular region complex determined by X-ray crystal structure analysis. For the CH2 domain and CH3 domain of the Fc portion, the left side is domain A and the right side is domain B. [Figure 45] The structure of the Fc (P208) / FcγRIIb extracellular region complex determined by X-ray crystallography and the structure of the Fc (WT) / FcγRIIa extracellular region complex (PDB code: 3RY6) were superimposed by least-squares fitting based on the Cα interatomic distances in the CH2 domain A of the Fc portion and compared. The thick line in the figure represents the Fc (P208) / FcγRIIb extracellular region complex, and the thin line represents the structure of the Fc (WT) / FcγRIIa extracellular region complex. Note that in the structure of the Fc (WT) / FcγRIIa extracellular region complex, only the CH2 domain A of the Fc portion is depicted. [Figure 46] This figure shows the detailed structure of the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, near Asp at position 237 (EU numbering) in the CH2 domain A of the Fc portion, which forms a hydrogen bond in the main chain with Tyr at position 160 of FcγRIIb. [Figure 47]FIG. 1 shows the structure of amino acid residues surrounding the side chain of Asp at position 237 (EU numbering) in CH2 domain A of the Fc portion, which forms a hydrogen bond in the main chain with Tyr at position 160 of FcγRIIb in the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex. [Figure 48] Figure 1 shows a comparison of the X-ray crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex shown in Reference Example 15 and the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, which were superimposed by the least-squares method based on the Cα interatomic distances in the CH2 domain B of the Fc portion, around the loop from positions 266 to 271 (EU numbering). Compared to Fc (P238D), within this loop, Fc (P208) has an H268D alteration at position 268 (EU numbering) and a P271G alteration at position 271 (EU numbering). [Figure 49] FIG. 1 shows the structure around Ser239 in CH2 domain B of the Fc portion in the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density obtained by X-ray crystal structure analysis with 2Fo-Fc coefficients. [Figure 50] Figure 1 shows a comparison of the three-dimensional structures of the Fc (P208) / FcγRIIaR extracellular region complex determined by X-ray crystal structure analysis and the Fc (P208) / FcγRIIb extracellular region complex, superimposed by the least-squares method based on the Cα interatomic distances. [Figure 51] Figure 1 shows a comparison of the X-ray crystal structures of the Fc (P208) / FcγRIIaR extracellular region complex and the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density (2Fo-Fc coefficients) obtained by X-ray crystal structure analysis near Asp at position 237 (EU numbering) in the CH2 domain A of the Fc portion. [Figure 52]Figure 1 shows a comparison of the X-ray crystal structures of the Fc (P208) / FcγRIIaR extracellular region complex and the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density (2Fo-Fc coefficients) obtained by X-ray crystal structure analysis near Asp at position 237 (EU numbering) in the CH2 domain B of the Fc portion. [Figure 53] 1 shows a comparison of the constant region sequences of G1d and G4d, in which the amino acids enclosed in bold frames indicate positions where the amino acid residues differ between G1d and G4d. [Figure 54] Figure 1 shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody determined by X-ray crystal structure analysis. (i) shows the heavy chain CDR3 crystal structure obtained under crystallization conditions in the presence of calcium ions. (ii) shows the heavy chain CDR3 crystal structure obtained under crystallization conditions in the absence of calcium ions. [Figure 55] FIG. 1 shows the time course of antibody concentrations in plasma of normal mice administered with H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody. [Figure 56] FIG. 1 shows the time course of soluble human IL-6 receptor (hsIL-6R) concentration in plasma of normal mice administered with H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody. [Figure 57] 1 shows ion-exchange chromatograms of an antibody comprising the human Vk5-2 sequence and an antibody comprising the hVk5-2_L65 sequence, in which the glycosylation sequence in the human Vk5-2 sequence has been modified. The solid line represents the chromatogram of the antibody comprising the human Vk5-2 sequence (heavy chain: CIM_H (SEQ ID NO: 67), light chain: hVk5-2 (SEQ ID NO: 57)), and the dashed line represents the chromatogram of the antibody comprising the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 67), light chain: hVk5-2_L65 (SEQ ID NO: 70)). [Figure 58A]Figure 1 shows ion exchange chromatograms of an antibody comprising the LfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55), light chain: LfVk1_Ca (SEQ ID NO: 83)) and an antibody comprising a sequence in which the Asp (D) residue in the LfVk1_Ca sequence has been modified to an Ala (A) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is designated as the main peak, and the y-axis is normalized to the main peak. Figure 2 shows a chromatogram of an antibody comprising LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 58B] FIG. 10 shows a chromatogram of an antibody comprising LfVk1_Ca1 (SEQ ID NO: 85) as a light chain. [Figure 58C] FIG. 10 shows a chromatogram of an antibody comprising LfVk1_Ca2 (SEQ ID NO: 86) as a light chain. [Figure 58D] FIG. 10 shows a chromatogram of an antibody comprising LfVk1_Ca3 (SEQ ID NO: 87) as a light chain. [Figure 59A] Figure 1 shows ion exchange chromatograms of an antibody comprising the LfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55), light chain: LfVk1_Ca (SEQ ID NO: 83)) and an antibody comprising the LfVk1_Ca6 sequence (heavy chain: GC_H (SEQ ID NO: 55), light chain: LfVk1_Ca6 (SEQ ID NO: 88)) in which the Asp (D) residue at position 30 (Kabat numbering) in the LfVk1_Ca sequence has been altered to a Ser (S) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is designated as the main peak, and the y-axis is normalized to the main peak. Figure 2 shows a chromatogram of an antibody comprising LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 59B] FIG. 10 shows a chromatogram of an antibody comprising LfVk1_Ca6 (SEQ ID NO: 88) as a light chain. [Figure 60]This figure shows the relationship between the amino acid distribution (labeled Library) and the designed amino acid distribution (labeled Design) of the sequence information of 290 clones isolated from E. coli transformed with a Ca-dependent antigen-binding antibody gene library. The horizontal axis shows the amino acid position represented by Kabat numbering. The vertical axis shows the ratio of the amino acid distribution. [Figure 61] This figure shows sensorgrams of the anti-IL-6R antibody (tocilizumab), the 6RC1IgG_010 antibody, the 6RC1IgG_012 antibody, and the 6RC1IgG_019 antibody under high calcium ion concentration conditions (1.2 mM). The horizontal axis represents time, and the vertical axis represents RU value. [Figure 62] This figure shows sensorgrams of the anti-IL-6R antibody (tocilizumab), the 6RC1IgG_010 antibody, the 6RC1IgG_012 antibody, and the 6RC1IgG_019 antibody under low calcium ion concentration conditions (3 μM). The horizontal axis represents time, and the vertical axis represents RU value. [Figure 63] This figure shows the relationship between the amino acid distribution (labeled Library) and the designed amino acid distribution (labeled Design) of the sequence information of 132 clones isolated from E. coli into which a pH-dependent antigen-binding antibody gene library was introduced. The horizontal axis shows the amino acid position represented by Kabat numbering, and the vertical axis shows the ratio of the amino acid distribution. [Figure 64] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 7.4. The horizontal axis represents time, and the vertical axis represents RU value. [Figure 65] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 6.0. The horizontal axis represents time, and the vertical axis represents RU value. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides an antigen-binding molecule that reduces an antigen concentration in plasma, the antigen-binding molecule having the following characteristics (1) to (6): (1) The antigen-binding molecule comprises an antigen-binding domain and a receptor-binding domain; (2) The receptor-binding domain has the activity of binding to human FcRn (Neonatal Fc Receptor) under acidic pH conditions; (3) the binding activity of the receptor-binding domain to a human Fc receptor is higher than that of native human IgG under a neutral pH range condition; (4) The antigen-binding activity of the antigen-binding domain changes depending on the ion concentration conditions. (5) The antigen (to be bound) has two or more physiological activities, (6) Upon binding of the antigen-binding molecule, one or more physiological activities of the antigen (to which the antigen is bound) are inhibited, while at least one physiological activity is maintained.

[0017] In the present invention, "biological activity" refers to activities that bring about quantitative and / or qualitative changes or effects in living organisms, tissues, cells, proteins, DNA, RNA, etc., such as activities that regulate biological functions such as metabolism, growth, reproduction, homeostasis, mental activity, and biodefense. More specifically, examples of such activities include activities that regulate cell proliferation and maturation, endocrine-mediated metabolism, signal transduction in the nervous system, blood circulation, wound healing, immune response, and cell migration. Biological activity can also be referred to as "biological activity." A bioactive substance is a substance that possesses such physiological activity. A bioactive substance exerts its physiological activity by acting on specific biological molecules (target molecules) to cause some kind of change or effect. In the present invention, a bioactive substance is also referred to as a bioactive antigen. The bioactive substance in the present invention may be any substance that has physiological activity, but is preferably a bioactive polypeptide or a modified version thereof (bioactive peptide). The target molecule for a bioactive substance to exert its physiological activity is preferably a receptor present on the cell surface or within the cell. By binding to the specific receptor, the bioactive substance transmits a signal to the cell and exerts its physiological activity. When a physiologically active substance is converted from a precursor that does not have physiological activity to a mature form that has physiological activity, the enzyme that performs such conversion is also included in the physiologically active substance of the present invention. In this case, the target molecule is the substrate (precursor) of the enzyme. The physiologically active substance may be a substance produced by an organism (human or non-human organism) or may be an artificially synthesized substance. In the present invention, a physiologically active substance that is thought to be one of the causes of disease in an organism (preferably human) is preferred.

[0018] Examples of physiologically active peptides include cell growth factors such as fibroblast growth factors (FGFs), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), epidermal growth factors (EGFs), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and bone morphogenetic proteins (BMPs); cytokines such as interferons (IFNs), interleukins (ILs), colony-stimulating factors (CSFs), erythropoietin, and tumor necrosis factor (TNF); various hormones such as insulin and parathyroid hormone (PTH); enzymes including proteases such as matrix metalloproteinases (MMPs); and enzyme inhibitors such as TIMPs (Tissue Inhibitor of Metalloproteases).

[0019] The physiologically active substance in the present invention is preferably a physiologically active substance derived from a mammal, and particularly preferably a physiologically active substance derived from a human.

[0020] Physiologically active substances can be obtained, for example, by purification from living organisms. They can also be produced by chemical synthesis. When the physiologically active substance is a physiologically active peptide, recombinant peptides can be produced using genetic engineering techniques. That is, nucleic acids encoding the physiologically active peptide can be synthesized from the amino acid sequence of the physiologically active peptide or the nucleic acid sequence encoding it by gene cloning methods or nucleic acid synthesis methods known to those skilled in the art. The nucleic acid is inserted into a known expression vector to transform appropriate host cells, and the physiologically active peptide of interest can be purified from the host cells or culture supernatant by known methods. Purification can be performed by using multiple chromatographic methods, such as conventional ion chromatography and affinity chromatography, once or in combination, or independently. Furthermore, as long as the physiologically active peptide retains its inherent physiological activity, it can also be produced as a partial peptide containing a portion of the physiologically active peptide, or as a fusion peptide fused with a different polypeptide, such as a peptide tag or Fc fragment. Fusion peptides can be produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above (Sambrook J et al., Molecular Cloning 2014). nd (ed. (1989) 9.47-9.58, Cold Spring Harbor Lab. Press). Target molecules that bind to physiologically active substances can also be obtained by a similar method.

[0021] As a method for measuring the physiological activity of a physiologically active substance, in vitro physiological activity can be measured using a method that prepares the physiologically active substance and its target molecule and detects their binding, such as ELISA, FACS, or Biacore. Alternatively, physiological activity can be measured by reacting the physiologically active substance with receptor-expressing cells and detecting changes that occur in the cells (e.g., changes in cell proliferation or morphology, changes in gene or protein expression, etc.). In vivo physiological activity can be measured by administering the physiologically active substance to an animal and observing changes that occur in the animal (e.g., changes related to biological functions such as metabolism, growth, and homeostasis).

[0022] In the present invention, "having two or more types of physiological activity" means that a physiologically active substance has the property of binding to two or more different target molecules. The physiologically active substance may directly bind to the target molecule, or the physiologically active substance may bind to a substance other than the target molecule that promotes binding to the target molecule, and then indirectly bind to the target molecule. A physiologically active substance has a domain for binding to a target molecule, and it is preferable that the physiologically active substance has multiple different binding domains corresponding to two or more different target molecules. In particular, it is preferable that the binding domains are located at positions separated in the three-dimensional structure to such an extent that binding of the antigen-binding molecule of the present invention to the physiologically active substance is not simultaneously inhibited.

[0023] In the present invention, "having activity" means that in a system in which the activity can be measured, the measured value is higher than the background value in that system (or the value when a negative control is measured). For example, having binding activity means that the measured value is higher than the background value in a system in which the binding activity can be measured, such as ELISA, FACS, or Biacore. In the present invention, the measured value is preferably at least 2-fold higher than the background value, more preferably at least 3-fold higher, even more preferably at least 5-fold higher, and particularly preferably at least 10-fold higher.

[0024] In the present invention, "inhibiting activity" means that in a system capable of measuring the activity, the measured value after addition of a certain substance is lower than the measured value before addition of the certain substance (or the value when a negative control is added). For example, inhibiting binding activity means that in a system capable of measuring binding activity, such as ELISA, FACS, or Biacore, the measured value after addition of a certain substance is lower than the measured value before addition of the certain substance. In the present invention, the decrease in the measured value after addition of a certain substance relative to the measured value before addition of the certain substance (or when a negative control is added) is preferably 80% or less, more preferably 50% or less, even more preferably 30% or less, and particularly preferably 10% or less.

[0025] In the present invention, "maintaining activity" means that in a system capable of measuring the activity, the measured value after addition of a certain substance is 80% or more of the measured value before addition of the certain substance (or the value when a negative control is added). For example, maintaining binding activity means that in a system capable of measuring binding activity, such as ELISA, FACS, or Biacore, the measured value after addition of a certain substance is 80% or more of the measured value before addition of the certain substance. In the present invention, the measured value after addition of a certain substance is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more of the measured value before addition of the certain substance (or when a negative control is added).

[0026] In the present invention, "reducing the antigen concentration in plasma" means that, when an antigen-binding molecule of the present invention is administered to a subject, the concentration of the antigen present in plasma is lower than when a negative control is administered to a subject. The percentage of reduction is not particularly limited, but is preferably 80% or less, more preferably 50% or less, even more preferably 30% or less, and most preferably 10% or less. In the present invention, "reducing the antigen concentration in plasma" can also be rephrased as "promoting the elimination (clearance) of antigens from plasma," "reducing the residence time of antigens in plasma," "shortening the plasma half-life of antigens," etc. Furthermore, "in plasma" may also refer to serum. The antigen-binding molecules provided by the present invention can be administered to subjects (living bodies) by, for example, intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, or intramuscular injection. Subjects to which the antigen-binding molecules of the present invention are administered are preferably animals, more preferably mammals, and even more preferably humans.

[0027] The reduction in antigen concentration may be achieved by promoting the uptake of the antigen into cells. Preferably, the reduction in antigen concentration reduces the physiological activity of the antigen in vivo, and particularly preferably reduces all types of physiological activity of the antigen.

[0028] The concentration of an antigen can be measured by an appropriate method known to those skilled in the art. When the antigen is a physiologically active peptide, a standard sample of known concentration and a system capable of quantitatively measuring it (e.g., ELISA or Biacore) are prepared, and a standard curve showing the relationship between each concentration and the measured value is created, thereby enabling the concentration of a physiologically active peptide of unknown concentration contained in a sample to be measured. Examples of samples include biological plasma, cell culture medium, and cell extract.

[0029] In the present invention, the term "uptake of an antigen into cells" means that the antigen is taken up into cells by endocytosis. Whether or not the uptake of an antigen into cells has been promoted can be determined, for example, by adding the antigen to a cell culture medium and then measuring whether the antigen concentration in the culture medium has decreased compared to a control, or whether the antigen concentration in the cells has increased compared to a control. Promotion of the uptake of an antigen into cells means that the elimination of the antigen from plasma in the body is promoted. Therefore, whether or not the uptake of an antigen into cells has been promoted can also be determined, for example, by administering the antigen to a body and then measuring whether the antigen concentration in plasma has decreased compared to a control.

[0030] The antigen-binding molecules provided by the present invention are not particularly limited as long as they have the characteristics described in (1) to (6) above, but are preferably polypeptides that specifically bind to antigens, human FcRn, and human Fc receptors, more preferably antibodies, and particularly preferably IgG. The antibodies may be chimeric antibodies, humanized antibodies, human antibodies, etc. They may also be bispecific antibodies, modified antibodies bound to various molecules, or polypeptides containing antibody fragments. The antigen-binding molecules provided by the present invention comprise an antigen-binding domain and a receptor-binding domain. Here, the term "domain" refers to a constituent unit that can be isolated by dividing it, and its size is not particularly limited. Each domain comprises a polypeptide. The antigen-binding domain of the present invention is not particularly limited as long as it has the property of specifically binding to an antigen. Preferred examples include antibodies and fragments thereof (variable regions, Fab, F(ab')2, Fv, CDR, etc.), antibody-like molecules called scaffolds (DARPins (WO2002 / 020565), Affibodies (WO1995 / 001937), Avimers (WO2004 / 044011, WO2005 / 040229), Adnectins (WO2002 / 032925), etc.), and target molecules (receptors) and fragments thereof (soluble receptors) that bind to physiologically active substances. A particularly preferred example is the variable region of an antibody. Furthermore, the receptor-binding domain of the present invention is not particularly limited as long as it has the property of specifically binding to human FcRn and / or human Fc receptors. Preferred examples include antibodies (IgG) and fragments thereof (constant region, Fc, etc.), albumin and fragments thereof (domain 3), anti-FcRn antibodies and fragments thereof (variable region, Fab, F(ab')2, Fv, CDR, etc.), anti-FcRn antibody-like molecules (DARPins (WO2002 / 020565), Affibodies (WO1995 / 001937), Avimers (WO2004 / 044011, WO2005 / 040229), Adnectins (WO2002 / 032925), etc.), and anti-FcRn peptides. A more preferred example is the Fc region of IgG.The human Fc receptor of the present invention is not particularly limited as long as it is a receptor to which the Fc region of human IgG, particularly human IgG, binds, but is preferably human FcRn or human Fcγ receptor. The IgG may be derived from a non-human animal or a human, but is preferably human IgG, and particularly preferably human IgG1. The IgG Fc region preferably has amino acid modifications as described below.

[0031] The antigen-binding molecules of the present invention are sufficient as long as they contain at least one receptor-binding domain. For example, a single antigen-binding molecule may contain one antigen-binding domain and one receptor-binding domain, or one antigen-binding domain and multiple receptor-binding domains. When a single antigen-binding molecule contains multiple receptor-binding domains, these receptor-binding domains may all bind to the same type of human Fc receptor, or may each bind to different types of human Fc receptor. Since one of the requirements that the antigen-binding molecules of the present invention must satisfy is that they have the activity to bind to human FcRn, it is preferred that at least one receptor-binding domain contained in the antigen-binding molecules of the present invention is a domain that binds to human FcRn. Examples of embodiments in which a single antigen-binding molecule contains two receptor-binding domains include, but are not limited to, antigen-binding molecules in which both domains bind to human FcRn, and antigen-binding molecules in which one domain binds to human FcRn and the other domain binds to human Fcγ receptor. Furthermore, when an antigen-binding molecule contains one receptor-binding domain, the receptor-binding domain must at least bind to human FcRn, and one receptor-binding domain may also have the property of simultaneously binding to other types of human Fc receptors. An example of such a receptor-binding domain is the Fc region of IgG. The Fc region of IgG has the property of binding to human FcRn and human Fcγ receptors.

[0032] As used herein, native human IgG refers to naturally occurring human IgG, preferably having a fucose-containing sugar chain bound to position 297 (EU numbering) of the Fc region. Naturally occurring human IgG1, IgG2, IgG3, or IgG4 can be used as native human IgG, with naturally occurring human IgG1 being preferred. Whether the bound sugar chain is a fucose-containing sugar chain can be determined, for example, by the following method: The sugar chain is released by reacting the test human IgG with N-Glycosidase F (Roche diagnostics) (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), and the resulting concentrate is fluorescently labeled with 2-aminopyridine (Bigge et al., Anal. Biochem. (1995) 230 (2) 229-238). The fluorescently labeled 2-AB-modified glycans are removed by solid-phase extraction using a cellulose cartridge and analyzed by normal-phase chromatography. By observing the peaks detected in the chromatogram, it is possible to determine whether the glycans bound to the Fc region of the test human IgG are fucose-containing glycans.

[0033] A chimeric antibody is an antibody produced by combining sequences derived from different animals. A specific example of a chimeric antibody is an antibody consisting of the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody.

[0034] Humanized antibodies, also known as reshaped human antibodies, are antibodies derived from non-human mammals, such as mice, in which the complementarity-determining regions (CDRs) of the antibodies are grafted onto the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General genetic recombination techniques for CDR grafting are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0035] A bispecific antibody is an antibody that has two variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

[0036] Examples of polypeptides containing antibody fragments include Fab fragments, F(ab')2 fragments, scFv (Nat Biotechnol. 2005 Sep;23(9):1126-36), domain antibodies (dAbs) (WO2004 / 058821, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, and Fc fusion proteins. Molecules containing an Fc region can be used as receptor-binding domains. The Fc region refers to the heavy chain constant region of an antibody molecule, extending from the N-terminus of the hinge region at the papain cleavage site to the hinge, CH2, and CH3 domains. The Fc region of an IgG refers to, for example, the region from cysteine ​​at position 226 to the C-terminus, or from proline at position 230 to the C-terminus, according to EU numbering, but is not limited thereto. Although not particularly limited, examples of IgG Fc regions include the Fc regions of human IgG1 (SEQ ID NO: 49), IgG2 (SEQ ID NO: 50), IgG3 (SEQ ID NO: 51), or IgG4 (SEQ ID NO: 52). The Fc region of human IgG1 is preferred.

[0037] An antibody-like molecule (scaffold molecule) is a general term for molecules that have a common skeletal structure and have the property of being able to specifically bind to any antigen (Current Opinion in Biotechnology 2006, 17:653-658, Current Opinion in Biotechnology 2007, 18:1-10, Current Opinion in Structural Biology 1997, 7:463-469, Protein Science 2006, 15:14-27). Examples of such molecules include DARPins (WO2002 / 020565), affibodies (WO1995 / 001937), avimers (WO2004 / 044011, WO2005 / 040229), and adnectins (WO2002 / 032925).

[0038] The antibody may contain a modified sugar chain. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (WO99 / 54342, etc.), antibodies lacking fucose attached to the sugar chain (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913, etc.), and antibodies with sugar chains containing bisecting GlcNAc (WO02 / 79255, etc.).

[0039] The binding between the antigen-binding molecule of the present invention and an antigen, or the binding between a physiologically active antigen and a target molecule, can be measured using methods known to those skilled in the art, such as ELISA, FACS, or Biacore. By setting the measurement conditions to extracellular or intracellular conditions, it is also possible to examine the difference in binding activity under these conditions. Furthermore, by combining this with the above-mentioned method for measuring the physiological activity of a physiologically active substance, it is possible to determine whether the binding of the antigen-binding molecule of the present invention to a physiologically active antigen inhibits the physiological activity of the antigen or whether the physiological activity is maintained.

[0040] The present invention provides antigen-binding molecules that, upon binding to an antigen, inhibit one or more of the antigen's binding activities with target molecules while maintaining its binding activity with at least one target molecule. That is, it is preferred that, upon binding to an antigen with two or more physiological activities, one or more of the physiological activities of the antigen are inhibited. "Inhibiting one or more physiological activities" (or "physiological activities are inhibited") means that, among the antigen's binding activities with multiple target molecules, the antigen's binding activity with one or more target molecules is inhibited (or the binding activity is inhibited). Furthermore, even when the antigen-binding molecule of the present invention binds to an antigen with two or more physiological activities, it is preferred that at least one of the physiological activities of the antigen is maintained (or the physiological activity is maintained). "Maintaining at least one physiological activity" (or "physiological activity is maintained") means that, among the antigen's binding activities with multiple target molecules, the antigen's binding activity with at least one target molecule is maintained (or the binding activity is maintained).

[0041] When the excessive presence of a physiologically active antigen in the body is the cause of a certain disease, molecules that inhibit the physiological activity of the antigen by binding to the antigen, such as neutralizing antibodies, are thought to be useful in treating the disease. However, when an antigen has two or more physiological activities, a neutralizing antibody can only inhibit one physiological activity. On the other hand, the antigen-binding molecules of the present invention are extremely useful compared to general neutralizing antibodies because they can promote the elimination of the antigen from the blood (serum or plasma) even if at least one physiological activity is maintained, thereby ultimately reducing the physiological activity in vivo.

[0042] In the present invention, when the human Fc receptor is human FcRn, the receptor-binding domain of the antigen-binding molecule is preferably the Fc region of IgG, and more preferably a variant Fc region in which at least one amino acid in the Fc region of IgG has been altered. The IgG may be derived from a non-human animal or a human, but is preferably human IgG (IgG1, IgG2, IgG3, IgG4), and particularly preferably human IgG1. Examples of amino acid alterations include amino acid substitution, insertion, deletion, etc., with amino acid substitution being preferred. The number of amino acids to be altered is not particularly limited; only one amino acid may be altered, or two or more amino acids may be altered. The amino acid alteration may be at any amino acid position, as long as the altered Fc region retains human FcRn-binding activity under acidic and neutral pH conditions and has stronger human FcRn-binding activity under neutral pH conditions than human IgG. It is generally known that in vivo, the extracellular pH (e.g., in plasma) is neutral, while the intracellular pH (e.g., in endosomes) is acidic. It is also known that IgG-FcRn binding is observed only under acidic (intracellular) pH conditions, and is hardly observed under neutral (extracellular) pH conditions. For the antigen-binding molecules of the present invention, the acidic pH is the pH in endosomes, and the neutral pH is preferably the pH in plasma.

[0043] If the receptor-binding domain of an antigen-binding molecule provided by the present invention can be conferred with properties such that it has human FcRn-binding activity under intracellular and extracellular pH conditions and that its human FcRn-binding activity under extracellular pH conditions is stronger than that of human IgG, the antigen-binding molecule of the present invention that has bound to an antigen extracellularly will further bind to FcRn on the cell surface and be translocated into the cell, thereby promoting the uptake of the antigen from the extracellular into the cell. The antigen-binding molecule provided by the present invention is useful because administering such an antigen-binding molecule to a living body reduces the concentration of antigen present in plasma and reduces the physiological activity of the antigen in vivo.

[0044] Human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22-29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18 (12), 592-598). FcRn is expressed as a heterodimer consisting of a soluble β-chain (or light chain), β2-microglobulin, and a transmembrane α-chain (or heavy chain). The α-chain of FcRn consists of three extracellular domains (α1, α2, and α3), and the α1 and α2 domains interact with the FcRn-binding domain in the Fc region of antibodies (Raghavan et al., Immunity (1994) 1, 303-315).

[0045] The gene sequence and amino acid sequence of human FcRn have been registered under GenBank accession numbers NM_001136019 (SEQ ID NO: 16) and NP_001129491 (SEQ ID NO: 17), respectively. In addition to humans, the gene sequence and amino acid sequence of mouse FcRn have been registered under GenBank accession numbers NM_010189 (SEQ ID NO: 18) and NP_034319 (SEQ ID NO: 19), and the gene sequence and amino acid sequence of rat FcRn have been registered under GenBank accession numbers NM_033351 (SEQ ID NO: 20) and NP_203502 (SEQ ID NO: 21), respectively.

[0046] Human FcRn (SEQ ID NO: 17) forms a complex with human β2-microglobulin (SEQ ID NO: 38) in vivo. A complex of soluble human FcRn and β2-microglobulin can be produced using standard recombinant expression methods. The binding activity of the receptor-binding domain of the present invention can be assessed using such soluble human FcRn / β2-microglobulin complexes. In the present invention, unless otherwise specified, human FcRn refers to a form that is capable of binding to the receptor-binding domain of the present invention, and examples include a complex of human FcRn and human β2-microglobulin.

[0047] Receptor-binding domains that bind to FcRn more strongly than native human IgG under neutral pH conditions can be prepared by modifying the amino acids in the Fc region of human IgG. Examples of modifications include substitution, insertion, or deletion of one or more amino acids. Alternatively, antigen-binding domains that are characterized by FcRn binding can also be used as receptor-binding domains. Whether the FcRn-binding activity of a receptor-binding domain is higher than that of the Fc region of native human IgG can be determined appropriately using the methods described above.

[0048] In the present invention, "human FcRn-binding activity under an acidic pH range condition" refers to human FcRn-binding activity at pH 4.0 to pH 6.5, preferably at pH 5.0 to pH 6.5, more preferably at any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably at pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Furthermore, in the present invention, "human FcRn-binding activity under a neutral pH range condition" refers to human FcRn-binding activity at pH 6.7 to pH 10.0. Preferably, it refers to human FcRn-binding activity at pH 7.0 to pH 9.0, more preferably refers to human FcRn-binding activity at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and particularly preferably refers to human FcRn-binding activity at pH 7.4, which is close to the pH of plasma in vivo.

[0049] When the binding affinity between a receptor-binding domain and human FcRn is too low at pH 7.4, making accurate measurement of the affinity difficult, pH 7.0 can be used instead of pH 7.4. The binding affinity between a receptor-binding domain and human FcRn may be measured 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 receptor-binding domain and human FcRn. More preferably, a temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C is used. While not particularly limited, 25°C is a preferred embodiment.

[0050] When modifying the Fc region of IgG to obtain a receptor-binding domain that binds to human FcRn, preferred positions for modification include, for example, positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 260, 262 to 272, 274, 276, 278 to 289, and 291 to 320 (EU numbering). The amino acids at positions 324 to 341, 343, 345, 360 to 362, 370, 375 to 378, 380, 382, ​​384 to 387, 389 to 391, 396, 413, 414, 416, 422, 423, 424, 426 to 438, 440, and 442 are preferred. More specifically, examples of such amino acid modifications include those listed in Table 1. The use of these modifications can strengthen the binding of the IgG Fc region to human FcRn under neutral pH conditions.

[0051] [Table 1]

[0052] Furthermore, examples of alterations that can enhance the binding to human FcRn under acidic pH conditions compared to native human IgG are shown in Table 2. Among these alterations, alterations that can enhance the binding to human FcRn even under neutral pH conditions can be appropriately selected and used in the present invention. Particularly preferred positions for modification when modifying the IgG Fc region include, for example, EU numbering positions 234, 235, 236, 237, 238, 239, 244, 245, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 267, 270, 272, 274, 279, 280, 282, 283, 284, 285, 286, 288, 289, 293, 295, 297th, 298th, 303rd, 305th, 307th, 308th, 309th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 325th, 326th, 327th, 328th, 329th, 330th, 332th, 334th, 338th, 339th, 340th, 341st, 343rd, 345th, 360th, 361st, 362th, 375th, 376th, 377th, 378th, 380th, 382nd, 384th, 385th, 386th, Examples of suitable amino acids include those at positions 387, 389, 390, 391, 413, 422, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 437, 438, 440, and 442. Other preferred positions include positions 252, 254, 256, 309, 311, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 327, 328, 430, 431, 433, 434, 435, 436, 437, 438, 440, and 442 (EU numbering) as described in WO1997 / 034631.amino acids at positions 433 and / or 434, and amino acids at positions 253, 310, 435 and / or 426 in combination with these amino acids; amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​386, 388, 400, 413, 415, amino acids at positions 424, 433, 434, 435, 436, 439 and / or 447; amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and / or 436 (EU numbering) as described in WO2002 / 060919; amino acids at positions 250, 314 and 428 (EU numbering) as described in WO2004 / 092219; amino acids at positions 238, 244, 245 and 249 (EU numbering) as described in WO2006 / 020114; 252nd, 256th, 257th, 258th, 260th, 262th, 270th, 272th, 279th, 283th, 285th, 286th, 288th, 293th, 307th, 311th, 312th, 316th, 317th, 318th, 332th, 339th, 341st, 343th, 375th, 376th, 377th, 378th, 380th, 382th, 423rd, 427th, 430th, 431st, 434th, 436th, 438th, Amino acids at positions 440 and / or 442, positions 251, 252, 307, 308, and 378 (EU numbering) as described in WO2010 / 045193,Examples of amino acids that can be modified include those at positions 428, 430, 434, and / or 436. By modifying at least one amino acid selected from these amino acids, the human FcRn-binding activity in a neutral pH range can be increased. 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.

[0053] [Table 2]

[0054] Furthermore, examples of receptor-binding domains that have human FcRn-binding activity under acidic and neutral pH conditions include the Fc region of IgG, which is represented by EU numbering: The 234th amino acid is Arg, The 235th amino acid is Gly, Lys, or Arg; The 236th amino acid is Ala, Asp, Lys, or Arg; The 237th amino acid is Lys, Met, or Arg; The amino acid at position 238 is Ala, Asp, Lys, Leu, or Arg; The 239th amino acid is Asp or Lys, The 244th amino acid is Leu, The 245th amino acid is Arg, The 248th amino acid is Ile or Tyr, The 249th amino acid is Pro, The 250th amino acid is Ala, Glu, Phe, Ile, Met, Gln, Ser, Val, Trp, Gly, His, Leu, Asn, or Tyr; The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, Trp, or Tyr; The 253rd amino acid is Val, The 254th amino acid is Ala, Gly, His, Ile, Gln, Ser, Val, or Thr; The 255th amino acid is Ala, Asp, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Gly, Ser, Trp, Tyr, or Glu; The 256th amino acid is Ala, Asp, Glu, Arg, Asn, Pro, Thr, Ser, or Gln; The 257th amino acid is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The 258th amino acid is Asp or His, The 260th amino acid is Ser, The 262nd amino acid is Leu, The 265th amino acid is Ala, The 267th amino acid is Met or Leu, The 270th amino acid is Lys or Phe, The 272nd amino acid is Ala, Leu, or Arg; The 274th amino acid is Ala, The 279th amino acid is Leu, Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 280th amino acid is Ala, Gly, His, Lys, Asn, Gln, Arg, Ser, Thr, or Glu; The 282nd amino acid is Ala or Asp, The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 284th amino acid is Lys, The 285th amino acid is Asn, The 286th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr or Glu; The 288th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Val, Trp, Tyr or Ser; The 289th amino acid is His, The 293rd amino acid is Val, The 295th amino acid is Met, The 297th amino acid is Ala, The 298th amino acid is Gly. The 303rd amino acid is Ala, The 305th amino acid is Ala or Thr, The 307th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The 308th amino acid is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The 309th amino acid is Ala, Asp, Glu, Pro, His, or Arg; The 311th amino acid is Ala, His, Glu, Lys, Leu, Met, Ser, Val, Trp, or Ile; The 312th amino acid is Ala, Asp, Pro, or His; The 313th amino acid is Tyr or Phe, The 314th amino acid is Ala, Leu, Lys, or Arg; The 315th amino acid is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, Tyr or His; The 316th amino acid is Ala, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Asp; The 317th amino acid is Ala or Pro, The 318th amino acid is Asn or Thr, The 325th amino acid is Ala, Gly, Met, Leu, Ile, or Ser; The 326th amino acid is Asp, The 327th amino acid is Gly, The amino acid at position 328 is Arg, Asp, Glu, or Tyr; The 329th amino acid is Lys or Arg, The 330th amino acid is Leu, The 332nd amino acid is Glu, Phe, His, Lys, Leu, Met, Arg, Ser, Trp, or Val; The 334th amino acid is Leu, The 338th amino acid is Ala, The 339th amino acid is Asn, Thr, or Trp; The 340th amino acid is Ala, The 341st amino acid is Pro, The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 345th amino acid is Ala, The 360th amino acid is His, The 361st amino acid is Ala, The 362nd amino acid is Ala, The 375th amino acid is Ala or Arg, The 376th amino acid is Ala, Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys, The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Thr, or Ser; The 382nd amino acid is Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Trp, Tyr, or Val; The 384th amino acid is Ala, The 385th amino acid is Ala, Gly, Lys, Ser, Thr, Asp, His, or Arg; The amino acid at position 386 is Arg, Asp, Ile, Met, Ser, Thr, Lys, or Pro; The 387th amino acid is Ala, Arg, His, Pro, Ser, Thr, or Glu; The 389th amino acid is Ala, Asn, Pro, or Ser; The 390th amino acid is Ala, The 391st amino acid is Ala, The 413th amino acid is Ala, The 423rd amino acid is Asn, The 424th amino acid is Ala or Glu, The 427th amino acid is Asn, The 428th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The 431st amino acid is His or Asn, The 433rd amino acid is Arg, Gln, His, Ile, Pro, Ser, or Lys; The 434th amino acid is Ala, Phe, Gly, Met, His, Ser, Trp, or Tyr; The 435th amino acid is Lys, Arg, or Asn; The 436th amino acid is Ala, His, Ile, Leu, Glu, Phe, Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp, or Val; The 437th amino acid is Arg, Amino acid 438 is Lys, Leu, Thr, or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys, The Fc region may have amino acids selected from one or more positions. The amino acids selected may be selected from one or more positions. Examples of combinations of amino acids at two or more positions include the amino acid combinations listed in Table 3, Tables 4-1 to 4-5, and Tables 13-1 to 13-14.

[0055] [Table 3]

[0056] [Table 4-1]

[0057] Table 4-2 is a continuation of Table 4-1. [Table 4-2]

[0058] Table 4-3 is a continuation of Table 4-2. [Table 4-3]

[0059] Table 4-4 is a continuation of Table 4-3. [Table 4-4]

[0060] Table 4-5 is a continuation of Table 4-4. [Table 4-5]

[0061] Herein, "having a higher human FcRn-binding activity than native human IgG" means, for example, that the human FcRn-binding activity is 105% or more, preferably 110% or more, 115% or more, 120% or more, or 125% or more, and particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 7.5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-fold or more of that of native human IgG.

[0062] These amino acid modifications can be appropriately carried out using known techniques, and are described, for example, in Drug Metab Dispos. 2007 Jan;35(1):86-94, Int Immunol. 2006 Dec;18(12):1759-69, J Biol Chem. 2001 Mar 2;276(9):6591-604, J Biol Chem. 2007;282(3):1709-17, J Immunol. 2002;169(9):5171-80, J Immunol. 2009;182(12):7663-71, Molecular Cell, Vol. 7, 867-877, April, 2001, Nat Biotechnol. 1997 Jul;15(7):637-40, Nat Biotechnol. 2005 Oct;23(10):1283-8, Proc Natl Acad Sci US A. 2006 Dec 5;103(49):18709-14, EP2154157, US20070141052, WO2000 / 042072, WO2002 / 060919, WO2006 / 020114, WO2006 / 031370, WO2010 / 033279, WO2006 / 053301, and WO2009 / 086320 have been reported in which the Fc region of human IgG1 has been modified.

[0063] According to Yeung et al. (The Journal of Immunology, 2009, 182: 7663-7671), the human FcRn-binding activity of human IgG1 in the acidic pH range (pH 6.0) is KD 1.7 μM, whereas the human FcRn-binding activity of human IgG1 is barely detectable in the neutral pH range. Thus, preferred embodiments of the antigen-binding molecules provided by the present invention include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 20 μM or stronger and whose human FcRn-binding activity in the neutral pH range is equal to or stronger than that of human IgG. More preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 2.0 μM or stronger and whose human FcRn-binding activity in the neutral pH range is KD 40 μM or stronger. More preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in an acidic pH range is a KD of 0.5 μM or stronger and whose human FcRn-binding activity in a neutral pH range is a KD of 15 μM or stronger. The KD values ​​shown here are values ​​measured by the method described in The Journal of Immunology, 2009, 182: 7663-7671 (in which the antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0064] In a preferred embodiment, the antigen-binding molecule provided by the present invention has a human FcRn-binding activity higher than that of human IgG at pH 7.0 and 25°C. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25°C is 28-fold higher than that of human IgG, or a KD of 3.2 μM or stronger. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25°C is 38-fold higher than that of human IgG, or a KD of 2.3 μM or stronger.

[0065] Although the dissociation constant (KD) can be used as a measure of human FcRn-binding activity, the human FcRn-binding activity of human IgG is barely detectable in the neutral pH range (pH 7.4), making it difficult to calculate the KD. One method for determining whether the human FcRn-binding activity at pH 7.4 is higher than that of human IgG is to determine the magnitude of the binding response in Biacore when analytes are applied at the same concentration. Specifically, if the response when human FcRn is applied to a chip on which an antigen-binding molecule provided by the present invention is immobilized is greater than the response when human FcRn is applied to a chip on which human IgG is immobilized, the human FcRn-binding activity of the antigen-binding molecule can be determined to be higher than that of human IgG.

[0066] An Fcγ receptor (FcγR) refers to a receptor that can bind to the Fc region of IgG (for example, IgG1, IgG2, IgG3, IgG4, etc.), and refers to substantially any member of the Fcγ receptor family. 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), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR isoforms or allotypes, but is not limited to these. Organisms from which FcγRs are derived include, but are not limited to, humans, mice, rats, rabbits, and monkeys, and may be derived from any organism. 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 FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI are shown in SEQ ID NOs: 39 (NM_000566.3) and 40 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of human FcγRIIa (allotype H131) are shown in SEQ ID NOs: 41 (BC020823.1) and 42 (AAH20823.1), respectively (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 42 is substituted with Arg). The polynucleotide sequence and amino acid sequence of FcγRIIb are shown in SEQ ID NOs: 42 (BC020823.1) and 42 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIa are set forth in SEQ ID NOs: 43 (BC146678.1) and 44 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIa are set forth in SEQ ID NOs: 45 (BC033678.1) and 46 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are set forth in SEQ ID NOs: 47 (BC128562.1) and 48 (AAI28563.1), respectively (RefSeq accession numbers are shown in parentheses). Whether an Fcγ receptor has binding activity to the Fc region of IgG can be confirmed by FACS, ELISA, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE using the surface plasmon resonance (SPR) phenomenon, and the like (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0067] Additionally, "Fc ligand" or "effector ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form a complex. Binding of the Fc ligand to the Fc region preferably induces one or more effector functions. Examples of Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, staphylococcal protein G, and viral FcγR. Examples of Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to FcγRs (Davis et al., (2002) Immunological Reviews 190, 123-136). Examples of Fc ligands also include unidentified molecules that bind to Fc.

[0068] 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 two subunits: an α chain that binds to the Fc region of IgG and a common γ chain with ITAMs that transduce activation signals intracellularly. Meanwhile, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its intracellular 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 the activation of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are referred to as activating Fcγ receptors in the present invention.

[0069] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transduces 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 B cells. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic function and the production of inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are referred to as inhibitory Fcγ receptors in the present invention.

[0070] Receptor-binding domains that bind to Fcγ receptors more strongly than native human IgG under neutral pH conditions can be prepared by modifying amino acids in the Fc region of human IgG. Examples of modifications include substitution, insertion, or deletion of one or more amino acids. Furthermore, antigen-binding domains that bind to Fcγ receptors can also be used as receptor-binding domains. Examples of such receptor-binding domains include FcγRIIIa-binding Fab fragments, camel-derived single-domain antibodies, and single-chain Fvs, as described in Protein Eng Des Sel. 2009 Mar;22(3):175-88, Protein Eng Des Sel. 2008 Jan;21(1):1-10, and J Immunol. 2002 Jul 1;169(1):137-44, and the FcγRI-binding cyclic peptide described in FASEB J. 2009 Feb;23(2):575-85. Whether the binding activity of a receptor-binding domain to an Fcγ receptor is higher than the binding activity of the Fc region of native human IgG to an Fcγ receptor can be determined appropriately using the methods described above.

[0071] In the present invention, "human Fcγ receptor-binding activity under an acidic pH range condition" refers to human Fcγ receptor-binding activity between pH 4.0 and pH 6.5. Preferably, it refers to human Fcγ receptor-binding activity between pH 5.0 and pH 6.5, more preferably to human Fcγ receptor-binding activity at any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably to human Fcγ receptor-binding activity between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo. Furthermore, in the present invention, "human Fcγ receptor-binding activity under a neutral pH range condition" refers to human Fcγ receptor-binding activity between pH 6.7 and pH 10.0. Preferably, it refers to human Fcγ receptor binding activity at pH 7.0 to pH 9.0, more preferably to human Fcγ receptor binding activity at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and particularly preferably to human Fcγ receptor binding activity at pH 7.4, which is close to the pH of plasma in vivo.

[0072] The binding affinity between a receptor-binding domain and a human Fcγ receptor may be measured at any temperature between 10°C and 50°C as a measurement condition. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between a receptor-binding domain and a human Fcγ receptor. More preferably, a temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C is used. Although not particularly limited, a temperature of 25°C is one preferred embodiment.

[0073] A suitable example of a receptor-binding domain of the present invention is the Fc region of human IgG. The origin of the Fc region is not limited, and it can be obtained from any non-human animal or human. Preferred non-human animals include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels, and non-human primates. In another embodiment, the receptor-binding domain can be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. The Fc region is preferably obtained from the Fc region of human IgG1, but is not limited to a particular class of IgG. That is, the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as the receptor-binding domain, as appropriate. Examples of naturally occurring or artificially modified IgG variants 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, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0074] As long as the receptor-binding domain binds to Fcγ receptors more strongly than native human IgG under neutral pH conditions, amino acids at any positions can be modified. When the receptor-binding domain is prepared by modifying the Fc region of human IgG1, amino acid modifications that enhance Fcγ receptor-binding activity under neutral pH conditions include, for example, the amino acid modifications described in 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.

[0075] When the Fc region of IgG is modified to form a receptor-binding domain that binds to a human Fcγ receptor, preferred amino acids include, for example, 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, 247, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272th, 273th, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282th, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 292th, 293th, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 307th, 308th, 309th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 339th, 341st, 343rd, 375th, 376th, 377th, 378th, 379th, 380th, 382nd, 385th, 386th, 387th, 389th, 392nd, 396th, 421st, 423rd, 427th, 428th, 429th, 430th, 431st, 433rd, 434th, 436th, 438th,The amino acid sequence is at least one amino acid selected from the group consisting of amino acids 440 and 442. Modification of these amino acids enhances the binding activity of the IgG Fc region to Fcγ receptors under neutral pH conditions.

[0076] Particularly preferred modifications for enhancing binding to Fcγ receptors under neutral pH conditions include, for example, modifications represented by EU numbering of the Fc region: The 221st amino acid is Lys or Tyr, Amino acid 222 is Phe, Trp, Glu, or Tyr; The 223rd amino acid is Phe, Trp, Glu, or Lys; Amino acid 224 is Phe, Trp, Glu, or Tyr; The 225th amino acid is Glu, Lys, or Trp, The 227th amino acid is Glu, Gly, Lys, or Tyr; The 228th amino acid is Glu, Gly, Lys, or Tyr; The 230th amino acid is Ala, Glu, Gly, or Tyr; The 231st amino acid is Glu, Gly, Lys, Pro, or Tyr; The 232nd amino acid is Glu, Gly, Lys, or Tyr; The 233rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 234th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 235th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 236th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 237th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 238th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 239th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; The 240th amino acid is Ala, Ile, Met, or Thr; The 241st amino acid is Asp, Glu, Leu, Arg, Trp, or Tyr; The 243rd amino acid is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The 244th amino acid is His, The 245th amino acid is Ala, The 246th amino acid is Asp, Glu, His, or Tyr; The 247th amino acid is Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The 249th amino acid is Glu, His, Gln, or Tyr; The 250th amino acid is Glu or Gln, The 251st amino acid is Phe, Amino acid 254 is Phe, Met, or Tyr; The 255th amino acid is Glu, Leu, or Tyr; The 256th amino acid is Ala, Met, or Pro; The 258th amino acid is Asp, Glu, His, Ser, or Tyr; The 260th amino acid is Asp, Glu, His, or Tyr, The amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; The 263rd amino acid is Ala, Ile, Met, or Thr; The 264th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 265th amino acid is Ala, Glu, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 266th amino acid is Ala, Phe, Ile, Leu, Met, or Thr; The 267th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; The 268th amino acid is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Trp; The 269th amino acid is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 270th amino acid is Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 271st amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 272nd amino acid is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Amino acid 273 is Phe or Ile; The 274th amino acid is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 275th amino acid is Leu or Trp, The 276th amino acid is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 278th amino acid is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The 279th amino acid is Ala, The 280th amino acid is Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr; The 281st amino acid is Asp, Lys, Pro, or Tyr; The 282nd amino acid is Glu, Gly, Lys, Pro, or Tyr; The 283rd amino acid is Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; The 284th amino acid is Asp, Glu, Leu, Asn, Thr, or Tyr; The 285th amino acid is Asp, Glu, Lys, Gln, Trp, or Tyr; The 286th amino acid is Glu, Gly, Pro, or Tyr; The 288th amino acid is Asn, Asp, Glu, or Tyr; The 290th amino acid is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr; The 291st amino acid is Asp, Glu, Gly, His, Ile, Gln, or Thr; The 292nd amino acid is Ala, Asp, Glu, Pro, Thr, or Tyr; The 293rd amino acid is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 294th amino acid is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 295th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 296th amino acid is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; The 297th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 298th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr; The 299th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The 300th amino acid is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The 301st amino acid is Asp, Glu, His, or Tyr, The 302nd amino acid is Ile, The 303rd amino acid is Asp, Gly, or Tyr; The 304th amino acid is Asp, His, Leu, Asn, or Thr; The 305th amino acid is Glu, Ile, Thr, or Tyr; The 311th amino acid is Ala, Asp, Asn, Thr, Val, or Tyr; The 313th amino acid is Phe, The 315th amino acid is Leu, The 317th amino acid is Glu or Gln, The 318th amino acid is His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; The amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; The 322nd amino acid is Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr; The 323rd amino acid is Ile, Leu, or Met; The 324th amino acid is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; The 325th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 326th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The 327th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; The 328th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 329th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 330th amino acid is Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 331st amino acid is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 332nd amino acid is Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 333rd amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, or Tyr; The 334th amino acid is Ala, Glu, Phe, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 335th amino acid is Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; The 336th amino acid is Glu, Lys, or Tyr; The 337th amino acid is Asp, Glu, His, or Asn, The 339th amino acid is Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr; The 376th amino acid is Ala or Val, The 377th amino acid is Gly or Lys, The 378th amino acid is Asp, The 379th amino acid is Asn, The 380th amino acid is Ala, Asn, or Ser; The 382nd amino acid is Ala or Ile, The 385th amino acid is Glu, The 392nd amino acid is Thr, The 396th amino acid is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr; The 421st amino acid is Lys. The 427th amino acid is Asn, Amino acid 428 is Phe or Leu, The 429th amino acid is Met, The 434th amino acid is Trp, The 436th amino acid is Ile, and 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 Tables 5-1 to 5-3.

[0077] [Table 5-1]

[0078] Table 5-2 is a continuation of Table 5-1. [Table 5-2]

[0079] Table 5-3 is a continuation of Table 5-2. [Table 5-3]

[0080] As used herein, "having a higher human Fcγ receptor-binding activity than native human IgG" means that the human Fcγ receptor-binding activity of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb is higher than that of native human IgG. For example, the activity of binding to human Fcγ receptors is 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% 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 of that of native human IgG.

[0081] Receptor-binding domains of the present invention may have the property that their binding activity to a specific Fcγ receptor is higher than their binding activity to other Fcγ receptors (selective binding to a specific Fcγ receptor), such as receptor-binding domains whose binding activity to inhibitory Fcγ receptors is higher than their binding activity to activating Fcγ receptors. Preferred examples include receptor-binding domains whose binding activity to the inhibitory Fcγ receptor FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) is higher than that to an activating Fcγ receptor selected from FcγRI (CD64) including the isoforms FcγRIa, FcγRIb, and FcγRIc, FcγRIII (CD16) including the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and FcγRII (CD32) including the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc. Particularly preferred are receptor-binding domains whose binding activity to FcγRIIb-1 and / or FcγRIIb-2 is higher than its binding activity to FcγRIIa (allotype H131).

[0082] Whether a receptor-binding domain has the property of selectively binding to a specific Fcγ receptor can be determined by measuring and comparing the KD values ​​of the receptor-binding domain for each Fcγ receptor. For example, if the KD value of the receptor-binding domain for an activating Fcγ receptor divided by the KD value for an inhibitory Fcγ receptor is 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, or 75 or more. Above, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more Above, 410 and above, 420 and above, 430 and above, 440 and above, 450 and above, 460 and above, 470 and above, 480 and above, 490 and above, 500 and above, 520 and above, 540 and above, 560 and above, 580 and above, 600 and above, 620 and above, 640 and above, 660 and above, 680 and above, 700 and above, 720 and above, 740 and above, 760 and above, 780 and above, 800 and above, 820 and above, 840 and above, 860 and above, 880 and above, 900 and above, 920 and above, 940 and above, 960 and above, 980 and above , 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, or 100,000 or more, the receptor-binding domain can be determined to selectively bind to inhibitory Fcγ receptors over activating Fcγ receptors.

[0083] Although not particularly limited, examples of receptor-binding domains whose binding activity to inhibitory Fcγ receptors is higher than that to activating Fcγ receptors (which selectively bind to inhibitory Fcγ receptors) preferably include IgG Fc region variants described in WO2012 / 115241, such as Fc regions in which the amino acids at positions 238 and / or 328 (EU numbering) have been altered to different amino acids, more preferably Fc regions in which amino acid 238 has been altered to Asp and / or amino acid 328 has been altered to Glu. Alternatively, IgG Fc region variants described in US2009 / 0136485 can also be selected as appropriate.

[0084] At least one other alteration may be added to the above alterations in the IgG Fc region, which preferably results in enhanced FcγRIIb-binding activity and maintained or reduced binding activity to FcγRIIa (allotype H131) and FcγRIIa (allotype R131). Addition of such alterations improves binding selectivity for FcγRIIb over FcγRIIa. Alterations that improve binding selectivity for FcγRIIb over FcγRIIa (allotype R131) are preferred, and alterations that improve binding selectivity for FcγRIIb over FcγRIIa (allotype R131) and FcγRIIa (allotype H131) are even more preferred. Non-limiting examples of such alterations include: The 233rd amino acid is Asp, The 234th amino acid is Trp or Tyr, Amino acid 235 is Phe, Trp, or Tyr; The 236th amino acid is Asp, The 237th amino acid is Ala, Asp, Glu, Phe, Leu, Met, Trp, or Tyr; Amino acid 238 is Phe or Leu, The 239th amino acid is Asp, Glu, Gly, Leu, or Asn, The 266th amino acid is Ile, Leu, or Met; The 267th amino acid is Ala, Asp, Glu, Ile, Met, Gln, or Val; The 268th amino acid is Ala, Asp, Glu, Gly, Asn, or Gln; The 271st amino acid is Gly or Leu, The 295th amino acid is Leu, The 296th amino acid is Asp, The 300th amino acid is Asp, Glu, or Gln, The 323rd amino acid is Ile, Leu, or Met; The 324th amino acid is Ile or Val, The 325th amino acid is Met or Ser, The 326th amino acid is Ala, Asp, Glu, Phe, His, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The 327th amino acid is Asp, Glu, Gly, or Asn, The 328th amino acid is Ala, Asp, Phe, Ile, Met, Gln, Ser, Thr, Val, Trp, or Tyr; The 330th amino acid is Lys, Met, or Arg; Amino acid 331 is Phe, Trp, or Tyr; The 332nd amino acid is Phe, The 333rd amino acid is Pro, The 334th amino acid is Ala, Trp, Glu, Phe, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr; The 335th amino acid is Asp, and The 337th amino acid is Asp, The modification of at least one amino acid selected from the group consisting of:

[0085] Furthermore, among these, preferred examples of modifications include EU numbering; The 233rd amino acid is Asp, The 234th amino acid is Trp or Tyr, The 237th amino acid is Ala, Asp, Glu, Phe, Leu, Met, Trp, or Tyr; The 239th amino acid is Asp, The 267th amino acid is Ala, Gln, or Val; The 268th amino acid is Asp, Glu, or Asn The 271st amino acid is Gly The 296th amino acid is Asp, The 323rd amino acid is Ile, Leu, or Met; The 326th amino acid is Ala, Asp, Glu, Leu, Met, Asn, Gln, Ser, or Thr; The 330th amino acid is Lys, Met, or Arg; The modification of at least one amino acid selected from the group consisting of:

[0086] The above modifications may be made at one position or in combination of two or more positions. Preferred examples of such modifications include the modifications shown in Tables 24 to 25, 27 to 34, and 36 to 37.

[0087] An embodiment of the receptor-binding domain contained in the antigen-binding molecules of the present invention includes, but is not limited to, an altered Fc region of human IgG1 (SEQ ID NO: 49), IgG2 (SEQ ID NO: 50), IgG3 (SEQ ID NO: 51), or IgG4 (SEQ ID NO: 52). An example of such an altered Fc region is an Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly. Fc regions of human IgG (IgG1, IgG2, IgG3, IgG4) in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) and antigen-binding molecules comprising such Fc regions have higher binding activity to inhibitory Fcγ receptors than to activating Fcγ receptors.

[0088] In the present invention, at least one further modification may be made to the Fc region in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 is Gly, preferably such that the binding activity to FcγRIIb-1 and / or FcγRIIb-2 is enhanced and the binding activity to FcγRIIa (allotype H131) and FcγRIIa (allotype R131) is maintained or reduced. Furthermore, it is preferable that the degree of enhancement of binding activity to inhibitory Fcγ receptors (FcγRIIb-1 and / or FcγRIIb-2) is higher than the degree of enhancement of binding activity to activating Fcγ receptors (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa (allotype V158), FcγRIIIa (allotype F158), FcγRIIIb (allotype FcγRIIIb-NA1), FcγRIIIb (allotype FcγRIIIb-NA2), FcγRIIa (allotype H131), FcγRIIa (allotype R131)). Such alterations improve binding selectivity for FcγRIIb over FcγRIIa.

[0089] Examples of selective receptor-binding domains include, but are not limited to, those in which the amino acid at position 238 (EU numbering) in the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) has been modified to Asp and the amino acid at position 271 (EU numbering) has been modified to Gly, and the following amino acids (EU numbering) have been modified: 233, 234, 237, 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 264, 265, 266, 267, 268, 269, 270, 272, 279, 283, 285, 286, 288, 293rd, 296th, 307th, 308th, 309th, 311th, 312th, 314th, 316th, 317th, 318th, 326th, 327th, 330th, 331st, 332th, 333th, 339th, 341st, 343th, 375th, 376th, 377th, 378th, 380th, 382nd, 385th, 386th, 387th, 389th, 396th, 423rd, 427th, 428th, 430th, 431st, 433rd, 434th, 436th, 438th, Examples include Fc regions in which one or more amino acids at positions 440 and 442 have been altered.

[0090] Furthermore, examples of selective receptor-binding domains include, but are not limited to, those in which the amino acid at position 238 in the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) is modified to Asp and the amino acid at position 271 in the EU numbering system is modified to Gly, and in which the amino acid at position 271 in the EU numbering system is modified to Asp and Gly, respectively; The 233rd amino acid is Asp, The 234th amino acid is Tyr, The 237th amino acid is Asp, The 264th amino acid is Ile, The 265th amino acid is Glu, Amino acid 266 is Phe, Met, or Leu; The 267th amino acid is Ala, Glu, Gly, or Gln; The 268th amino acid is Asp or Glu, The 269th amino acid is Asp, The amino acid at position 272 is Asp, Phe, Ile, Met, Asn, or Gln; The 296th amino acid is Asp, The 326th amino acid is Ala or Asp, The 327th amino acid is Gly, The 330th amino acid is Lys or Arg, The 331st amino acid is Ser, The 332nd amino acid is Thr, The 333rd amino acid is Thr, Lys, or Arg; The 396th amino acid is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr; The present invention also includes an Fc region in which one or more amino acids selected from the group consisting of:

[0091] Although not particularly limited, examples of the Fc region include the Fc regions listed in Tables 6-1 to 6-6.

[0092] [Table 6-1]

[0093] Table 6-2 is a continuation of Table 6-1. [Table 6-2]

[0094] Table 6-3 is a continuation of Table 6-2. [Table 6-3]

[0095] Table 6-4 is a continuation of Table 6-3. [Table 6-4]

[0096] Table 6-5 is a continuation of Table 6-4. [Table 6-5]

[0097] Table 6-6 is a continuation of Table 6-5. [Table 6-6]

[0098] It is preferred that the above-mentioned receptor-binding domain that binds to human Fcγ receptor further contains amino acid modifications that enhance FcRn binding under acidic pH conditions. Examples of amino acids that can be modified in this way include amino acids at positions 252, 254, 256, 309, 311, 315, 433, and / or 434 (EU numbering) as described in WO1997 / 034631, and amino acids at positions 253, 310, 435, and / or 426 (EU numbering) combined with these amino acids; and amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 370, 372, 374, 376, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 420, 421, 422, 423, 424, 425, 426, 428, 430, 431, 432, 433, 434, 435, 436, 438, 440, 445, 446, 447, 448, 449, 450, 45 Amino acids at positions 362, 376, 378, 380, 382, ​​386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and / or 447, and amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, EU numbering as described in WO2002 / 060919. amino acids at positions 434 and / or 436, amino acids at positions 250, 314 and 428 (EU numbering) as described in WO2004 / 092219, amino acids at positions 238, 244, 245, 249, 252, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316, 317, 318, 332, 339, 341 and 343 (EU numbering) as described in WO2006 / 020114,Examples of such amino acids include those at positions 375, 376, 377, 378, 380, 382, ​​423, 427, 430, 431, 434, 436, 438, 440, and / or 442, and those at positions 251, 252, 307, 308, 378, 428, 430, 434, and / or 436 (EU numbering) as described in WO2010 / 045193. Modification of these amino acids enhances the binding of the IgG Fc region to FcRn under acidic pH conditions.

[0099] More specifically, examples of such modifications include EU numbering; The 244th amino acid is Leu, The 245th amino acid is Arg, The 249th amino acid is Pro, The 250th amino acid is Gln or Glu, The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, or Tyr; The 254th amino acid is Ser or Thr, The 255th amino acid is Arg, Gly, Ile, or Leu; The 256th amino acid is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr; The 257th amino acid is Ala, Ile, Met, Asn, Ser, or Val; The 258th amino acid is Asp, The 260th amino acid is Ser, The 262nd amino acid is Leu, The 270th amino acid is Lys, The 272nd amino acid is Leu or Arg, The 279th amino acid is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 285th amino acid is Asn, The 286th amino acid is Phe, The 288th amino acid is Asn or Pro, The 293rd amino acid is Val, The 307th amino acid is Ala, Glu, Gln, or Met; The 308th amino acid is Ile, Pro, or Thr; The 309th amino acid is Pro, The 311th amino acid is Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp, The 312th amino acid is Ala, Asp, or Pro; The 314th amino acid is Ala or Leu, The 316th amino acid is Lys, The 317th amino acid is Pro, The 318th amino acid is Asn or Thr, Amino acid 332 is Phe, His, Lys, Leu, Met, Arg, Ser, or Trp; The 339th amino acid is Asn, Thr, or Trp; The 341st amino acid is Pro, The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 375th amino acid is Arg, The 376th amino acid is Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys, The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Ser, or Thr; Amino acid 382 is Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 385th amino acid is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; The 386th amino acid is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; The 387th amino acid is Ala, Arg, His, Pro, Ser, or Thr; The 389th amino acid is Asn, Pro, or Ser; The 423rd amino acid is Asn, The 427th amino acid 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 431st amino acid is His or Asn, The 433rd amino acid is Arg, Gln, His, Ile, Lys, Pro, or Ser; The 434th amino acid is Ala, Gly, His, Phe, Ser, Trp, or Tyr; The 436th amino acid is Arg, Asn, His, Ile, Leu, Lys, Met, or Thr; Amino acid 438 is Lys, Leu, Thr, or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys, The modification of at least one amino acid selected from the group consisting of:

[0100] Examples of modifications that can enhance binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Ile at amino acid 308, Pro at amino acid 309, and / or Glu at amino acid 311; modifications including Thr at amino acid 308, Pro at amino acid 309, Leu at amino acid 311, Ala at amino acid 312, and / or Ala at amino acid 314; Modifications including Ile or Thr at amino acid 308, Pro at amino acid 309, Glu, Leu, or Ser at amino acid 311, Ala at amino acid 312, and / or Ala or Leu at amino acid 314, and modifications including Thr at amino acid 308, Pro at amino acid 309, Ser at amino acid 311, Asp at amino acid 312, and / or Leu at amino acid 314; At least one or more modifications selected from the group:

[0101] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: modifications including Leu at amino acid 251, Tyr at amino acid 252, Ser or Thr at amino acid 254, Arg at amino acid 255, and / or Glu at amino acid 256; Examples include:

[0102] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: Modifications including Leu, Met, Phe, Ser, or Thr at amino acid 428, Arg, Gln, His, Ile, Lys, Pro, or Ser at amino acid 433, His, Phe, or Tyr at amino acid 434, and / or Arg, Asn, His, Lys, Met, or Thr at amino acid 436, and a modification including His or Met at amino acid 428 and / or His or Met at amino acid 434; At least one or more modifications selected from the group:

[0103] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Arg at amino acid 385, Thr at amino acid 386, Arg at amino acid 387, and / or Pro at amino acid 389; and a modification comprising Asp at amino acid 385, Pro at amino acid 386, and / or Ser at amino acid 389; At least one or more modifications selected from the group:

[0104] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: A modification containing Gln or Glu at amino acid 250, and a modification in which the amino acid at position 428 contains Leu or Phe; At least one or more modifications selected from the group:

[0105] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Gln at amino acid position 250 and / or Leu or Phe at amino acid position 428, and a modification including Glu at amino acid position 250 and / or Leu or Phe at amino acid position 428; At least one or more modifications selected from the group:

[0106] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Gln at amino acid position 307 and Ala or Ser at amino acid position 434; a modification containing Pro at amino acid 308 and Ala at amino acid 434; a modification comprising Tyr at amino acid 252 and Ala at amino acid 434; a modification containing Val at amino acid position 378 and Ala at amino acid position 434; a modification containing Leu at amino acid 428 and Ala at amino acid 434; a modification comprising Ala at amino acid 434 and Ile at amino acid 436; A modification comprising Pro at amino acid 308 and Tyr at amino acid 434, and a modification comprising Gln at amino acid position 307 and Ile at amino acid position 436; At least one or more modifications selected from the group:

[0107] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Gln at amino acid 307, Ala at amino acid 380, and Ser at amino acid 434; a modification comprising Gln at amino acid 307, Ala at amino acid 380, and Ala at amino acid 434; A modification comprising Tyr at amino acid 252, Pro at amino acid 308, and Tyr at amino acid 434, and a modification comprising Asp at amino acid 251, Gln at amino acid 307, and His at amino acid 434; At least one or more modifications selected from the group:

[0108] In addition to the above, examples of modifications that can enhance the binding to human FcRn under acidic pH conditions compared to human IgG include: a modification comprising Ile at amino acid 257 and Ile at amino acid 311; a modification comprising Ile at amino acid position 257 and His at amino acid position 434, and a modification containing Val at amino acid 376 and His at amino acid 434; At least one or more modifications selected from the group:

[0109] When an antigen-binding molecule of the present invention is produced using the Fc region of human IgG, the effect of the antigen-binding molecule of the present invention can be verified by using an antigen-binding molecule containing the Fc region of an IgG of the same subclass as the control. Examples of control human IgG Fc regions that can be used include the Fc regions of human IgG1 (SEQ ID NO: 49, RefSeq Accession No. AAC82527.1 with A added to the N-terminus), human IgG2 (SEQ ID NO: 50, RefSeq Accession No. AAB59393.1 with A added to the N-terminus), human IgG3 (SEQ ID NO: 51, RefSeq Accession No. CAA27268.1), and human IgG4 (SEQ ID NO: 52, RefSeq Accession No. AAB59394.1 with A added to the N-terminus).

[0110] Four Fcγ receptors have been identified in mice: FcγRI (CD64), FcγRIIb (CD32), FcγRIII (CD16), and FcγRIV (CD16-2 or FcγRIII-2). As in humans, FcγRIIb is considered the only inhibitory Fcγ receptor. Two splicing variants of FcγRIIb, FcγRIIb1 and FcγRIIb2, have been reported. In both humans and mice, FcγRIIb1 has a longer intracellular domain than FcγRIIb2. FcγRIIb1 has been confirmed to be expressed in B cells, while FcγRIIb2 has been confirmed to be expressed in macrophages, mast cells, dendritic cells, basophils, neutrophils, and eosinophils (J. Clin. Immunol. (2005) 25 (1), 1-18).

[0111] It has been reported that dysfunction and decreased expression of FcγRIIb are correlated with the onset of autoimmune diseases in humans. For example, some SLE patients have been reported to have reduced FcγRIIb expression due to a genetic polymorphism in the FcγRIIb promoter region, which weakens transcriptional activator binding (Hum. Genet. (2005) 117, 220-227; J. Immunol. (2004) 172, 7192-7199; J. Immunol. (2004) 172, 7186-7191). Furthermore, two genetic polymorphisms have been reported in SLE patients, where the 233rd amino acid of FcγRIIb is either Ile or Thr. This site is located in the transmembrane domain of FcγRIIb, and it has been reported that when the 233rd amino acid is Thr, FcγRIIb is less likely to reside in lipid rafts than when it is Ile, resulting in a decrease in the signaling function of FcγRIIb (Nat. Med. (2005) 11, 1056-1058, Hum. Mol. Genet., (2005) 14, 2881-2892). It has also been reported that knockout mice in which the FcγRIIb gene of C57BL / 6 mice was disrupted exhibit SLE-like symptoms, such as autoantibody production and glomerulonephritis (Immunity 13 (2000) 277-285, J. Exp. Med. (2002) 195, 1167-1174). Furthermore, decreased expression of FcγRIIb has been reported in mice, which have been considered a model of spontaneous SLE (Immunogenetics (2000) 51, 429-435, Int. Immunol. (1999) 11, 1685-1691, Curr. Biol. (2000) 10, 227-230, J. Immunol. (2002) 169, 4340-4346). These findings suggest that FcγRIIb regulates humoral immunity in mice, as in humans.

[0112] When antibodies comprising the Fc region of the present invention eliminate antigens via FcγRIIb, the endocytic function of FcγRIIb is thought to be the most important factor. As mentioned above, FcγRIIb has two splicing variants, FcγRIIb1 and FcγRIIb2, and it has been reported that the latter is primarily involved in the endocytosis of antibody-antigen immune complexes (J. Immunol. (1994), 152, 574-585; Science (1992) 256, 1808-1812; Cell (1989) 58, 317-327). It has previously been reported that mouse FcγRIIb2 is incorporated into clathrin-coated pits and undergoes endocytosis (Cell (1989) 58, 317-327). Furthermore, it has been reported that the dileucine motif is required for FcγRIIb2-mediated endocytosis, and this motif is conserved in both humans and mice (EMBO J. (1994) 13 (13), 2963-2969). This suggests that FcγRIIb2 has endocytic activity in humans as well as in mice.

[0113] On the other hand, it has been reported that FcγRIIb1, unlike FcγRIIb2, does not undergo endocytosis. FcγRIIb1 contains an insertion sequence in the intracellular domain that is not found in FcγRIIb2. This sequence is thought to inhibit the incorporation of FcγRIIb1 into clathrin-coated pits, resulting in the inhibition of endocytosis (J. Cell. Biol. (1992) 116, 875-888, J. Cell. Biol. (1989) 109, 3291-3302). Because human FcγRIIb1 also contains an insertion sequence, similar to that in mouse, it is predicted that a similar mechanism is responsible for the difference in the endocytic ability of FcγRIIb1 and FcγRIIb2. It has also been reported that approximately 40% of immune complexes on the cell surface are internalized within 20 minutes in both humans and mice (Mol. Immunol. (2011) 49, 329-337, Science (1992) 256, 1808-1812). Based on this, it is predicted that FcγRIIb2 also internalizes immune complexes into cells in humans at a rate similar to that observed in mice.

[0114] Among the Fcγ receptor family, FcγRIIb is the only one that contains intracellular ITIM in both humans and mice, and the distribution of expressing cells is the same, suggesting that its function in immune regulation is similar. Furthermore, considering the fact that immune complexes are internalized into cells at similar rates in both humans and mice, it is thought that the use of mice makes it possible to predict the effect of antibody-mediated antigen elimination via FcγRIIb in humans. Indeed, in the Examples described below, it was shown that when modified molecules (mF44 and mF46) with enhanced affinity for mouse FcγRIIb and FcγRIII compared to mIgG1 were administered to normal mice, antigen clearance was increased compared to administration of mIgG1.

[0115] In the Examples described below, similar experiments were performed using Fc receptor γ-chain-deficient mice. It has been reported that in mice, FcγRs other than FcγRIIb are expressed only in the presence of the γ-chain; therefore, Fc receptor γ-chain-deficient mice express only FcγRIIb. By administering mF44 or mF46 to Fc receptor γ-chain-deficient mice, it became possible to examine the effect on antigen clearance when selectively enhancing FcγRIIb binding activity. The results of the Examples showed that mF44 and mF46 administered to Fc receptor γ-chain-deficient mice increased antigen clearance compared to mIgG1 administered to the same mice. Furthermore, the results of the Examples revealed that mF44 and mF46, when administered to Fc receptor γ-chain-deficient mice, cleared antigens to a similar extent as when administered to normal mice.

[0116] Furthermore, in the Examples described below, similar experiments were performed using FcγRIII-deficient mice. Because mIgG1, mF44, and mF46 bind only to FcγRIIb and FcγRIII among mouse FcγRs, administering these antibodies to FcγRIII-deficient mice makes it possible to examine the effect on antigen clearance when FcγRIIb-selective binding activity is enhanced. The results of the Examples showed that mF44 and mF46 administered to FcγRIII-deficient mice increased antigen clearance compared to mIgG1 administered to the same mice. The results of the Examples also revealed that mF44 and mF46 administered to FcγRIII-deficient mice eliminated antigens to approximately the same extent as when administered to normal mice and when administered to Fc receptor γ chain-deficient mice.

[0117] These results demonstrated that antigen elimination can be accelerated by selectively enhancing binding activity only to FcγRIIb, without enhancing binding activity to activating Fcγ receptors.

[0118] In addition to the literature reports discussed above, the above-mentioned mouse verification results suggest that FcγRIIb-mediated internalization of immune complexes occurs in the human body as well, just as in mice, and that, as a result, antibodies having an Fc region with enhanced binding activity selectively to human FcγRIIb can accelerate antigen elimination. Furthermore, as discussed above, since FcγRIIb-mediated internalization of immune complexes is thought to occur at similar rates in mice and humans, it is thought that antibodies having an Fc region with enhanced affinity for human FcγRIIb can achieve the same effect in accelerating antigen elimination as antibodies having an Fc region with enhanced affinity for mouse FcγRIIb.

[0119] The Kabat numbering system is generally used when referring to residues in the variable region of an antibody (approximately residues 1 to 107 of the light chain and residues 1 to 113 of the heavy chain) (e.g., Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the heavy chain constant region of an antibody (e.g., the EU index reported in Kabat et al., supra). "Kabat's EU index" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable region of an antibody use the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant region of an antibody use the EU numbering system (see, e.g., WO2006 / 073941).

[0120] The antigen-binding domain of the present invention is characterized by its antigen-binding activity differing between intracellular and extracellular conditions. Intracellular and extracellular conditions refer to conditions that differ between cells in a living organism, and examples of categories of conditions include ion concentrations, more specifically, hydrogen ion concentration (pH) and calcium ion concentration. Intracellular conditions are preferably environments characteristic of the environment inside endosomes, and extracellular conditions are preferably environments characteristic of the environment in plasma.

[0121] Antigen-binding domains whose antigen-binding activity changes depending on ion concentration can be obtained by screening a large number of antigen-binding domains for domains with such properties. For example, when the antigen-binding molecules of the present invention are antibodies, antibodies with such properties can be obtained by producing a large number of antibodies with different sequences using hybridoma or antibody library methods and measuring the antigen-binding activity of these antibodies under different ion concentrations. The B cell cloning method exemplified in Example 1 and the like herein is particularly suitable as a method for screening such antibodies. Furthermore, as described below, by identifying at least one characteristic amino acid residue that can confer on an antigen-binding domain the property of changing its antigen-binding activity depending on ion concentration, and preparing a library of a large number of antigen-binding domains that share such a characteristic amino acid residue as a common structure but have different sequences, antigen-binding domains with such properties can be efficiently screened from such a library.

[0122] In one embodiment of the present invention, the ion concentration condition refers to the hydrogen ion concentration condition or the pH condition. In the present invention, the concentration condition of protons, i.e., the atomic nuclei of hydrogen atoms, is also treated as the hydrogen ion concentration exponent (pH) condition. When the activity of hydrogen ions in an aqueous solution is expressed as aH+, pH is defined as -log10aH+. When the ionic strength in an aqueous solution is (for example, 10 -3If the ionic strength of water is lower than 25°C, aH+ is approximately equal to the hydrogen ionic strength. For example, the ionic product of water at 25°C and 1 atmosphere is Kw = aH + aOH = 10 -14 Therefore, in pure water, aH+ = aOH = 10 -7 In this case, a pH of 7 is neutral, an aqueous solution with a pH of less than 7 is acidic, and an aqueous solution with a pH of more than 7 is alkaline. In the present invention, when pH conditions are used as ion concentration conditions, it is desirable that the antigen-binding activity under acidic pH conditions (i.e., high proton concentration or low pH) is lower than the antigen-binding activity under neutral pH conditions (i.e., low proton concentration or high pH).

[0123] The pH inside the cell is more acidic than outside the cell, and conversely, the pH outside the cell is more neutral than inside the cell. The present invention provides antigen-binding molecules that are in a neutral pH range outside the cell and in an acidic pH range inside the cell. In the present invention, the acidic pH range is preferably pH 4.0 to pH 6.5, more preferably pH 5.0 to pH 6.5, even more preferably any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. In the present invention, the neutral pH range is preferably pH 6.7 to pH 10.0, more preferably pH 7.0 to pH 9.0, still more preferably any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and particularly preferably pH 7.4, which is close to the pH in plasma (blood).

[0124] When the strength of antigen-binding activity of the antigen-binding domain provided by the present invention is compared under acidic pH conditions and neutral pH conditions, it is preferable that binding is stronger under neutral pH conditions than under acidic pH conditions. When the strength of binding activity is expressed as KD (Dissociation constant), the value of KD (acidic pH) / KD (neutral pH) is preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. There is no particular upper limit for the value of KD (acidic pH) / KD (neutral pH), and any value such as 100, 400, 1,000, or 10,000 may be used as long as it can be produced using the techniques of those skilled in the art. It is also possible to use kd (Dissociation rate constant) instead of KD. If it is difficult to calculate the KD value, it may be evaluated based on the magnitude of the binding response when analyte is injected at the same concentration on a Biacore. When an antigen is applied to a chip provided by the present invention on which an antigen-binding molecule is immobilized, the binding response under an acidic pH condition is preferably 1 / 2 or less, more preferably 1 / 3 or less, even more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less of the binding response under a neutral pH condition.

[0125] It is generally known that in vivo, the extracellular pH (e.g., in plasma) is neutral, while the intracellular pH (e.g., in endosomes) is acidic. If the antigen-binding domain of an antigen-binding molecule provided by the present invention can be conferred with a property that weakens its antigen-binding activity under intracellular pH conditions compared to extracellular pH conditions, the antigen bound to the antigen-binding molecule of the present invention will dissociate from the antigen-binding molecule of the present invention intracellularly, thereby promoting the uptake of the antigen from extracellular to intracellular. The antigen-binding molecule provided by the present invention is useful because administering such an antigen-binding molecule to a living body reduces the concentration of antigen present in plasma and reduces the physiological activity of the antigen in vivo.

[0126] The method for conferring to the antigen-binding domain of the present invention the property of weaker antigen binding under acidic pH conditions than under neutral pH conditions is not particularly limited, and any method may be used. Specifically, as described in WO2009 / 125825, examples include substituting at least one amino acid residue with histidine and / or inserting at least one histidine into the antigen-binding domain. A preferred embodiment of the antigen-binding molecule of the present invention is an antigen-binding molecule in which at least one amino acid residue in the antigen-binding domain is substituted with histidine and / or at least one histidine is inserted. It is already known that pH-dependent antigen-binding activity can be conferred by substituting amino acid residues in an antibody with histidine (Ito W et al., FEBS Lett. (1992) 309, 85-88). The position at which histidine substitution and / or insertion takes place is not particularly limited; any amino acid residue may be substituted with histidine, and any position at which histidine may be inserted, as long as the antigen-binding activity is weaker in the acidic pH range than in the neutral pH range. Preferred examples of positions at which histidine substitution and / or insertion takes place include sites that directly bind to antigens or sites that contribute to maintaining the three-dimensional structure of such sites. For example, when the antigen-binding domain is the variable region of an antibody, examples of such sites include the CDR region and regions that contribute to maintaining the three-dimensional structure. Specifically, the heavy chain positions include H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102, and the light chain positions include L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94. Of these, H32, H61, L53, L90, and L94 are considered to be highly universal (the positions of amino acid residues are shown using the Kabat numbering system (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH))).Preferred combinations of substitutions with histidine at multiple sites include, for example, the combination of H27, H31, and H35, the combination of H27, H31, H32, H35, H58, H62, and H102, the combination of L32 and L53, and the combination of L28, L32, and L53. Furthermore, a preferred example of a combination of substitution sites between the heavy chain and the light chain is the combination of H27, H31, L32, and L53.

[0127] In alanine scanning methods known to those skilled in the art, histidines can be randomly substituted and / or inserted into the antigen-binding domains of the antigen-binding molecules provided by the present invention by methods such as histidine scanning, in which alanine is replaced with histidine, and from these, antigen-binding domains that bind to antigens weaker in an acidic pH range than in a neutral pH range can be selected.

[0128] The number of histidines to be substituted and / or inserted can be determined appropriately by those skilled in the art, and only one site may be substituted with histidine, or only one site may be inserted with histidine. Alternatively, two or more sites may be substituted with histidine, or two or more sites may be inserted with histidine. Furthermore, a combination of substitution and insertion of histidine at two or more sites may be performed.

[0129] It is preferable that the antigen-binding domain has equivalent antigen-binding activity before and after histidine substitution and / or insertion, where equivalent activity means at least 10%, preferably at least 30%, even more preferably at least 50%, even more preferably at least 80%, and particularly preferably at least 90% of the original activity.

[0130] When the antigen-binding molecule provided by the present invention contains an antibody constant region, methods for imparting the property of weaker antigen-binding under acidic pH conditions compared to neutral pH conditions to the antigen-binding molecule provided by the present invention include modifying the antibody constant region. Examples of methods for modifying the antibody constant region include comparing constant region isotypes (IgG1, IgG2, IgG3, IgG4) and selecting an isotype that has lower antigen-binding activity under acidic pH conditions (faster dissociation rate under acidic pH conditions). Another method involves introducing amino acid modifications into the amino acid sequence of an isotype (IgG1, IgG2, IgG3, IgG4) to reduce antigen-binding activity under acidic pH conditions (faster dissociation rate under acidic pH conditions). The sequences of the hinge region of antibody constant regions vary significantly depending on the isotype (IgG1, IgG2, IgG3, IgG4), and differences in the amino acid sequence of the hinge region significantly affect antigen-binding activity. Therefore, by selecting an appropriate isotype depending on the type of antigen to be bound, it is possible to reduce antigen-binding activity under acidic pH conditions. Furthermore, when introducing amino acid modifications into the amino acid sequence of an isotype, the hinge region is considered to be the preferred site for the amino acid modification.

[0131] A library of many antigen-binding domains with different sequences that share a common structure of amino acid residues whose antigen-binding activity changes depending on the hydrogen ion concentration as described above is prepared, and screening from this library can be performed to efficiently obtain antigen-binding domains that have the activity to bind to a desired antigen and whose antigen-binding activity changes depending on the hydrogen ion concentration.

[0132] For example, by combining a light chain variable region containing in its framework sequence at least one amino acid residue whose antigen-binding activity changes depending on hydrogen ion concentration with a heavy chain variable region having a random sequence, a library containing multiple antigen-binding domains with different sequences that share the same amino acid residue whose antigen-binding activity changes depending on hydrogen ion concentration can be produced. When such amino acid residues are introduced into the light chain modified region, in a preferred embodiment, the amino acid residues may be contained in CDR1 of the light chain modified region, more preferably at positions 24, 27, 28, 31, 32, and / or 34 according to the Kabat numbering system in CDR1 of the light chain variable region. In another preferred embodiment, the amino acid residues may be contained in CDR2 of the light chain variable region, more preferably at positions 50, 51, 52, 53, 54, 55, and / or 56 according to the Kabat numbering system in CDR2 of the light chain variable region. In another preferred embodiment, these amino acid residues may be contained in the CDR3 of the light chain variable region, more preferably at positions 89, 90, 91, 92, 93, 94, and / or 95A according to the Kabat numbering system in the CDR3 of the light chain variable region. As long as the antigen-binding activity is altered depending on the hydrogen ion concentration, such amino acid residues may be contained alone or in combination of two or more.

[0133] When preparing an antigen-binding domain by combining a light chain variable region containing at least one amino acid residue whose antigen-binding activity changes depending on hydrogen ion concentration conditions with a heavy chain variable region having a random sequence, the light chain variable region can also be designed to contain flexible residues. As long as the antigen-binding activity of the antigen-binding domain of the present invention changes depending on hydrogen ion concentration conditions, the number and location of flexible residues are not particularly limited. That is, one or more flexible residues can be contained in the light chain CDR sequence and / or FR sequence. Suitable examples of flexible residues to be introduced into the light chain variable region sequence include, but are not limited to, the amino acid residues listed in Table 7 or Table 8. Furthermore, suitable light chain variable region sequences other than the amino acid residues whose antigen-binding activity changes depending on hydrogen ion concentration conditions and the flexible residues include, but are not limited to, germline sequences such as Vk1 (SEQ ID NO: 58), Vk2 (SEQ ID NO: 59), Vk3 (SEQ ID NO: 60), and Vk4 (SEQ ID NO: 61).

[0134] [Table 7] (Positions represent Kabat numbering)

[0135] [Table 8] (Positions represent Kabat numbering)

[0136] Any amino acid residue can be suitably used as an amino acid residue whose antigen-binding activity changes depending on hydrogen ion concentration conditions. Specific examples of such amino acid residues include those with a side chain pKa of 4.0-8.0. Suitable examples of such electron-donating amino acids include natural amino acids such as histidine and glutamic acid, as well as histidine analogs (US20090035836) and unnatural amino acids such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), or 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11(17), 3761-2768). Particularly suitable examples of such amino acid residues include those with a side chain pKa of 6.0-7.0. A suitable example of such electron-donating amino acid is histidine.

[0137] In one embodiment of the present invention, the ion concentration refers to the metal ion concentration. "Metal ions" refer to ions of elements belonging to Group I, such as alkali metals and copper group elements excluding hydrogen, Group II, such as alkaline earth metals and zinc group elements, Group III, such as boron, Group IV, such as carbon and silicon, Group VIII, such as iron group elements and platinum group elements, and each of the A subgroups of Groups V, VI, and VII, as well as metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become cations, which is called ionization tendency. Metals with a high ionization tendency are considered to be chemically active. An example of a suitable metal ion in the present invention is calcium ion. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction (e.g., skeletal muscle, smooth muscle, cardiac muscle), activation of leukocytes (e.g., motility and phagocytosis), activation of platelets (e.g., deformation and secretion), activation of lymphocytes, activation of mast cells (e.g., histamine secretion), cell responses mediated by catecholamine α receptors and acetylcholine receptors, exocytosis, release of transmitters from neuronal terminals, and axonal flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have multiple calcium ion-binding sites and are thought to have originated from a common molecular evolutionary origin, and many of their binding motifs are also known. Well-known examples include the cadherin domain, the EF hand found in calmodulin, the C2 domain found in protein kinase C, the Gla domain found in the blood coagulation protein Factor IX, the C-type lectins found in the asialoglycoprotein receptor and mannose-binding receptor, the A domain found in the LDL receptor, annexin, the thrombospondin type 3 domain, and the EGF-like domain. In the present invention, when the metal ion is a calcium ion, it is desirable that the antigen-binding activity under a low calcium ion concentration is lower than that under a high calcium ion concentration.

[0138] Furthermore, calcium ion concentrations are lower inside cells than outside cells, and conversely, higher outside cells than inside cells. A preferred low calcium ion concentration in the present invention is 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. A preferred high calcium ion concentration in the present invention is 100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). In the present invention, the low calcium ion concentration is preferably the calcium ion concentration in endosomes, and the high calcium ion concentration is preferably the calcium ion concentration in plasma.

[0139] When the strength of antigen-binding activity of an antigen-binding domain provided by the present invention is compared under low and high calcium ion concentration conditions, it is preferred that the binding is stronger under high calcium ion concentration conditions than under low calcium ion concentration conditions. In other words, it is preferred that the antigen-binding activity of the antigen-binding domain of the present invention is lower under low calcium ion concentration conditions than under high calcium ion concentration conditions.

[0140] When the strength of binding activity is expressed as KD (Dissociation constant), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (low calcium ion concentration) / KD (high calcium ion concentration) value is not particularly limited, and may be any value, such as 100, 400, 1,000, or 10,000, as long as it can be produced by those skilled in the art. It is also possible to use kd (Dissociation rate constant) instead of KD. When it is difficult to calculate the KD value, it may be evaluated based on the magnitude of the binding response when an analyte is flowed at the same concentration in a Biacore. When an antigen is flowed through a chip on which an antigen-binding molecule provided by the present invention is immobilized, the binding response under low calcium concentration conditions is preferably 1 / 2 or less, more preferably 1 / 3 or less, even more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less, of the binding response under high calcium concentration conditions.

[0141] It is generally known that in vivo, the calcium ion concentration is high outside cells (e.g., in plasma) and low inside cells (e.g., in endosomes). Therefore, in the present invention, it is preferable that the extracellular condition is a high calcium ion concentration condition and the intracellular condition is a low calcium ion concentration condition.

[0142] If the antigen-binding domain of an antigen-binding molecule provided by the present invention can be conferred with a property that weakens its antigen-binding activity under intracellular calcium ion concentration conditions compared to extracellular calcium ion concentration conditions, the antigen bound to the antigen-binding molecule of the present invention will dissociate from the antigen-binding molecule of the present invention intracellularly, thereby promoting the uptake of the antigen from extracellular to intracellular. The antigen-binding molecule provided by the present invention is useful because administering such an antigen-binding molecule to a living body reduces the antigen concentration present in plasma and reduces the physiological activity of the antigen in vivo.

[0143] Examples of screening methods for antigen-binding domains or antigen-binding molecules that have lower antigen-binding activity under low calcium ion concentrations than under high calcium ion concentrations include the methods described in WO2012 / 073992 etc. (e.g., paragraphs 0200-0213).

[0144] The method for imparting to the antigen-binding domain of the present invention the property of weaker antigen binding under low calcium ion concentrations than under high calcium ion concentrations is not particularly limited, and any method may be used. Specifically, as described in Japanese Patent Application No. 2011-218006, for example, methods include substituting at least one amino acid residue in the antigen-binding domain with an amino acid residue having metal chelating activity and / or inserting at least one amino acid residue having metal chelating activity. A preferred embodiment of the antigen-binding molecule of the present invention is an antigen-binding molecule in which at least one amino acid residue in the antigen-binding domain has been substituted with an amino acid residue having metal chelating activity and / or at least one amino acid residue having metal chelating activity has been inserted. Suitable examples of amino acid residues having metal chelating activity include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0145] Furthermore, preferred examples of amino acid residues that change the antigen-binding activity of an antigen-binding domain depending on calcium ion concentration conditions include amino acid residues that form a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490), Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J. (1990) 265, 261-265), Bairoch and Cox (FEBS Lett. (1990) 269, 454-456), Davis (New Biol. (1990) 2, 410-419), Schaefer et al. (Genomics (1995) 25, 638-643), Economou et al. (EMBO J. (1990) 9, 349-354), Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). For example, EF hands contained in troponin C, calmodulin, parvalbumin, and myosin light chain, the C2 domain contained in protein kinase C, the Gla domain contained in the blood coagulation protein Factor IX, asialoglycoprotein receptor, mannose-binding receptor, ASGPR, CD23, C-type lectin contained in DC-SIGN, the A domain contained in the LDL receptor, an annexin domain, a cadherin domain, a thrombospondin type 3 domain, and an EGF-like domain can be suitably used as a calcium-binding motif. In addition to the above, the calcium-binding motif contained in the antigen-binding domain set forth in SEQ ID NO: 57 can also be suitably used.

[0146] The antigen-binding domain of the present invention may contain amino acid residues whose antigen-binding activity changes depending on calcium ion concentration, such as the aforementioned amino acid residues with metal chelating activity and amino acid residues that form calcium-binding motifs. The position of such amino acid residues in the antigen-binding domain is not particularly limited, and may be any position as long as the antigen-binding activity changes depending on calcium ion concentration. Furthermore, as long as the antigen-binding activity changes depending on calcium ion concentration, such amino acid residues may be contained alone or in combination of two or more. Suitable examples of such amino acid residues include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. When the antigen-binding domain is an antibody variable region, these amino acid residues may be contained in the heavy chain variable region and / or light chain variable region. In a preferred embodiment, these amino acid residues may be contained in CDR3 of the heavy chain variable region, more preferably at positions 95, 96, 100a, and / or 101 according to the Kabat numbering system in CDR3 of the heavy chain variable region.

[0147] In another preferred embodiment, these amino acid residues may be contained in CDR1 of the light chain variable region, more preferably at positions 30, 31, and / or 32 according to the Kabat numbering of CDR1 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR2 of the light chain variable region, more preferably at position 50 according to the Kabat numbering of CDR2 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR3 of the light chain variable region, more preferably at position 92 according to the Kabat numbering of CDR3 of the light chain variable region.

[0148] The above embodiments may be combined. For example, the amino acid residue may be contained in two or three CDRs selected from CDR1, CDR2, and CDR3 of the light chain variable region. More preferably, the amino acid residue may be contained in one or more of positions 30, 31, 32, 50, and / or 92 according to the Kabat numbering system in the light chain variable region.

[0149] A library of many antigen-binding domains with different sequences that share a common structure of amino acid residues whose antigen-binding activity changes depending on calcium ion concentration as described above is prepared, and screening from this library can be performed to efficiently obtain antigen-binding domains that have the activity to bind to a desired antigen and whose antigen-binding activity changes depending on calcium ion concentration.

[0150] When the antigen-binding domain is an antibody variable region, in a particularly preferred embodiment, the framework sequences of the light chain and / or heavy chain variable region desirably have human germline framework sequences. Thus, in one aspect of the present invention, if the framework sequences are completely human sequences, the antigen-binding domain of the present invention is expected to provoke little or no immunogenic response when administered to humans (e.g., for the treatment of a disease). In this sense, "having a germline sequence" in 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 domain of the present invention that has a sequence that combines framework sequences from multiple different human germlines is also an antigen-binding domain of the present invention that "has a germline sequence."

[0151] Although the present invention is not bound by any particular theory, one reason why the use of germline sequences is expected to eliminate adverse immune responses in most individuals is believed to be as follows: As a result of the affinity maturation step that occurs during a normal immune response, somatic mutations frequently occur in the variable regions of immunoglobulins. These mutations primarily occur around the CDRs, whose sequences are hypervariable, but also affect residues in the framework regions. Because these framework region mutations are not present in germline sequences, they may 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 sequences are expected to be less immunogenic or non-immunogenic in patients. To maximize the likelihood of immune tolerance, genes encoding the variable regions may be selected from a collection of functional (normally expressed) germline genes.

[0152] Suitable examples of frameworks include currently known fully human framework region sequences, and such framework region sequences are listed 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 domain 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.

[0153] 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 domains of the present invention based on this sequence information. Completely human framework regions or framework sub-regions other than these can also be suitably used.

[0154] Fully human VK 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)).

[0155] Completely human VL 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).

[0156] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used with at least one amino acid residue that changes the antigen-binding activity depending on the ion concentration conditions. Other examples of framework sequences include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

[0157] For example, by combining a light chain variable region whose framework sequence contains at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration with a heavy chain variable region having a random sequence, a library containing multiple antigen-binding domains with different sequences but sharing the same amino acid residue whose antigen-binding activity changes depending on calcium ion concentration can be produced. A suitable example of an antigen-binding domain library is one that combines a light chain variable region belonging to the Vk5-2 family, as represented by SEQ ID NO: 57 (Vk5-2), with a heavy chain variable region having a random sequence. Another suitable example of an antigen-binding domain library is one that combines a light chain variable region sequence in which a specific amino acid residue in a germline sequence, such as SEQ ID NO: 58 (Vk1), SEQ ID NO: 59 (Vk2), SEQ ID NO: 60 (Vk3), or SEQ ID NO: 61 (Vk4), has been substituted with at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration, with a heavy chain variable region having a random sequence.

[0158] Furthermore, flexible residues can also be designed to be contained in a light chain variable region containing in its framework sequence at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration. The number and location of flexible residues are not particularly limited, as long as the antigen-binding activity of the antigen-binding domain of the present invention changes depending on ion concentration. That is, one or more flexible residues can be contained in the CDR sequences and / or FR sequences of the heavy chain variable region and / or light chain variable region. Non-limiting examples of flexible residues that can be introduced into the light chain variable region of SEQ ID NO: 57 (Vk5-2) include the amino acid residues listed in Table 9 or Table 10.

[0159] [Table 9]

[0160] [Table 10]

[0161] As used herein, "flexible residues" refers to amino acid residues present at positions in the light chain variable region and heavy chain variable region where the types of amino acids are highly diverse when comparing the amino acid sequences of several known and / or natural antibodies or antigen-binding domains. Highly diverse positions are generally present in the CDR regions. Data provided by, for example, 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 a collection of numerous human light chain and heavy chain sequences, and this sequence information is useful for determining highly diverse positions in the present invention. According to the present invention, a position is highly diverse if there are 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 different amino acids present at that position. Also, a 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 at least about 10, preferably at least about 12 different amino acids.

[0162] When preparing an antigen-binding domain by combining a light chain variable region containing at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration with a heavy chain variable region having a random sequence, it is also possible to design the light chain variable region to further contain flexible residues. The number and location of flexible residues are not particularly limited, as long as the antigen-binding activity changes depending on calcium ion concentration. That is, one or more flexible residues may be contained in the light chain CDR sequence and / or FR sequence. Suitable examples of flexible residues to be introduced into the light chain variable region include, but are not limited to, the amino acid residues listed in Table 9 or Table 10.

[0163] In the present invention, heavy and / or light chain variable regions having random sequences can be prepared as a randomized variable region library by appropriately combining known methods. In one embodiment, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, humans with increased blood antibody titers after vaccination, patients with infectious diseases, cancer patients, autoimmune disease patients, etc. is preferably used as the randomized variable region library.

[0164] In another embodiment, a synthetic library in which any CDR sequence in a V gene in genomic DNA or a reconstructed functional V gene is replaced with a synthetic oligonucleotide set encoding a codon set of appropriate length is preferably used as a randomized variable region library. In this case, since diversity is observed in the CDR3 sequence of the heavy chain variable region, it is also possible to replace only the CDR3 sequence. When diversifying the amino acid sequence of an antigen-binding molecule, it is preferable to diversify 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 in the variable region, it is generally the CDR. Surface-exposed positions can be determined from the coordinates of a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can also be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400) and Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Surface-exposed positions can also be determined using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. A suitable example of software available for this purpose is the SYBYL Biopolymer Module software (Tripos Associates). When an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is generally 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).

[0165] In another embodiment, a naive library constructed based on antibody genes derived from lymphocytes of healthy individuals is also particularly suitable for use 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)). Because the repertoire of antibody sequences derived from lymphocytes of healthy individuals is unbiased, high diversity is expected. In the present invention, an amino acid sequence containing a naive sequence refers to an amino acid sequence obtained from such a naive library.

[0166] In one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library containing multiple antigen-binding domains with different sequences, which are prepared by combining heavy chain variable regions containing at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration conditions with light chain variable regions having random sequences. A suitable example is, but is not limited to, a library of antigen-binding domains that combines the heavy chain variable region set forth in SEQ ID NO: 117 (6RL#9-IgG1) or SEQ ID NO: 119 (6KC4-1#85-IgG1) with a light chain variable region having a random sequence. Alternatively, a light chain variable region having a germline sequence may be appropriately selected and used instead of the light chain variable region having a random sequence. A suitable example is, but is not limited to, a library of antigen-binding domains that combines the heavy chain variable region set forth in SEQ ID NO: 117 (6RL#9-IgG1) or SEQ ID NO: 119 (6KC4-1#85-IgG1) with a light chain variable region having a germline sequence.

[0167] Furthermore, the heavy chain variable region containing at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration can also be designed to contain flexible residues. As long as the antigen-binding activity of the antigen-binding domain of the present invention changes depending on calcium ion concentration, the number and location of the flexible residues are not particularly limited. That is, one or more flexible residues can be contained in the CDR and / or FR sequences of the heavy and / or light chains. Examples of flexible residues that can be introduced into the heavy chain variable region of SEQ ID NO: 117 (6RL#9-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues other than positions 95, 96, and / or 100a in heavy chain CDR3. Examples of flexible residues that can be introduced into the heavy chain variable region of SEQ ID NO: 119 (6KC4-1#85-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues other than positions 95 and / or 101 in heavy chain CDR3.

[0168] Alternatively, a library containing multiple antigen-binding domains with different sequences can be prepared by combining a heavy chain variable region containing at least one amino acid residue whose antigen-binding activity changes depending on the calcium ion concentration with a light chain variable region having a random sequence or a germline sequence. A preferred example of an antigen-binding domain library is one that combines a heavy chain variable region in which a specific amino acid residue in the heavy chain variable region has been substituted with at least one amino acid residue whose antigen-binding activity changes depending on the calcium ion concentration with a light chain variable region having a random sequence or a germline sequence. Non-limiting examples of such amino acid residues include those in heavy chain CDR1, heavy chain CDR2, and positions 95, 96, 100a, and / or 101 of heavy chain CDR3. As long as these amino acid residues form a calcium-binding motif and / or change the antigen-binding activity depending on the calcium ion concentration, these amino acid residues may be contained alone or in combination.

[0169] Even when a heavy chain variable region containing at least one amino acid residue whose antigen-binding activity changes depending on calcium ion concentration is combined with a light chain variable region having a random sequence or a germline sequence, the heavy chain variable region can be designed to further contain flexible residues. As long as the antigen-binding activity of the antigen-binding domain of the present invention changes depending on calcium ion concentration, the number and location of flexible residues are not particularly limited. That is, one or more flexible residues may be contained in the heavy chain CDR sequence and / or FR sequence. Furthermore, the amino acid sequences of CDR1, CDR2, and / or CDR3 of the heavy chain variable region other than the amino acid residue whose antigen-binding activity changes depending on calcium ion concentration may be randomized, as in the synthetic library described above. When a germline sequence is used for the light chain variable region, suitable examples include, but are not limited to, germline sequences such as SEQ ID NO: 58 (Vk1), SEQ ID NO: 59 (Vk2), SEQ ID NO: 60 (Vk3), and SEQ ID NO: 61 (Vk4).

[0170] In the present invention, 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 to modify amino acids. Furthermore, several known methods can be used to modify 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 to 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, is preferably used.

[0171] The KD value of the antigen-binding domain provided by the present invention can be measured using methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, flow cytometers, etc. Specifically, when using Biacore, the KD value can be measured by immobilizing an antigen-binding molecule comprising the antigen-binding domain provided by the present invention on a chip and then flowing an antigen as an analyte across the chip. By performing measurements under acidic pH conditions and neutral pH conditions, it is possible to calculate the KD(acidic pH) / KD(neutral pH), and by performing measurements under low calcium ion concentration conditions and high calcium ion concentration conditions, it is possible to calculate the KD(low calcium ion concentration) / KD(high calcium ion concentration).

[0172] The antigen-binding domains provided by the present invention may have the property that their antigen-binding activity changes depending on ion concentration conditions under multiple conditions simultaneously. For example, the antigen-binding domains provided by the present invention may have the property that their antigen-binding activity is lower in an acidic pH range than in a neutral pH range, and that their antigen-binding activity is lower under a low calcium ion concentration than under a high calcium ion concentration.

[0173] Specific examples of antigens having two or more types of physiological activity in the present invention include activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, adiponectin, aFGF, AGE, allergen, amyloid beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, anti-Id, antithrombin III, anthrax, apoA1, apo-serum amyloid A, apo-SAA, beta-2-microglobulin, bFGF, B lymphocyte stimulating factor (BLyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), and BMP-7. (OP-1), BMP-8 (BMP-8a), C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), 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, CCL, CCL1 / I-309, CCL11 / eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / E LC, CCL2 / MCP-1, CCL20 / MIP-3-α, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-l-β, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-γ, Clostridium botulinum botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, CTGF (Connective Tissue Growth Factor)Growth Factor), CTLA-4, CX3CL1 / fractalkine, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF- l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL16, CXCL2 / Gro-β CXCL3 / Gro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCL10 / IP-10, DC-SIGN, digoxin, EGF-like domain containing protein 7 7), endotoxin, RSV F protein, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, Factor VIII, Factor VIIIc, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, fibronectin, GRO / MGSA, GRO-β, GRO-γ, Helicobacter pylori (H. pylori), haptens (NP-cap or NIP-cap), HB-EGF, HCMV gB envelope glycoprotein, Hep B gp120, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E virus, hepcidin, Herpes simplex virus (HSV) gB glycoprotein, HIV envelope proteins such as GP120, HIV MIB gp120 V3 loop, HLA, HLA-DR, HMGB1 (High Mobility Group Box 1), HSP47, Hsp90, HSV gD glycoprotein, human cytomegalovirus (HCMV), human serum albumin, human tissue-type plasminogen activator (t-PA), IFN-α, IFN-β, IFN-γ, IgE, IGF, immunoglobulin immune complex, immunoglobulin, influenza, inhibin, inhibin α, inhibin β, laminin 5, latency-associated peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDL, leptin, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, lipoprotein, L-selectin, nonstructural protein type 3 from hepatitis C virus(NS3), oncostatin M, osteopontin, oxidized LDL, polyglycol chains of different sizes (e.g., PEG-20, PEG-30, PEG40), prekallikrein, prion protein, procalcitonin, proinsulin, prolactin, proprotein convertase PC9, prorelaxin, respiratory syncytial virus (RSV) F, rheumatoid factor, RSV Fgp, sclerostin, serum amyloid P, serum albumin, Shiga-like toxin II, syndecan-1, tenascin, TGF, TGF-α, TGF-β, TGF-β pan-specific (TGF-β Pan) Examples of antigens include TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thyroxine-binding globulin, TNF-α, TNF-β, TNIL-I, toxic metabolites, transforming growth factors (TGFs) such as TGF-α and TGF-β, VEGF, viral antigens, and von Willebrand factor (vWF). Particularly preferred examples include HMGB1, CTGF, and IgE. These antigens are preferably derived from mammals, and particularly preferably from humans.

[0174] The gene and amino acid sequences of human HMGB1 have been registered under GenBank accession numbers NM_002128 (SEQ ID NO: 22) and NP_002119 (SEQ ID NO: 23), respectively. Outside of humans, the gene and amino acid sequences of mouse HMGB1 have been registered under GenBank accession numbers NM_010439 (SEQ ID NO: 24) and NP_034569 (SEQ ID NO: 25), and the gene and amino acid sequences of rat HMGB1 have been registered under GenBank accession numbers NM_012963 (SEQ ID NO: 26) and NP_037095 (SEQ ID NO: 27), respectively.

[0175] The gene and amino acid sequences of human CTGF are registered under GenBank accession numbers NM_001901 (SEQ ID NO: 28) and NP_001892 (SEQ ID NO: 29), respectively. Outside of humans, the gene and amino acid sequences of mouse CTGF are registered under GenBank accession numbers NM_010217 (SEQ ID NO: 30) and NP_034347 (SEQ ID NO: 31), respectively, and the gene and amino acid sequences of rat CTGF are registered under GenBank accession numbers NM_022266 (SEQ ID NO: 32) and NP_071602 (SEQ ID NO: 33), respectively.

[0176] The gene sequence of the human IgE constant region is registered under GenBank accession number L00022 (SEQ ID NO: 34), and the gene sequence of the mouse IgE constant region is registered under GenBank accession number X01857 (SEQ ID NO: 35).

[0177] Target molecules to which HMGB1 binds include RAGE (Receptor for Advanced Glycation Endproducts), TLR4 (Toll-Like Receptor 4), IL-1 receptor, TLR2 (Toll-Like Receptor 2), thrombospondin, TREM-1 (Triggering Receptor Expressed on Myeloid cells-1), and CD24. Substances that promote the binding of HMGB1 to these target molecules include DNA, RNA, LPS (Lipopolysaccharide), IL-1β (Interleukin-1β), CXCL12 (chemokine (CXC motif) Ligand 12), and nucleosomes. Preferred examples of target molecules in the present invention include RAGE and TLR4.

[0178] Reported target molecules to which CTGF binds include IGF-1 (Insulin-like Growth Factor-1), IGF-2 (Insulin-like Growth Factor-2), integrin αvβ3, TGF-β (Transforming Growth Factor-β), BMP-4 (Bone Morphogenetic Protein-4), LRP-1 (LDL receptor-related protein-1), VEGF (vascular endothelial growth factor), Wnt, HSPG (Heparan Sulfate Proteoglycans), integrins, LRP-5 (LDL receptor-related protein-5), and LRP-6 (LDL receptor-related protein-6).

[0179] FcεRI, FcεRII, etc. have been reported as target molecules to which IgE binds, and these are also preferred examples of target molecules in the present invention.

[0180] The present invention provides polynucleotides encoding the antigen-binding molecules of the present invention. Polynucleotides are primarily composed of DNA, RNA, and other nucleic acid analogs.

[0181] The present invention provides vectors containing the polynucleotides of the present invention. There are no particular limitations on the type of vector that can be used, as long as it stably retains the inserted nucleic acid, and various commercially available vectors can be used. Examples of vectors for gene cloning include M13-based vectors and pUC-based vectors. When vectors are used for the purpose of producing the antigen-binding molecules of the present invention, expression vectors are particularly useful. Expression vectors are not particularly limited, as long as they express polypeptides in test tubes, E. coli, cultured cells, or individual organisms. For example, in vitro expression vectors include the pBEST vector (Promega), E. coli expression vectors include the pGEX, pET, and pBluescript vectors (Stratagene), cultured cell expression vectors include the pME18S-FL3 vector (GenBank Accession No. AB009864), animal cell expression vectors include pcDNA, and in individual organism expression vectors include the pME18S vector (Mol Cell Biol. 8:466-472 (1988)). The polynucleotide of the present invention can be inserted into a vector using, for example, the In-Fusion Advantage PCR Cloning Kit (Clontech).

[0182] The present invention provides host cells harboring the vectors provided by the present invention. There are no particular limitations on the host cells that can be used, and for example, Escherichia coli and various animal cells can be suitably used. The host cells can be used, for example, as production systems for producing and expressing the antigen-binding molecules of the present invention. Production systems include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0183] Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108: 94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero; amphibian cells such as Xenopus oocytes (Valle et al., Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, and Tn5. CHO-DG44, CHO-DX11B, COS7, HEK293, and BHK are preferred. CHO is particularly preferred for large-scale expression. Vectors can be introduced into host cells by techniques known to those skilled in the art, such as the calcium phosphate method, the DEAE-dextran method, a method using the cationic liposome DOTAP (Boehringer Mannheim), electroporation, lipofection, microinjection, etc. Alternatively, the FreeStyle 293 Expression System (Invitrogen) can be used to carry out processes from gene introduction to polypeptide expression.

[0184] Plant cells, such as cells derived from Nicotiana tabacum and duckweed (Lemna minor), are known as protein production systems, and the antigen-binding molecules provided by the present invention can be produced by callus culture of these cells. Protein expression systems using fungal cells, such as yeast cells of the genus Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces pombe), and filamentous fungi, such as Aspergillus cells (e.g., Aspergillus niger), are known.

[0185] When prokaryotic cells are used, there are production systems using bacterial cells, such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis, which are known as protein production systems.

[0186] On the other hand, systems for producing polypeptides in vivo include, for example, production systems using animals and production systems using plants. A target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in the animal or plant body and recovered. The host in the present invention includes these animals and plants.

[0187] When animals are used, production systems using mammals and insects are available. Examples of mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Furthermore, when mammals are used, transgenic animals can be used. For example, a polynucleotide encoding an antigen-binding molecule provided by the present invention is prepared as a fusion gene with a gene encoding a polypeptide that is specifically produced in milk, such as caprine β-casein. A polynucleotide fragment containing this fusion gene is then injected into a goat embryo, and the embryo is then implanted into a female goat. The antigen-binding molecule of interest can be obtained from the milk produced by the transgenic goat born to the goat that received the embryo, or from the milk produced by its offspring. To increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat, an appropriate hormone may be administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12: 699-702).

[0188] Furthermore, silkworms, for example, can be used as insects for producing the antigen-binding molecules of the present invention. When using silkworms, the antigen-binding molecules of interest can be obtained from the body fluids of the silkworms by infecting them with a baculovirus into which a polynucleotide encoding the antigen-binding molecule of interest has been inserted.

[0189] Furthermore, when using plants to produce the antigen-binding molecules provided by the present invention, tobacco can be used, for example. When using tobacco, a polynucleotide encoding the antigen-binding molecule of interest is inserted into a plant expression vector, such as pMON 530, and this vector is introduced into bacteria such as Agrobacterium tumefaciens. This bacterium is then used to infect tobacco, such as Nicotiana tabacum, and the desired antigen-binding molecule can be isolated from the tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-8). Alternatively, the same bacteria can be used to infect duckweed (Lemna minor), clone the desired antigen-binding molecule, and then isolate it from the duckweed cells (Cox KM et al. Nat. Biotechnol. 2006 Dec;24(12):1591-1597).

[0190] The antigen-binding molecules obtained in this manner can be isolated from inside or outside host cells (culture medium, milk, etc.) and purified as substantially pure, homogeneous molecules. The antigen-binding molecules provided by the present invention can be separated and purified using any separation and purification method commonly used for purifying polypeptides, and are not limited in any way. For example, separation and purification can be performed by appropriately selecting and combining methods such as column chromatography, filtering, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0191] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be performed using liquid-phase chromatography such as HPLC and FPLC. Examples of columns used in affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0192] If necessary, the antigen-binding molecules of the present invention can be subjected to the action of an appropriate protein-modifying enzyme, such as trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, or glucosidase, to optionally modify or partially remove peptides.

[0193] The present invention also provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention as an active ingredient. Pharmaceutical compositions can be used to treat diseases, but the pharmaceutical compositions provided by the present invention are preferably used to treat diseases thought to be caused in part by physiologically active antigens. The antigen is preferably an antigen with two or more physiological activities, and the physiological activity is preferably reduced in vivo by the antigen-binding molecules provided by the present invention. As used herein, "treatment" refers to obtaining a pharmacological and / or physiological effect. The effect can be preventive, in that it completely or partially prevents the symptoms of a disease, or therapeutic, in that it completely or partially treats the symptoms of a disease. As used herein, "treatment" includes all disease treatments in mammals, particularly humans. Furthermore, "treatment" also includes preventing the onset of a disease in a subject who has not yet been diagnosed with the disease, and inhibiting or alleviating the progression of disease symptoms.

[0194] Pharmaceutical compositions provided by the present invention can be formulated by methods known to those skilled in the art (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). If necessary, antigen-binding molecules provided by the present invention can also be formulated in combination with other pharmaceutical ingredients. For example, they may also contain pharmaceutically acceptable carriers and additives. Furthermore, pharmaceutical compositions provided by the present invention can be used parenterally, for example, in the form of a sterile solution or suspension injection in water or other pharmaceutically acceptable liquid. Dosage forms for oral and parenteral administration and their preparation methods are well known to those skilled in the art. Pharmaceutical compositions can be prepared by conventional methods, for example, by mixing pharmaceutically acceptable carriers with pharmaceutical compositions provided by the present invention. In the present invention, examples of commonly used carriers include, but are not limited to, sterile water, physiological saline, vegetable oil, emulsifiers, surfactants, excipients, vehicles, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavorings, and flavoring agents. Other commonly used carriers can also be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. Pharmaceutical compositions provided by the present invention can be formulated by appropriately combining these and blending them in a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is set so as to obtain an appropriate dosage within the specified range.

[0195] The pharmaceutical compositions provided by the present invention can be administered orally or parenterally, but are preferably administered parenterally, specifically by injection, nasal administration, pulmonary administration, transdermal administration, etc. Examples of injection administration include intravenous administration, intramuscular administration, intraperitoneal administration, and subcutaneous administration. The dosage can be appropriately selected from the range of 0.0001 mg to 1000 mg per kg of patient body weight or the range of 0.001 mg to 10,000 mg per patient, but is not limited thereto. The subject to be administered is a mammal, preferably a human.

[0196] The present invention also provides kits containing the antigen-binding molecules or pharmaceutical compositions provided by the present invention, and kits for use in various methods provided by the present invention. The kits provided by the present invention can also be packaged with instructions describing the method of use, as appropriate. Furthermore, the kits of the present invention can be suitably used for (i) methods for reducing antigen concentrations in plasma, (ii) methods for promoting antigen uptake into cells, or (iii) methods for reducing the physiological activity of antigens in vivo.

[0197] A preferred example of an antigen in the present invention is HMGB1. Diseases for which HMGB1 is thought to be one of the causes include sepsis, trauma, acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury in the brain, heart, liver, kidney, etc., pancreatitis, nephritis, hepatitis, colitis, meningitis, endophthalmitis, myopathy, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, multiple sclerosis (MS), colon cancer, osteosarcoma, cervical cancer, liver cancer, lymphoma, nasopharyngeal cancer, prostate cancer, skin cancer, urothelial cancer, lung cancer, autism, seizures, sleep apnea syndrome, HIV infection, pulmonary fibrosis, and burns. Another preferred example of an antigen is CTGF. Diseases for which CTGF is thought to be one of the causes include fibrotic diseases such as pulmonary fibrosis and hepatic fibrosis. Another preferred example of an antigen is IgE. Diseases in which IgE is thought to be one of the causes include allergic diseases such as bronchial asthma, atopic dermatitis, and hay fever.

[0198] The present invention also provides a method for producing the antigen-binding molecule of the present invention, comprising the steps of: (a) selecting antigens having two or more types of physiological activity; (b) obtaining an antigen-binding domain; (c) obtaining at least one receptor-binding domain; (d) selecting, from the antigen-binding domains obtained in step (b), domains whose antigen-binding activity changes depending on ion concentration conditions; (e) selecting, from the receptor-binding domains obtained in step (c), domains that have human FcRn-binding activity under acidic pH conditions and whose human Fc receptor-binding activity under neutral pH conditions is higher than the binding activity of native human IgG to human Fc receptors; (f) preparing an antigen-binding molecule in which the antigen-binding domain selected in step (d) and the receptor-binding domain selected in step (e) are linked; and (g) selecting, from the antigen-binding molecules prepared in step (f), antigen-binding molecules that, upon binding to an antigen, inhibit one or more physiological activities of the antigen while maintaining at least one physiological activity.

[0199] Antigen-binding domains of the present invention may be obtained by any method. For example, when the antigen-binding domain is an antibody or a fragment thereof (variable region, Fab, F(ab')2, Fv, CDR, etc.), it can be obtained by antibody library methods, hybridoma methods, B cell cloning methods (Bernasconi et al., Science (2002) 298, 2199-2202 or WO2008 / 081008), etc. Furthermore, antibodies or fragments thereof obtained in this manner may have at least one amino acid modified.

[0200] Many methods for constructing antibody libraries are already known. For example, those skilled in the art can construct phage display libraries with reference to literature such as Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 324.0-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14, Kang AS et al., Proc Natl Acad Sci USA (1991) 88, 4363-4366, and Published Japanese Translation of PCT International Publication No. 10-504970. If a phage display library is used in which antibody variable regions are expressed on the surface of phages as single-chain fragments (scFv), phages that bind to an antigen can be selected by panning, and the DNA sequence encoding the variable region of the antibody that binds to the antigen can be determined by analyzing the genes of the selected phages. Once the DNA sequence of the antibody variable region is known, it can be ligated to DNA encoding a desired constant region, inserted into an appropriate expression vector, and then introduced into host cells for expression to produce the antibody as a recombinant protein. These methods are already well known, and references such as WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388 can be used.In addition to phage display libraries, there are also ribosome display libraries (Schaffitzel C et al., J. Immunol. Methods (1999) 231, 119-135.), cell display libraries (Fuchs P et al., Biotechnology (1991) 9, 1369-1372, Boder ET & Wittrup KD, Nat Biotechnol (1997) 15, 553-557, WO95 / 15393), nucleotide display libraries (Cull MG et al., Proc Natl Acad Sci USA (1992) 89, 1865-1869, Roberts RD & Szostak JW, Proc Natl Acad Sci USA (1997) 94, 12297-12302), and eukaryotic virus display libraries (Grabherr R & Ernst W, Comb Chem High It is possible to use a known antibody library such as Throughput Screen (1991) 4, 185-192).

[0201] Methods for obtaining antibodies using the hybridoma method basically use known techniques, such as using a desired antigen or cells expressing the desired antigen as a sensitizing antigen, immunizing a subject using this antigen according to a conventional immunization method, fusing the resulting immune cells with known parent cells using a conventional cell fusion method, and screening for monoclonal antibody-producing cells (hybridomas) using a conventional screening method. Furthermore, cDNA encoding the variable region of the antibody can be obtained from the mRNA of the resulting hybridoma using reverse transcriptase. This can be ligated to DNA encoding the desired constant region, inserted into an appropriate expression vector, and then introduced into host cells for expression, thereby producing the antibody as a recombinant protein.

[0202] More specifically, although not limited to the following examples, sensitizing antigens can include both immunogenic complete antigens and incomplete antigens containing non-immunogenic haptens. For example, full-length proteins or partial peptides of a target protein can be used. It is also known that substances composed of polysaccharides, nucleic acids, lipids, etc. can serve as antigens, and the antigens are not particularly limited. Antigens can be prepared by methods known to those skilled in the art, such as methods using baculovirus (e.g., WO98 / 46777). Hybridomas can be produced, for example, by the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). When the antigen has low immunogenicity, it can be conjugated to an immunogenic macromolecule such as albumin and then immunized. Furthermore, the antigen can be conjugated to other molecules as needed.

[0203] Hybridomas can be obtained by immunizing animals with the appropriate sensitizing antigen described above. Alternatively, antibody-producing lymphocytes can be immortalized in vitro to produce antibody-producing cells. Various mammals can be used as immunized animals, but rodents, lagomorphs, and primates are commonly used. Examples of such animals include rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as cynomolgus monkeys, rhesus monkeys, hamadryas baboons, and chimpanzees. Transgenic animals carrying a repertoire of human antibody genes are also known, and human antibodies can be obtained by immunizing such animals with the desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735; Mendez et al., Nat. Genet. 1997, 15: 146-56). Instead of using such transgenic animals, a desired human antibody having binding activity to the antigen can be obtained, for example, by sensitizing human lymphocytes in vitro with a desired antigen and fusing the sensitized lymphocytes with human myeloma cells, such as U266 cells (see Japanese Patent Publication No. 1-59878).

[0204] Animals are immunized, for example, by appropriately diluting and suspending the sensitizing antigen in phosphate-buffered saline (PBS) or physiological saline, mixing it with an adjuvant if necessary, emulsifying it, and then injecting it intraperitoneally or subcutaneously into the animal. Thereafter, the sensitizing antigen, preferably mixed with Freund's incomplete adjuvant, is administered several times every 4 to 21 days. Antibody production can be confirmed by measuring the titer of the target antibody in the animal's serum using a conventional method.

[0205] Hybridomas can be produced by fusing antibody-producing cells, such as lymphocytes, obtained from an animal immunized with a desired antigen with myeloma cells using a conventional fusing agent (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). Hybridomas are cultured and grown as needed, and the binding specificity of antibodies produced by the hybridomas is measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Hybridomas producing antibodies with the desired specificity, affinity, or activity can then be subcloned, as needed, by limiting dilution or other techniques.

[0206] Subsequently, the gene encoding the antibody can be cloned from the hybridoma or antibody-producing cells (sensitized lymphocytes, etc.) using a probe that can specifically bind to the antibody gene (e.g., an oligonucleotide complementary to a sequence encoding the antibody constant region). Cloning from mRNA is also possible by RT-PCR. Immunoglobulins are classified into five different classes: IgA, IgD, IgE, IgG, and IgM. These classes are further divided into several subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, etc.).

[0207] Receptor-binding domains of the present invention may be obtained by any method. For example, when the receptor-binding domain is an anti-FcRn antibody or a fragment thereof (variable region, Fab, F(ab')2, Fv, CDR, etc.), it can be obtained by the antibody library method or hybridoma method described above. Other examples of receptor-binding domains include the Fc region of an antibody (IgG) and regions containing the Fc region (antibody constant region or full-length antibody). When the receptor-binding domain is the Fc region of an IgG, at least one amino acid may be modified.

[0208] Methods for adding, deleting, and / or substituting any amino acid in a polypeptide can be performed by techniques known to those skilled in the art, such as site-directed mutagenesis (Hashimoto-Gotoh T. et al., Gene (1995) 152, 271-275; Zoller MJ & Smith M., Methods Enzymol (1983) 100, 468-500; Kramer W. et al., Nucleic Acids Res (1987) 12, 9441-9456; Kramer W. & Fritz HJ, Methods Enzymol (1987) 154, 350-367; Kunkel TA, Proc Natl Acad Sci USA (1985) 82, 488-492).

[0209] The production of chimeric antibodies is known. For example, in the case of a human-mouse chimeric antibody, a chimeric antibody can be obtained by linking DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host for production.

[0210] Humanized antibodies, also known as reshaped human antibodies, are antibodies in which the complementarity-determining regions (CDRs) of an antibody derived from a non-human mammal, such as a mouse antibody, are grafted onto the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General genetic recombination techniques for CDR grafting are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576). Humanized antibodies can be produced by known methods, for example, by determining the CDRs of a mouse antibody, obtaining DNA encoding an antibody in which the CDRs are linked to the framework regions (FRs) of a human antibody, and then using a system using a conventional expression vector. Such DNA can be synthesized by assembly PCR using as primers several oligonucleotides engineered to have overlapping portions at the terminal regions of both the CDRs and FRs (see the method described in WO98 / 13388). The FRs of a human antibody linked via the CDRs are selected so that the CDRs form a favorable antigen-binding site. If necessary, amino acids in the FRs in the variable regions of the antibody may be modified so that the CDRs of the reshaped human antibody form a suitable antigen-binding site (Sato et al., Cancer Res. (1993) 53: 10.01-6). Amino acid residues in the FR that can be modified include those that bind directly to the antigen via non-covalent bonds (Amit et al., Science (1986) 233: 747-53), those that affect or influence the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and those that are involved in VH-VL interactions (EP239400 Patent Publication).

[0211] In addition to the above-mentioned humanization, modifications may also be made to improve the biological properties of antibodies, such as antigen binding. Modifications in the present invention can be performed by methods such as site-directed mutagenesis (see, for example, Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, and cassette mutagenesis. Generally, antibody variants with improved biological properties have an amino acid sequence identity and / or similarity of 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96%, 97%, 98%, 99%, etc.) to the amino acid sequence of the variable region of the original antibody. Herein, sequence identity and / or similarity is defined as the percentage of amino acid residues that are homologous (the same residues) and / or similar (residues classified into the same group based on the properties of general amino acid side chains) to the amino acid residues of the original sequence, after aligning the sequences and introducing gaps as necessary to maximize sequence identity. Generally, natural amino acid residues are classified based on the properties of their side chains. (1) Hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) Neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) Acidic: aspartic acid and glutamic acid; (4) Basic: arginine, histidine and lysine; (5) residues that influence chain orientation: glycine and proline; and (6) Aromatic: tyrosine, tryptophan, and phenylalanine are classified into the following groups.

[0212] In the methods for producing antigen-binding molecules provided by the present invention, multiple antigen-binding domains may be obtained and one domain whose antigen-binding activity changes depending on ion concentration may be selected from among them, or multiple domains may be prepared by modifying any antigen-binding domain by adding, deleting, and / or substituting at least one amino acid, and one domain whose antigen-binding activity changes depending on ion concentration may be selected from among them. Furthermore, an antigen-binding domain that inhibits one or more physiological activities of the antigen upon binding to the antigen but maintains at least one physiological activity may be selected from among multiple antigen-binding domains, as appropriate.

[0213] In the methods for producing antigen-binding molecules provided by the present invention, whether the antigen-binding molecule or its antigen-binding domain binds to an antigen and thereby inhibits one or more physiological activities of the antigen or maintains at least one physiological activity can also be confirmed by measuring whether one or more target molecule-binding activities of the antigen are inhibited or whether the activity of binding to at least one target molecule is maintained.

[0214] In the methods for producing antigen-binding molecules provided by the present invention, multiple receptor-binding domains may be obtained and from these, a domain may be selected that has human FcRn-binding activity under acidic pH conditions and has higher human Fc receptor-binding activity under neutral pH conditions than native human IgG; alternatively, multiple domains may be prepared by modifying any receptor-binding domain by adding, deleting, and / or substituting at least one amino acid, and from these, a domain may be selected that has human FcRn-binding activity under acidic pH conditions and has higher human Fc receptor-binding activity under neutral pH conditions than native human IgG.

[0215] In the methods for producing antigen-binding molecules provided by the present invention, antigen-binding molecules in which the antigen-binding domain and receptor-binding domain are linked can be produced by separately preparing a polynucleotide encoding the antigen-binding domain and a polynucleotide encoding the receptor-binding domain, ligating them in-frame, introducing them into an expression vector, and expressing the vector in host cells. The antigen-binding domain and receptor-binding domain may be linked directly or indirectly using any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., the linkers disclosed in Protein Engineering (1996) 9, 299-305). The length and amino acid sequence of the peptide linker are not particularly limited; however, peptides of 100 amino acids or less, preferably 50 amino acids or less, more preferably 30 amino acids or less, and particularly preferably 10 amino acids or less are typically used.

[0216] The present invention also provides a method for screening for an antibody whose antigen-binding activity changes depending on conditions, the method comprising the following steps: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) selecting cells that bind to a certain amount or more of the antigen from the cells of step (b); (d) subjecting the cells of step (c) to second conditions; and (e) A step of selecting cells from the cells of step (d) that have a reduced amount of antigen binding compared to step (c).

[0217] A more preferred embodiment of the above method includes the following steps: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) contacting the cells of step (b) with an anti-IgG antibody; (d) selecting cells from the cells of step (c) that bind to a certain amount or more of antigen and a certain amount or more of anti-IgG antibody; (e) subjecting the cells of step (d) to second conditions; and (f) A step of selecting cells from the cells of step (e) that have a reduced amount of antigen binding compared to step (d).

[0218] A further preferred embodiment of the above method includes the following steps: (a) preparing antibody-producing cells; (b) contacting the cells of (a) with an antigen under first conditions; (c) concentrating antigen-bound cells from among the cells of step (b); (d) contacting the cells of step (c) with an anti-IgG antibody; (e) selecting cells from the cells of step (d) that have bound to a certain amount or more of antigen and a certain amount or more of anti-IgG antibody; (f) subjecting the cells of step (e) to second conditions; and (g) A step of selecting cells from the cells of step (f) that have a reduced amount of antigen binding compared to step (e).

[0219] In the present invention, "antibody-producing cells" are not particularly limited as long as they contain an antibody gene and express an antibody protein. However, they are preferably cells naturally occurring in an animal body that produce antibodies, more preferably lymphocytes, and even more preferably B cells. Animals can be appropriately selected from various mammals (e.g., mice, rats, hamsters, rabbits, cynomolgus monkeys, rhesus monkeys, hamadryas baboons, chimpanzees, and humans), with rabbits being particularly preferred in the present invention. It is also preferable to use animals immunized with a desired antigen. Antibody-producing cells may be naturally occurring cells or artificially produced cells such as hybridomas and genetically engineered cells. The antibody-producing cells used in the present invention preferably have the property of secreting antibodies extracellularly (secretory antibodies) and / or the property of presenting antibodies on the cell membrane (membrane-type antibodies). Naturally occurring antibody-producing cells in an animal body can be suitably collected from, for example, the spleen, lymph nodes, blood (peripheral blood mononuclear cells), etc.; such methods are well known to those skilled in the art and are also described in the Examples below.

[0220] In the present invention, the "first condition" and "second condition" refer to two different types of conditions, and any conditions can be set as long as it is desired to obtain antibodies with different antigen-binding activities under those conditions. Preferred examples in the present invention are extracellular conditions and intracellular conditions. The categories of conditions are not particularly limited as long as they are conditions to which antibody-producing cells are exposed, and examples include temperature, pH, and compositions contained in the culture medium and their concentrations. Preferred are ion concentrations, with hydrogen ion concentration (pH) and calcium ion concentration being particularly preferred. Intracellular conditions preferably refer to conditions characteristic of the environment inside the endosome, and extracellular conditions preferably refer to conditions characteristic of the environment in plasma.

[0221] The pH outside the cell is neutral compared to the pH inside the cell, and conversely, the pH inside the cell is acidic compared to the pH outside the cell. In the present invention, the neutral pH range is preferably pH 6.7 to pH 10.0, more preferably pH 7.0 to pH 9.0, even more preferably any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and particularly preferably pH 7.4, which is close to the pH in plasma (blood). In the present invention, the acidic pH range is preferably pH 4.0 to pH 6.5, more preferably pH 5.0 to pH 6.5, even more preferably any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo.

[0222] Furthermore, calcium ion concentrations are higher outside cells than inside cells, and conversely, calcium ion concentrations are lower inside cells than outside cells. In the present invention, preferred high calcium ion concentration conditions are 100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). In the present invention, preferred low calcium ion concentration conditions are 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. Low calcium ion concentration conditions can also be achieved by adding a chelating agent such as EDTA instead of reducing the amount of calcium added.

[0223] The "first condition" and "second condition" may include multiple conditions simultaneously; for example, the "first condition" may be a neutral pH condition and a high calcium ion concentration condition, and the "second condition" may be an acidic pH condition and a low calcium ion concentration condition.

[0224] The antigen may be any substance that can produce an antibody against it, and the type is not particularly limited, but it is preferably an antigen that contains a polypeptide. Furthermore, in the above-described antibody screening method provided by the present invention, it is preferable that the antigen is labeled with some substance that can be detected with high sensitivity. The labeling substance may be directly linked to the antigen, or may be indirectly linked to the antigen using an antigen-antibody reaction, a biotin-avidin reaction, or the like. Examples of labeling substances include radioisotopes, chemiluminescent compounds, fluorescent compounds, phosphorescent compounds, magnetic particles, enzymes, etc., with fluorescent compounds being particularly preferred. Examples of fluorescent compounds include fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Cy5 (PE-Cyanin5), PE-Cy5.5 (PE-Cyanin5.5), PE-Cy5 (PE-Cyanin7), rhodamine isothiocyanate, Texas Red (Texas Red), ECD (PE-Texas Red-x), allophycosanin (APC), APC-Cy7 (APC-Cyanin7, PharRed), Per-CP (Peridinin Chlorophyll Protein), and PerCP-Cy5.5 (Per-CP-Cyanin5.5).

[0225] Furthermore, in the antibody screening method provided by the present invention, it is preferable that the anti-IgG antibody is also labeled with some substance that can be detected with high sensitivity. By selecting cells to which anti-IgG antibodies bind from antibody-producing cells, the proportion of cells expressing IgG as a subclass can be increased. The expression of IgG in B cells indicates that class switching to IgG has occurred, and enriching cells producing such mature antibodies is considered advantageous in screening for antibodies with high binding activity. The labeling substance may be directly linked to the antigen or indirectly linked to the antigen using an antigen-antibody reaction or a biotin-avidin reaction. Examples of labeling substances are as described above. When both an antigen and an anti-IgG antibody are used, it is desirable that the substances labeled for each antibody are different and that the detection methods are different (so that they can be detected separately). Labeling of the antigen and anti-IgG antibody can be performed with reference to methods known to those skilled in the art (e.g., US Pat. Nos. 5,057,313 and 5,156,840).

[0226] In the antibody screening method provided by the present invention, the step of selecting cells bound to a certain amount or more of antigen and / or anti-IgG antibody is preferably carried out by detecting the labeling substance. For example, when the antigen and / or anti-IgG antibody are each labeled with a different type of fluorescent compound, whether or not a certain amount or more of antigen and / or anti-IgG antibody is bound to the cells can be evaluated by whether or not the fluorescence emitted by each fluorescent compound is detected with an intensity equal to or greater than a certain amount. The certain amount can be determined arbitrarily by those skilled in the art depending on the purpose. In the present invention, the selection step is preferably carried out using FACS (Fluorescence Activated Cell Sorting).

[0227] In the antibody screening method provided by the present invention, the step of enriching cells bound to the antigen is preferably carried out by detecting the labeling substance. For example, when the antigen is labeled with magnetic particles, antigen-bound and non-bound cells can be separated using a magnet device, and antigen-bound cells can be enriched by removing non-antigen-bound cells. In the present invention, the enrichment step is preferably carried out using MACS (Magnetic Activated Cell Sorting, registered trademark).

[0228] By using the above-described antibody screening method provided by the present invention, it becomes possible to simply and efficiently screen a large number of antibody-producing cells for antibodies whose antigen-binding activity changes depending on conditions. Because the number of cells that can be screened can be significantly increased compared to conventional methods, the probability of finding rare antibodies that have not been found before is significantly increased, making the above-described antibody screening method provided by the present invention useful.

[0229] The present invention also provides methods for reducing plasma antigen concentrations, methods for promoting antigen uptake into cells, and methods for reducing the physiological activity of antigens in vivo, by administering the antigen-binding molecules of the present invention.

[0230] The present invention also provides therapeutic agents for diseases, comprising the antigen-binding molecules of the present invention as an active ingredient, such as the aforementioned diseases thought to be caused in part by HMGB1, diseases thought to be caused in part by CTGF, or diseases thought to be caused in part by IgE. The present invention also provides kits for use in methods for reducing plasma antigen concentrations, methods for promoting antigen uptake into cells, or methods for reducing the physiological activity of antigens in vivo, which kits comprise the antigen-binding molecules of the present invention. Furthermore, the present invention provides methods for treating diseases thought to be caused in part by physiologically active antigens, methods for reducing plasma antigen concentrations, methods for promoting antigen uptake into cells, and methods for reducing the physiological activity of antigens in vivo, which comprise the step of administering the antigen-binding molecules of the present invention. The present invention also provides therapeutic agents for diseases thought to be caused in part by physiologically active antigens, agents for reducing plasma antigen concentrations, agents for promoting antigen uptake into cells, or agents for reducing the physiological activity of antigens in vivo, which contain the antigen-binding molecules of the present invention as an active ingredient. The present invention also provides antigen-binding molecules of the present invention for use in methods for treating diseases thought to be caused in part by physiologically active antigens, methods for reducing plasma antigen concentrations, methods for promoting antigen uptake into cells, or methods for reducing the physiological activity of antigens in vivo. The present invention also provides use of the antigen-binding molecules of the present invention in the production of therapeutic agents for diseases thought to be caused in part by physiologically active antigens, agents for reducing plasma antigen concentrations, agents for promoting antigen uptake into cells, or agents for reducing the physiological activity of antigens in vivo. The present invention also provides processes for producing therapeutic agents for diseases thought to be caused in part by physiologically active antigens, agents for reducing plasma antigen concentrations, agents for promoting antigen uptake into cells, or agents for reducing the physiological activity of antigens in vivo, which processes comprise the step of using the antigen-binding molecules of the present invention. Examples of diseases include diseases thought to be caused in part by HMGB1, CTGF, or IgE.

[0231] It should be noted that the amino acids contained in the amino acid sequences described in the present invention may be modified after translation (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art), and even when such amino acids are modified after translation, they are naturally included in the amino acid sequences described in the present invention.

[0232] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0233] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0234] Example 1: Production of anti-HMGB1 antibodies by rabbit B cell cloning Preparation of HMGB1 The antigen, HMGB1, was prepared as follows. An animal cell expression vector was constructed by inserting a DNA sequence encoding human HMGB1 (GenBank accession number NP002119, SEQ ID NO: 7). Full-length human HMGB1 protein was expressed in the culture supernatant using this expression vector and FreeStyle293 (Invitrogen). HMGB1 protein was purified from the resulting culture supernatant using cation exchange column chromatography, anion exchange column chromatography, and gel filtration column chromatography.

[0235] Immunization of animals with antigens Rabbits were immunized with HMGB1. The first immunization consisted of an intradermal injection of 100 μg of HMGB1 protein in complete Freund's adjuvant (CFA). Two or more booster immunizations were then administered, each with 50 μg of HMGB1 protein in incomplete Freund's adjuvant (IFA), at intervals of at least one week. Antibody titers were measured to confirm antibody production within the animals.

[0236] Tissue collection from immunized animals and preparation of single cell suspensions Animals confirmed to produce antibodies were euthanized, and their spleens, lymph nodes, and blood were collected. Peripheral blood mononuclear cells (PBMCs) were prepared from the blood. An equal volume of blood was carefully placed on top of Histpaque-1077 (Sigma) in a 50 mL centrifuge tube and centrifuged at 400 × g for 30 minutes at 25°C. After centrifugation, the PBMC layer was carefully collected using a glass Pasteur pipette and transferred to a sterile 50 mL tube. Approximately 10 times the volume of the collected cell suspension was added to RPMI-1640 containing 2% FBS, and the cells were washed by centrifugation at 1000 × g for 5 minutes and removing the supernatant. The same washing procedure was repeated to prepare PBMCs. PBMCs were stained with trypan blue, and cell density was determined using a hemocytometer.

[0237] After harvesting, the spleen and lymph nodes were passed through a 70 μm cell strainer (BD Falcon) using the plunger of a 5 mL syringe to generate a single-cell suspension. The cells were collected in a sterile 50 mL tube with RPMI medium containing 2% FBS. The cells were washed by centrifugation at 1000 × g for 5 minutes and the supernatant was removed. Another 50 mL of RPMI-1640 containing 2% FBS was added for washing. After the final wash, cells were stained with trypan blue and the cell density was determined using a hemocytometer.

[0238] Recovery of antigen-binding B cells Single cell suspensions of blood, spleen, and lymph nodes prepared as described above were centrifuged at 1000 × g for 5 minutes and washed twice with HBSS (20 mM HEPES, 5.3 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 0.3 mM Na2HPO4, 0.1% BSA, 2 mM CaCl2, 5 mM glucose, 138 mM NaCl, pH 7.4). Biotinylated human HMGB1 was diluted to 500 nM in HBSS and added to the cells to a density of 1E08 cells / 100 μL. Biotinylated HMGB1 was prepared by labeling HMGB1 protein using the EZ-Link NHS-PEG4-Biotin and Biotinylation Kit (Thermo Scientific) according to the attached protocol, followed by dialysis against TBS 300 mM NaCl (10 mM Tris-HCl, 300 mM NaCl) using a microdialyzer (TOMY). The cell suspension was incubated on ice for 30 minutes. The cells were then washed with 50 mL of HBSS to remove unbound biotinylated HMGB1. MACS® Streptavidin Beads (Miltenyi Biotech) were diluted 10-fold with HBSS, and added to the cells at a density of 1E08 cells / 500 μL. The cell suspension was incubated on ice for 30 minutes. After washing with 50 mL of HBSS, the cells were resuspended in HBSS at a density of 1E08 cells / 500 μL. The cell suspension was subjected to an autoMACS Pro Separator to collect the positive cell fraction bound to MACS streptavidin beads.

[0239] The harvested cells were resuspended by diluting biotinylated HMGB1 in HBSS and adding it to a density of 1E08 cells / 100 μL or less. The cell suspension was incubated on ice for 30 minutes and then washed with 50 mL of HBSS. In some cases, this additional incubation with biotinylated human HMGB1 was not required. Streptavidin-FITC (BD) and mouse anti-rabbit IgG-PE (Southern Biotech) were diluted in HBSS and added to a density of 1E08 cells / 100 μL or less. The cell suspension was incubated on ice for 30 minutes and then washed with 50 mL of HBSS. The cells were then resuspended in HBSS at a density of 1E07 cells / 100 μL or less. The cell suspension was analyzed using a FACSAria (BD) to collect cells with high FITC and PE fluorescence.

[0240] pH or Ca 2+ Recovery of antigen-binding antibody-expressing B cells whose dissociation ability changes with changes in ionic concentration pH or Ca 2+We concentrated and recovered B cells expressing antibodies whose dissociation ability changes with ion concentration as follows. After preparation, single-cell suspensions of blood, spleen, and lymph nodes were centrifuged at 1000 × g for 5 minutes. The cells were then washed twice with HBSS (20 mM HEPES, 5.3 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 0.3 mM Na2HPO4, 0.1% BSA, 2 mM CaCl2, 5 mM glucose, 138 mM NaCl, pH 7.4). Biotinylated HMGB1 was diluted to 500 nM in HBSS and added to the cells to a density of 1E08 cells / 100 μL. The cell suspension was then incubated on ice for 30 minutes. Unbound biotinylated HMGB1 was then removed by washing with 50 mL of HBSS. MACS Streptavidin Beads (Miltenyi Biotech) were diluted 10-fold with HBSS and added to the cells to a density of 1E08 cells / 500 μL. The cell suspension was incubated on ice for 30 minutes. After washing with 50 mL of HBSS, the cells were resuspended in HBSS to a density of 1E08 cells / 500 μL. The cell suspension was then used to collect the positive cell fraction bound to MACS Streptavidin Beads using an autoMACS Pro Separator.

[0241] The harvested cells were resuspended by diluting biotinylated HMGB1 to 500 nM in HBSS. This solution was added to the cells at a density of 1E08 cells / 100 μL or less. The cell suspension was incubated on ice for 30 minutes and then washed with 50 mL of HBSS. Streptavidin-FITC (BD) and mouse anti-rabbit IgG-PE (Southern Biotech) were diluted in HBSS and added to the cells at a density of 1E08 cells / 100 μL or less. The cell suspension was incubated on ice for 30 minutes and then washed with 50 mL of HBSS. The cells were then resuspended in HBSS at a density of 1E07 cells / 100 μL or less. The cell suspension was analyzed using a FACSAria (BD) and gated on fractions with high FITC and PE fluorescence. At the time of collection, MBSS (20 mM MES, 5.3 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 0.3 mM Na2HPO4, 0.1% BSA, 2 mM EDTA, 5 mM Glucose, 138 mM NaCl, pH 5.8) was added to the collection tube. After that, the tube was left in MBSS solution for 30 minutes. 2+ For antibodies that are prone to antigen dissociation due to a decrease in ionic concentration, leaving the sample in the low pH of MBSS and the presence of EDTA will cause the antigen to dissociate, resulting in a decrease in FITC fluorescence intensity. After leaving the sample for 30 minutes, the FACSAria was used again to gate and collect cells that had the same PE fluorescence intensity as the first sorting but lower FITC fluorescence intensity than the first sorting. The results are shown in Figure 1. (A) shows the dot plot results for the first sorting. Cells in gate 1 were collected. (B) shows the dot plot results for the second sorting. Cells were collected in gates 1, 2, and 3 for the second sorting.

[0242] This cell recovery method allows for subsequent pH or Ca 2+There is no need to expand the scale of screening for antibodies whose dissociation ability changes with changes in ion concentration, and the dissociation ability changes with changes in pH or Ca 2+ Antibodies whose dissociation ability changes depending on the ion concentration can be efficiently obtained.

[0243] B cell culture The harvested cells were seeded into 96-well microtiter plates at ≤1 cell per well. Activated rabbit T cell conditioned medium was added to a final concentration of 5%. EL4 cells (European Collection of Cell Cultures) were added to a final concentration of approximately 25,000 cells per well. Activated rabbit T cell conditioned medium was prepared by filtering rabbit thymus tissue through a 70 μm cell strainer (BD Falcon) using a 5 mL syringe plunger. The cells were cultured in RPMI-1640 containing PHA (Roche), phorbol 12-myristate 13-acetate (Sigma), and 2% FBS. The culture supernatant was frozen at -70°C or below. To use EL4 cells as feeder cells, mytomycin C (Sigma) was added to 10 μg / mL to stop cell growth, and the cells were cultured at 37°C and 5% CO2 for at least 2 hours. These cultures were incubated at 37°C with 5% CO2 for 5-7 days, after which a portion of the supernatant containing the secreted antibodies was collected. The collected supernatant was used to assess the HMGB1 binding activity of the antibodies using the method described below. The cells were incubated at 37°C with 5% CO2 until the antibody HMGB1 binding activity was assessed.

[0244] Screening culture supernatants for monoclonal antibodies with desired specificity Antibody antigen recognition was screened using ELISA. Biotinylated HMGB1 was captured on a streptavidin-coated 384-well plate. The supernatant containing the secreted antibody was applied to the plate. After incubation at room temperature for 1 hour, the plate was washed three times with 80 μL of TBS (TAKARA) containing 2 mM CaCl2 and 0.05% Tween-20. A 40,000-fold dilution of Goat anti-rabbit IgG Fc HRP conjugate (BETHYL) in TBS containing 2 mM CaCl2 was added and incubated at room temperature for 1 hour. The plate was washed three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20, and 40 μL of chromogenic substrate (ABTS peroxidase substrate (KPL)) was added per well. After incubation for 1 hour, the absorbance at 405 nm was measured using a Molecular Devices SpectraMax. The results of measuring absorbance at 405 nm were analyzed to determine wells in which the secreted antibody was thought to recognize the HMGB1 protein.

[0245] pH or Ca 2+ Screening of antibodies whose dissociation ability changes with changes in ion concentration ELISA was performed using the culture supernatant to evaluate pH / Ca-dependent dissociation. Goat anti-rabbit IgG-Fc (BETHYL) diluted with PBS(-) to 1 μg / mL was added to a 384-well MAXISorp (Nunc) plate and left at room temperature for at least 1 hour. The PBS(-) diluted Goat anti-rabbit IgG-Fc was then removed from the plate, and TBS (pH 7.4) containing 1% BSA and 2 mM CaCl2 was added and left at room temperature for at least 1 hour. The TBS (pH 7.4) containing 1% BSA and 2 mM CaCl2 was then removed from the plate, and the culture supernatant was added. At this time, pH or Ca was not affected. 2+To evaluate antibodies whose dissociation ability changes with ion concentration, two wells of each B cell culture supernatant were added to an ELISA plate. The culture supernatant was added and incubated at room temperature for at least 1 hour or overnight at 4°C, allowing the antibodies in the culture supernatant to be trapped with goat anti-rabbit IgG-Fc. The plate was then washed three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20. Biotinylated HMGB1 was then added and incubated at room temperature for at least 1 hour. This allowed the rabbit antibody trapped by the goat anti-rabbit IgG-Fc to bind to the biotinylated HMGB1. The plate was then washed three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20 to wash away any biotinylated HMGB1 that had not bound to the rabbit antibody. Subsequently, 20 mM MES, 150 mM NaCl, 2 mM CaCl2, pH 7.4 (Buffer A) was added to one of the two wells containing the same culture supernatant, and 20 mM MES, 150 mM NaCl, 2 mM EDTA, pH 5.8 (Buffer B) was added to the other well, and the wells were left at 37°C or below for at least 1 hour. While Buffer A and Buffer B were added, biotinylated human HMGB1 dissociated from the rabbit antibody. However, if the antibody is prone to dissociation at low pH, or if CaCl2 is necessary to maintain the antigen-antibody bond, the antibody may dissociate. 2+When ionization was required, the antigen was more easily released from the antibody in wells containing Buffer B than in wells containing Buffer A. After washing three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20, 25 ng / mL Streptavidine-HRP (Genscript) prepared in TBS containing 2 mM CaCl2 was added and incubated at room temperature for 1 hour. Streptavidine-HRP bound to any remaining biotinylated HMGB1 that had not dissociated from the antibody. After washing three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20, a colorimetric substrate (ABTS peroxidase substrate) was added. After 1 hour of incubation, the absorbance at 405 nm was measured using a Molecular Devices SpectraMax. The absorbance at 405 nm was analyzed, and the wells containing Buffer A showed a stronger color than the wells containing Buffer B. This indicates that the antibody in the culture supernatant is affected by pH or Ca. 2+ It was thought that the antibody's dissociation ability changed with changes in ion concentration.

[0246] This ELISA system has two features. First, for each type of B cell culture supernatant, two wells of the culture supernatant are prepared on an ELISA plate. After the antigen and antibody are allowed to bind, one of the wells is heated to a high pH of around pH 7.0 or higher and contains Ca. 2+ The other was placed under conditions where the pH was around 6.0 or lower and Ca was present in the presence of about 1 mM or more of Ca. 2+ The key point is to perform incubation for dissociation of antigen and antibody under conditions of low ion concentration. By adding this incubation step, it is possible to efficiently control pH or Ca 2+It is possible to screen for antibodies whose dissociation ability changes with changes in ion concentration. Another feature is that rabbit antibodies in the culture supernatant are trapped with goat anti-rabbit IgG-Fc and then bound to an antigen. When the rabbit antibody is reacted after binding the antigen to the plate, a strong binding reaction occurs because each of the two arms of the antibody binds to the antigen immobilized on the plate, making it difficult for the antibody to dissociate even if it shows pH / Ca-dependent dissociation, and only antibodies showing strong dependency can be obtained. However, by trapping rabbit antibodies in the culture supernatant with goat anti-rabbit IgG-Fc and then binding the antigen to it, binding between the antigen and antibody tends to occur monovalently. Therefore, it is possible to detect antibodies that do not dissociate easily even when the pH or Ca is changed. 2+ Even when the change in dissociation ability due to changes in ion concentration is weak, it becomes possible to determine whether there is pH / Ca dependency.

[0247] The results are shown in Figures 2 and 3. Figure 2 shows the relationship between pH and Ca 2+ This is a dot plot of the results of screening for antibodies whose dissociation ability changes with changes in ion concentration. The Y axis represents the OD (405 nm) value after incubation in 20 mM MES, 150 mM NaCl, 2 mM CaCl2, pH 7.4. The X axis represents the OD (405 nm) value after incubation in 20 mM MES, 150 mM NaCl, 2 mM EDTA, pH 5.8. (A) shows the results of culturing B cells derived from gate 1 in Figure 1(B) and examining the dissociation ability of antibodies with changes in pH or CaCl2. 2+ (B) shows the results of screening for antibodies whose dissociation ability changes with changes in ion concentration. B cells from gate 2 in Figure 1(B) were cultured and then subjected to changes in pH or Ca. 2+ (C) shows the results of screening for antibodies whose dissociation ability changes with changes in ion concentration. B cells from gate 3 in Figure 1(B) were cultured and then subjected to changes in pH or Ca. 2+ This is the result of screening for antibodies whose dissociation ability changes with changes in ion concentration. Incubation at pH 5.8 in the presence of 2 mM EDTA showed a significant difference from incubation at pH 7.4 in the presence of 2 mM Ca. 2+The antibodies that dissociate more easily from the antigen than when incubated in the presence of EDTA are marked with a circle. 2+ The values ​​that did not change significantly under the conditions containing pH are indicated by squares. Figure 3 shows the number of clones in Figure 2 (A), (B), and (C). The gray areas indicate changes in pH or Ca. 2+ The number of clones (indicated by squares in (A), (B), and (C)) in which no change in antibody-antigen dissociation was observed even with changes in ion concentration was counted. The black areas indicate the number of clones in which no change in antibody-antigen dissociation was observed when pH was decreased or Ca 2+ This represents the number of clones (indicated by a circle in (A), (B), and (C)) that are susceptible to dissociation of the antibody and antigen when the ion concentration is decreased. In the recovery of antigen-binding B cells, as in the result of (A), the pH or Ca content of the antigen-binding antibody is important. 2+ The percentage of antigen-binding antibodies whose dissociation ability changes with changes in ion concentration is at most a few percent. 2+ By concentrating and recovering B cells expressing antibodies whose dissociation ability changes with changes in ion concentration, we were able to determine the pH or Ca content of antigen-binding antibodies, as shown in the results of (C). 2+ It is possible to increase the percentage of antigen-binding antibodies whose dissociation ability changes with changes in ion concentration to several tens of percent. 2+ It is possible to efficiently obtain extremely rare antibodies whose dissociation ability changes with changes in ion concentration and which still have physiological activity.

[0248] Identification of B cell variable region light and heavy chain sequences and expression of recombinant antibodies The results of screening for monoclonal antibodies with desired specificity or pH or Ca concentrations were obtained from cell culture plates incubated at 37°C and 5% CO2. 2+Based on the results of screening for antibodies whose dissociation ability changes with ion concentration, cells and culture supernatants were harvested into new 96-well plates using an MS2000 (J-Tek). The culture supernatant alone was transferred from the plate containing the harvested cells and culture supernatant to another 96-well plate. Meanwhile, the plate containing the cells harvested using the MS2000 was frozen and stored at -70°C or below. Antibody cDNA was isolated from the cells stored at -70°C, and antibody expression vectors were constructed. Primers used in the PCR reaction to isolate cDNA were designed to anneal to conserved regions (H and L regions) of the rabbit immunoglobulin sequence. Antibody cDNA was obtained using a two-step nested PCR recovery process. RNA purification was performed using the MagMax 96 RNA purification kit for microarray (Ambion). The purified RNA was then reverse-transcribed and subjected to first PCR using the OneStep RT-PCR kit (TAKARA). The primer sequences used are listed in Table 11. The first PCR product was then subjected to nested PCR using PrimeSTAR HS (TAKARA). The primer sequences used for PCR are shown in Table 11. In the table, R represents a mixture of A and G, V represents a mixture of A, C, and G, W represents a mixture of A and T, and Y represents a mixture of C and T.

[0249] [Table 11]

[0250] A cassette vector was constructed by incorporating antibody constant region sequences into an animal cell expression vector, and antibody expression vectors were constructed using this cassette vector. Two types of cassette vectors were constructed: one carrying a rabbit antibody heavy chain constant region sequence and the other carrying a rabbit antibody light chain constant region sequence. The vector carrying the heavy chain constant region sequence contained an ampicillin resistance gene. The vector carrying the light chain constant region sequence contained a kanamycin resistance gene. These two vectors contain sequences that partially overlap with the nested PCR primer sequences. Using the In-Fusion PCR cloning kit (Clontech), the nested PCR product was integrated into the cassette vector carrying the antibody constant region sequence, generating an expression vector carrying the full-length rabbit antibody gene. The nested PCR product was inserted into the vector using the Clontech In-Fusion PCR cloning kit. The vector was then transformed into bacteria for plasmid propagation and production. The transformed bacteria were cultured in LB medium containing ampicillin or kanamycin, and the plasmid was purified from the grown bacteria using a 96-well EndoFree ezFilter Plasmid Miniprep Kit (Biomiga). Then, antibodies were obtained according to the procedure described in Reference Example 1.

[0251] Example 2 Measurement of affinity and pH / Ca dependence of anti-HMGB1 antibodies Generation of MedG4-IgG1 antibody MedG4H-IgG1 (SEQ ID NO: 36) and MedG4L-CK (SEQ ID NO: 37) were designed by linking the human IgG1 constant region and human Igκ constant region to the VH region (SEQ ID NO: 19, SEQ ID NO: 17, SEQ ID NO: 19) and VL region (SEQ ID NO: 17, SEQ ID NO: 17) of the G4 antibody described in WO2007 / 084253. DNA encoding these antibodies was prepared using genetic engineering techniques, and the anti-HMGB1 antibody MedG4-IgG1 was produced by expressing these in animal cells using methods known to those skilled in the art.

[0252] Evaluation of the pH- and pH / Ca-dependent binding ability of the obtained antibodies to human HMGB1 The antibodies obtained were evaluated using Biacore T100 and T200 (GE Healthcare) to determine whether they possessed pH- and pH / Ca-dependent binding activity. Plasma conditions were pH 7.4 and a calcium ion concentration of 1.2 mM. Two endosomal conditions were established: pH 5.8 and a calcium ion concentration of 1.2 mM, and pH 5.8 and a calcium ion concentration of 3 μM. An appropriate amount of protein A (Invitrogen) was immobilized on a Sensor chip CM4 (GE Healthcare) using the amine coupling method, and the target antibody was captured onto the immobilized protein. Human HMGB1 was used as the antigen. Measurements were performed using three different running buffers: 1; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, 2 mmol / L CaCl2, pH 7.4; 2; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, 2 mmol / L CaCl2, pH 5.8; 3; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, 3 μmol / L CaCl2, pH 5.8. Human HMGB1 was diluted in each running buffer.

[0253] HMG233-IgG1, HMG236-IgG1, HMG481-IgG1, HMG487-IgG1 Antibody diluted in running buffer was injected at a flow rate of 10 μL / min for 1 minute to capture the antibody on the sensor chip. Subsequently, a diluted human HMGB1 solution (500 nM) and running buffer (as a reference solution) were injected at a flow rate of 10 μL / min for 1 minute to allow interaction with the captured antibody. The running buffer was then injected at a flow rate of 10 μL / min for another 1 minute to observe dissociation of human HMGB1. Finally, 10 mmol / L glycine-HCl, pH 1.5, was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the sensor chip.

[0254] The sensorgrams obtained from the measurements are shown in Figures 4-1 and 4-2. The capture amount of each antibody is converted to 100 RU. Because all antibodies rapidly reach equilibrium, the equilibrium value (= binding amount) during human HMGB1 injection reflects the dissociation constant KD (M). For HMG233-IgG1 and HMG236-IgG1, the amount of human HMGB1 binding to each antibody was significantly reduced at pH 5.8, 3 μM Ca compared to pH 7.4, 1.2 mM Ca and pH 5.8, 1.2 mM Ca. For HMG481-IgG1 and HMG487-IgG1, the amount of human HMGB1 binding to each antibody was significantly reduced at pH 5.8, 1.2 mM Ca and pH 5.8, 3 μM Ca compared to pH 7.4, 1.2 mM Ca.

[0255] HMG446-IgG1, MedG4-IgG1 For HMG446-IgG1 and MedG4-IgG1, antibodies diluted in running buffer were injected at a flow rate of 10 μL / min for 1 minute and captured on the sensor chip. Then, human HMGB1 diluted solution and running buffer (as a reference solution) were injected at a flow rate of 10 μL / min for 1 minute to allow interaction with the captured antibody. The running buffer was then injected at a flow rate of 10 μL / min for 2 minutes to observe dissociation of human HMGB1. Finally, 10 mmol / L glycine-HCl, pH 1.5, was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the sensor chip.

[0256] For MedG4-IgG1, the sensorgrams obtained were analyzed by curve fitting. A 1:1 binding model was used. The kinetic parameters, the binding rate constant (ka) (1 / Ms) and the dissociation rate constant (kd) (1 / s), were calculated, and the dissociation constant (KD) (M) of each antibody for human HMGB1 was calculated based on these values. For HMG446-IgG1, the dissociation constant (KD) (M) was calculated using a steady-state affinity model for the sensorgrams obtained. Each parameter was calculated using Biacore T200 Evaluation Software (GE Healthcare). The pH dependence was calculated by dividing the KD (M) at pH 5.8, 1.2 mM Ca by the KD (M) at pH 7.4, 1.2 mM Ca, and the pH / Ca dependence was calculated by dividing the KD (M) at pH 5.8, 3 μM Ca by the KD (M) at pH 7.4, 1.2 mM Ca.

[0257] The analysis results are summarized in Table 12. The KD(M) of HMG446-IgG1 was calculated to be 220 nM at pH 7.4, 1.2 mM Ca. The KD(M) for human HMGB1 increased 50-fold (50-fold decrease in affinity) when changed from pH 7.4, 1.2 mM Ca to pH 5.8, 1.2 mM Ca, and increased 82-fold (82-fold decrease in affinity) when changed to pH 5.8, 3 μM Ca, indicating that affinity for human HMGB1 is lower under endosomal conditions than under serum conditions. On the other hand, the KD(M) of MedG4-IgG1 was calculated to be 96 nM at pH 7.4, 1.2 mM Ca, and 15 nM at pH 5.8, 1.2 mM Ca, and 3 μM Ca. It was shown that the affinity for human HMGB1 was increased under endosomal conditions compared to serum conditions, suggesting that MedG4-IgG1 is less likely to dissociate from HMGB1 within endosomes.

[0258] [Table 12]

[0259] [Example 3] Evaluation of binding between HMGB1 and cell surface receptors Binding of HMGB1 to RAGE-Fc by ELISA A 5 μg / ml solution of recombinant human RAGE-Fc fusion protein (R&D SYSTEMS) in PBS was added to each well of the ELISA plate at 20 μl / well and incubated overnight at 4°C. The plate was then blocked with 100 μl of 5% skim milk at 37°C for 1 hour and washed four times with PBS / Tween. In a separate plate, 4 μg / mL HMGB1 was preincubated with 100 μg / mL anti-HMGB1 antibody or buffer in the presence of 2.5% skim milk at room temperature for 1 hour and then transferred to the blocked RAGE-coated plate. The plate was then incubated overnight at 4°C and washed four times with PBS / Tween. To detect HMGB1 bound to the immobilized RAGE-Fc, peroxidase-labeled mouse anti-HMGB1 monoclonal antibody was added to each well and the plate was incubated at room temperature for 2 hours. The plate was then washed five times, 20 μl of TMB color developer was added, and the absorbance of the plate at 450 nm was measured.

[0260] We confirmed that the anti-HMGB1 antibody did not inhibit the binding of the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody used for detection to HMGB1 by allowing the anti-HMGB1 antibody to compete with the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody on a plate on which HMGB1 had been immobilized.

[0261] Binding of HMGB1 to TLR4 / MD-2 by ELISA A 5 μg / ml solution of recombinant human TLR4 / MD-2 protein (R&D SYSTEMS) in PBS was added to each well of the ELISA plate at 20 μl / well and incubated overnight at 4°C. The plate was then blocked with 100 μl of 5% skim milk at 37°C for 1 hour and washed four times with PBS / Tween. In a separate plate, 10 μg / mL HMGB1 was preincubated with 100 μg / mL anti-HMGB1 antibody or buffer in the presence of 2.5% skim milk at room temperature for 1 hour, and then transferred to the blocked TLR4 / MD-2-coated plate. The plate was then incubated at room temperature for 2 hours and washed four times with PBS / Tween. To detect HMGB1 bound to the immobilized TLR4 / MD-2, 1 μg / mL of peroxidase-labeled mouse anti-HMGB1 monoclonal antibody was added to each well, and the plate was incubated at room temperature for 2 hours. The plate was then washed five times with PBS / Tween, 20 μl of TMB color developer was added, and the absorbance of the plate at 450 nm was measured.

[0262] We confirmed that the anti-HMGB1 antibody did not inhibit the binding of the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody used for detection to HMGB1 by allowing the anti-HMGB1 antibody to compete with the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody on a plate on which HMGB1 had been immobilized.

[0263] result Both RAGE and TLR4 have been identified as putative receptors for HMGB1. Several anti-HMGB1 antibodies were evaluated for their ability to inhibit the binding of RAGE-Fc fusion or TLR4 / MD-2 fusion to HMGB1 in an ELISA assay. For RAGE and TLR4, the measured values ​​without anti-HMGB1 antibody were set at 100, and the measured values ​​with each anti-HMGB1 antibody were calculated as relative values. These values ​​are shown in Figures 5 and 6. Anti-HMGB1 antibodies showing values ​​lower than 100 were determined to have the ability to inhibit binding to the respective receptors. Among the antibodies used in the RAGE ELISA, HMG233-IgG1 and HMG236-IgG1 inhibited HMGB1 binding to RAGE. The inhibition rates were 53.1% for HMG233-IgG1 and 64.1% for HMG236-IgG1. Among the antibodies used in the TLR4 / MD-2 ELISA, HMG481-IgG1, HMG487-IgG1, and HMG446-IgG1 inhibited HMGB1 binding to TLR4 / MD-2. The inhibitory rates were 93.5% for HMG481-IgG1, 75.7% for HMG487-IgG1, and 81.3% for HMG446-IgG1. HMG233-IgG1 and HMG236-IgG1 did not inhibit the binding of HMGB1 to TLR4 / MD-2. Furthermore, HMG481-IgG1, HMG487-IgG1, and HMG446-IgG1 did not inhibit the binding of HMGB1 to RAGE.

[0264] Among anti-HMGB1 antibodies, we demonstrated that one antibody inhibits the binding of HMGB1 to RAGE but not to TLR4, and another antibody inhibits the binding of HMGB1 to TLR4 / MD-2 but not to RAGE.

[0265] Example 4: Examination of the improvement of the effect of pH-dependent anti-human HMGB1 antibodies in accelerating human HMGB1 elimination In vivo test using normal mice The pharmacokinetics of human HMGB1 and anti-human HMGB1 antibodies were evaluated after co-administration of human HMGB1 and anti-human HMGB1 antibodies in normal mice (C57BL / 6J mice, Charles River Japan). A single 10 mL / kg dose of a mixture of human HMGB1 (0.1 mg / mL) and anti-human HMGB1 antibodies (MedG4-IgG1 1 mg / mL, HMG446 2.05 mg / mL) was administered via the tail vein. The antibody concentration in the mixture was determined so that 99.0% or more of the human HMGB1 in the mixture bound to the antibody. Blood samples were collected 5, 10, and 15 minutes, 1 hour, 4 hours, 2 days, and 7 days after administration. The collected blood was allowed to stand for 2 hours, then centrifuged at 12,000 rpm at 4°C for 5 minutes to obtain serum. The separated serum was stored in a freezer at ≤-20°C until analysis. In this specification, MedG4-IgG1 may also be referred to as med G4, and HMG446-IgG1 may also be referred to as HMG446-G1.

[0266] Measurement of serum anti-human HMGB1 antibody concentrations by ELISA Anti-human HMGB1 antibody concentrations in mouse serum were measured by ELISA. First, anti-human IgG (γ-chain specific) F(ab')2 fragment of antibody (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International) and left overnight at 4°C to prepare an anti-human IgG-coated plate. Standard curve samples with serum concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 μg / mL and mouse serum samples diluted 100-fold or more were prepared. 150 μL of 2000 ng / mL human HMGB1 was added to 150 μL of these standard curve samples and serum samples and left to incubate at room temperature for 1 hour. The mixture was then dispensed onto the anti-human IgG-coated plate and left to incubate at room temperature for another hour. The plate was then incubated with Goat Anti-Human IgG (γ-chain specific) Biotin (BIOT) Conjugate (Southern Biotech Association) for 1 hour at room temperature, followed by Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 1 hour at room temperature. A color reaction was developed using TMB One Component HRP Microwell Substrate (BioFX Laboratories). The reaction was stopped with 1N-Sulfuric acid (Showa Chemical), and the absorbance at 450 nm was measured using a microplate reader. The anti-human HMGB1 antibody concentration in mouse serum was calculated from the absorbance of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of anti-human HMGB1 antibody concentration in mouse serum after intravenous administration, measured using this method, is shown in Figure 8.

[0267] Measurement of human HMGB1 concentrations in serum by ELISA Human HMGB1 concentrations in mouse serum were measured using the HMGB1 ELISA kit II (Shino-Test). Standard curve samples with serum concentrations of 12,800, 6,400, 3,200, 1,600, 800, 400, and 200 μg / mL and mouse serum measurement samples diluted 100-fold or more were prepared. These samples were mixed in equal volumes with 40 μg / mL HMG446 solution (in the presence of HMG446-IgG1 or HMG446-F1) or 20 μg / mL MedG4-IgG1 solution (in the presence of MedG4-IgG1) and incubated at room temperature for 1 hour. The mixture was then dispensed onto the provided solid-phase plate and incubated at 37°C for 20–24 hours. The included labeled antibody solution was then reacted at 25°C for 2 hours, followed by a 30-minute reaction with a colorimetric reagent. The reaction was then stopped by adding a stop solution. The absorbance at 450 nm was then measured using a microplate reader. The concentration of human HMGB1 in mouse serum was calculated from the absorbance of the standard curve using the analytical software SOFTmax PRO (Molecular Devices). The time course of human HMGB1 concentration in mouse serum after intravenous administration, measured using this method, is shown in Figure 7.

[0268] Effect of pH / Ca-dependent binding on human HMGB1 HMG446-IgG1, whose binding activity to human HMGB1 is reduced under acidic pH or low calcium ion concentrations, and MedG4-IgG1, whose binding activity to human HMGB1 is not reduced under acidic pH or low calcium ion concentrations, were tested in vivo and the results were compared. As shown in Figure 8, the pharmacokinetics of both antibodies were linear. On the other hand, as shown in Figure 7, HMGB1 administered simultaneously with HMG446-IgG1, which binds to human HMGB1 in a pH-dependent manner, was found to accelerate the elimination of HMGB1 compared to HMGB1 administered simultaneously with MedG4-IgG1. Thus, it was demonstrated that conferring pH-dependent human HMGB1 binding ability reduced serum HMGB1 concentrations by approximately 4.0-fold one day after administration.

[0269] Effect of FcRn binding under neutral conditions (pH 7.4) In addition to HMG446-IgG1, HMG446-F1, which was generated by introducing amino acid substitutions into the IgG Fc region of HMG446-IgG1, was also tested in vivo in mice, and the test results were compared with those of HMG446-IgG1. As shown in Figure 8, the serum antibody concentration of HMG446-F1, which had increased binding to mouse FcRn under neutral conditions (pH 7.4), was approximately 1.2-fold lower than that of HMG446-IgG1 15 minutes after administration. As shown in Figure 7, HMGB1 administered simultaneously with HMG446-F1, which has increased binding to mouse FcRn under neutral conditions (pH 7.4), was shown to clear more rapidly than HMGB1 administered simultaneously with HMG446-IgG1. HMG446-F1 reduced serum HMGB1 concentrations by approximately 2.4-fold compared to HMG446-IgG1 after 15 minutes. Thus, it was demonstrated that conferring mouse FcRn-binding ability under neutral conditions (pH 7.4) can reduce serum human HMGB1 concentrations. As previously described, conferring mouse FcRn-binding ability under neutral conditions (pH 7.4) reduced serum antibody concentrations. However, the effect of reducing serum HMGB1 concentrations was greater than the reduction in antibody concentrations.

[0270] These results indicate that administering an antibody whose binding activity to human HMGB1 is reduced under acidic pH or low calcium ion concentration conditions can accelerate the elimination of human HMGB1 compared to administering an antibody whose binding activity to human HMGB1 is not reduced under acidic pH or low calcium ion concentration conditions, and that this effect is enhanced by the mouse FcRn binding ability under neutral conditions (pH 7.4).

[0271] Example 5: Generation of various antibody Fc variants with increased binding affinity to human FcRn at neutral pH Generation of Fc variants Various substitutions were introduced into antibody Fv4-IgG1, which contains VH3-IgG1 and VL3-CK as the H and L chains, respectively, as shown in SEQ ID NOs: 6 and 7 in WO2009 / 125825, in order to increase the binding affinity for human FcRn in the neutral pH range. Specifically, the amino acid substitutions shown in Tables 13-1 to 13-14 were introduced into the heavy chain constant region of Fv4-IgG1 to generate Fc variants (amino acid numbers at the mutation sites are based on EU numbering). The amino acid substitutions were introduced according to the method known to those skilled in the art, as described in Reference Example 1. The heavy chains thus produced and variants containing the light chain L (WT) described as SEQ ID NO: 5 in WO2009 / 125825 were expressed and purified by methods known to those skilled in the art and described in Reference Example 1.

[0272] Assessment of human FcRn binding The binding of the antibodies to human FcRn under neutral conditions (pH 7.0) was analyzed by Biacore. The results are shown in Tables 13-1 to 13-14.

[0273] [Table 13-1]

[0274] Table 13-2 is a continuation of Table 13-1. [Table 13-2]

[0275] Table 13-3 is a continuation of Table 13-2. [Table 13-3]

[0276] Table 13-4 is a continuation of Table 13-3. [Table 13-4]

[0277] Table 13-5 is a continuation of Table 13-4. [Table 13-5]

[0278] Table 13-6 is a continuation of Table 13-5. [Table 13-6]

[0279] Table 13-7 is a continuation of Table 13-6. [Table 13-7]

[0280] Table 13-8 is a continuation of Table 13-7. [Table 13-8]

[0281] Table 13-9 is a continuation of Table 13-8. [Table 13-9]

[0282] Table 13-10 is a continuation of Table 13-9. [Table 13-10]

[0283] Table 13-11 is a continuation of Table 13-10. [Table 13-11]

[0284] Table 13-12 is a continuation of Table 13-11. [Table 13-12]

[0285] Table 13-13 is a continuation of Table 13-12. [Table 13-13]

[0286] Table 13-14 is a continuation of Table 13-13. [Table 13-14]

[0287] Reference Example 1: Construction of antibody expression vectors and antibody expression and purification Full-length genes encoding the base sequences of the antibody variable region heavy and light chains were synthesized using methods known to those skilled in the art, such as assembly PCR. Amino acid substitutions were introduced using PCR or other methods known to those skilled in the art. The resulting plasmid fragments were inserted into animal cell expression vectors to prepare heavy and light cha...

Claims

1. A method for producing an antigen-binding molecule that reduces an antigen concentration in plasma, the antigen-binding molecule having the following characteristics (1) to (6): (1) The antigen-binding molecule comprises an antigen-binding domain and at least one receptor-binding domain; (2) the receptor-binding domain has the activity of binding to human neonatal Fc receptor (FcRn) under a pH 5.8 condition; (3) the receptor-binding domain is a receptor-binding domain shown in (a) or (b) below, and the human Fc receptor-binding activity of the receptor-binding domain at pH 7.0 is higher than the human Fc receptor-binding activity of native human IgG; (a) the receptor is human FcRn, and the receptor-binding domain comprises an Fc region in which at least one amino acid in the Fc region of IgG has been modified, and the amino acid modification is selected from the group consisting of 234, 235, 236, 237, 238, 239, 244, 245, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 267, 270, 272, 274, 279, 280, 282, 283, 284, 285, 286, 288, 289, 293, 295, 297, 298, 303, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 325, 326, 327, 328, 329, 330, 332, 334, 338, 339, 340, 341, 343, 345, 360, 361, 362, 375, 376, 377, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 391, 413, 422, 423, 424, 427, a receptor binding domain having at least one amino acid modification selected from 428, 430, 431, 433, 434, 435, 436, 437, 438, 440, and 442; (b) the receptor is a human Fcγ receptor, and the receptor binding domain comprises an Fc region in which at least one amino acid in the Fc region of IgG has been modified, and the amino acid modification is selected from the group consisting of 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, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, a receptor binding domain comprising an alteration of at least one amino acid selected from the following: 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 341, 343, 375, 376, 377, 378, 379, 380, 382, ​​385, 386, 387, 389, 392, 396, 421, 423, 427, 428, 429, 430, 431, 433, 434, 436, 438, 440, and 442; (4) The antigen-binding domain is an antigen-binding domain shown in the following (c) or (d), and the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions; (c) an antigen-binding domain, in which the ion concentration is hydrogen ion concentration (pH), and the ratio of antigen-binding activity under pH 5.8 to that under pH 7.4, expressed as KD(pH 5.8) / KD(pH 7.4), is 2 or greater; (d) an antigen-binding domain, wherein the ion concentration is a calcium ion concentration, and the ratio of antigen-binding activity at a calcium ion concentration of 3 μM to that at a calcium ion concentration of 1.2 mM is expressed as KD(calcium ion concentration: 3 μM) / KD(calcium ion concentration: 1.2 mM) of 2 or more; (5) The antigen has two or more types of physiological activity, (6) Upon binding of the antigen-binding molecule, one or more physiological activities of the antigen are inhibited while at least one physiological activity is maintained; wherein the method comprises at least the following steps: (A) obtaining the antigen-binding domain; and (B) obtaining the at least one receptor-binding domain.

2. The method according to claim 1, wherein the antigen-binding molecule binds to an antigen and thereby inhibits one or more types of binding activity of the antigen to a target molecule while maintaining binding activity to at least one type of target molecule.

3. The method according to claim 1 or 2, wherein the reduction in the antigen concentration in the plasma is due to promotion of uptake of the antigen into cells.

4. The method according to any one of claims 1 to 3, wherein the antigen concentration in plasma is reduced, thereby reducing the physiological activity of the antigen in the living body.

5. The method according to any one of claims 1 to 4, wherein the antigen is HMGB1 (High Mobility Group Box 1).

6. The method according to claim 5, wherein the antigen-binding molecule inhibits the binding of HMGB1 to RAGE (Receptor for Advanced Glycation Endproducts).

7. The method according to claim 5 or 6, wherein the antigen-binding molecule inhibits the binding of HMGB1 to TLR4 (Toll-Like Receptor 4).

8. The method according to any one of claims 1 to 4, wherein the antigen is CTGF (Connective Tissue Growth Factor).

9. Amino acid modifications in the Fc region of IgG are shown in EU numbering; The 234th amino acid is Arg; The 235th amino acid is Gly, Lys, or Arg; The 236th amino acid is Ala, Asp, Lys, or Arg; The 237th amino acid is Lys, Met, or Arg; The 238th amino acid is Ala, Asp, Lys, Leu, or Arg; The 239th amino acid is Asp or Lys; The 244th amino acid is Leu. The 245th amino acid is Arg; The 248th amino acid is Ile or Tyr; The 249th amino acid is Pro. The 250th amino acid is Ala, Glu, Phe, Ile, Met, Gln, Ser, Val, Trp, Gly, His, Leu, Asn, or Tyr; The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, Trp, or Tyr; The 253rd amino acid is Val, The 254th amino acid is Ala, Gly, His, Ile, Gln, Ser, Val, or Thr; The 255th amino acid is Ala, Asp, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Gly, Ser, Trp, Tyr, or Glu; The 256th amino acid is Ala, Asp, Glu, Arg, Asn, Pro, Thr, Ser, or Gln; The 257th amino acid is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The 258th amino acid is Asp or His; The 260th amino acid is Ser; The 262nd amino acid is Leu. The 265th amino acid is Ala. The 267th amino acid is Met or Leu, The 270th amino acid is Lys or Phe; The 272nd amino acid is Ala, Leu, or Arg; The 274th amino acid is Ala. The 279th amino acid is Leu, Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 280th amino acid is Ala, Gly, His, Lys, Asn, Gln, Arg, Ser, Thr, or Glu; The 282nd amino acid is Ala or Asp; The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 284th amino acid is Lys. The 285th amino acid is Asn, The 286th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or Glu; The 288th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Val, Trp, Tyr, or Ser; The 289th amino acid is His; The 293rd amino acid is Val; The 295th amino acid is Met; The 297th amino acid is Ala. The 298th amino acid is Gly. The 303rd amino acid is Ala. The 305th amino acid is Ala or Thr; The 307th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The 308th amino acid is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The 309th amino acid is Ala, Asp, Glu, Pro, His, or Arg; The 311th amino acid is Ala, His, Glu, Lys, Leu, Met, Ser, Val, Trp, or Ile; The 312th amino acid is Ala, Asp, Pro, or His; The 313th amino acid is Tyr or Phe; The 314th amino acid is Ala, Leu, Lys, or Arg; The 315th amino acid is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or His; The 316th amino acid is Ala, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Asp; The 317th amino acid is Ala or Pro; The 318th amino acid is Asn or Thr; The 325th amino acid is Ala, Gly, Met, Leu, Ile, or Ser; The 326th amino acid is Asp, The 327th amino acid is Gly; The amino acid at position 328 is Arg, Asp, Glu, or Tyr; The 329th amino acid is Lys or Arg; The 330th amino acid is Leu. The 332nd amino acid is Glu, Phe, His, Lys, Leu, Met, Arg, Ser, Trp, or Val; The 334th amino acid is Leu. The 338th amino acid is Ala. The 339th amino acid is Asn, Thr or Trp; The 340th amino acid is Ala. The 341st amino acid is Pro. The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 345th amino acid is Ala. The 360th amino acid is His, The 361st amino acid is Ala. The 362nd amino acid is Ala. The 375th amino acid is Ala or Arg; The 376th amino acid is Ala, Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys. The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Thr, or Ser; The 382nd amino acid is Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Trp, Tyr, or Val; The 384th amino acid is Ala. The 385th amino acid is Ala, Gly, Lys, Ser, Thr, Asp, His, or Arg; The 386th amino acid is Arg, Asp, Ile, Met, Ser, Thr, Lys, or Pro; The 387th amino acid is Ala, Arg, His, Pro, Ser, Thr, or Glu; Amino acid 389 is Ala, Asn, Pro, or Ser; The 390th amino acid is Ala. The 391st amino acid is Ala. The 413th amino acid is Ala. The 423rd amino acid is Asn, The 424th amino acid is Ala or Glu; The 427th amino acid is Asn, The 428th amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The 430th amino acid is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The 431st amino acid is His or Asn, The 433rd amino acid is Arg, Gln, His, Ile, Pro, Ser, or Lys; The 434th amino acid is Ala, Phe, Gly, Met, His, Ser, Trp, or Tyr; The amino acid at position 435 is Lys, Arg, or Asn; The 436th amino acid is Ala, His, Ile, Leu, Glu, Phe, Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp, or Val; The 437th amino acid is Arg; Amino acid at position 438 is Lys, Leu, Thr or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys. The method according to any one of claims 1 to 8, wherein the modification is of at least one amino acid selected from the group consisting of:

10. The method according to any one of claims 1 to 9, wherein the Fc region of IgG is an Fc region of IgG derived from a non-human animal.

11. The method according to any one of claims 1 to 9, wherein the Fc region of IgG is an Fc region of IgG of human origin.

12. Amino acid modifications in the Fc region of IgG are shown in EU numbering; The 221st amino acid is Lys or Tyr; Amino acid 222 is Phe, Trp, Glu, or Tyr; The 223rd amino acid is Phe, Trp, Glu, or Lys; Amino acid 224 is Phe, Trp, Glu, or Tyr; The 225th amino acid is Glu, Lys, or Trp; The 227th amino acid is Glu, Gly, Lys, or Tyr; The 228th amino acid is Glu, Gly, Lys, or Tyr; The 230th amino acid is Ala, Glu, Gly, or Tyr; The 231st amino acid is Glu, Gly, Lys, Pro, or Tyr; The 232nd amino acid is Glu, Gly, Lys, or Tyr; The 233rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 234th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 235th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 236th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 237th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 238th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 239th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; The 240th amino acid is Ala, Ile, Met, or Thr; The 241st amino acid is Asp, Glu, Leu, Arg, Trp, or Tyr; The 243rd amino acid is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The 244th amino acid is His, The 245th amino acid is Ala. The 246th amino acid is Asp, Glu, His, or Tyr; The 247th amino acid is Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The 249th amino acid is Glu, His, Gln, or Tyr; The 250th amino acid is Glu or Gln, The 251st amino acid is Phe. Amino acid 254 is Phe, Met or Tyr; The 255th amino acid is Glu, Leu, or Tyr; The 256th amino acid is Ala, Met, or Pro; The 258th amino acid is Asp, Glu, His, Ser, or Tyr; The 260th amino acid is Asp, Glu, His, or Tyr; Amino acid 262 is Ala, Glu, Phe, Ile, or Thr; The 263rd amino acid is Ala, Ile, Met, or Thr; The 264th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 265th amino acid is Ala, Glu, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 266th amino acid is Ala, Phe, Ile, Leu, Met, or Thr; The 267th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; The 268th amino acid is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Trp; The 269th amino acid is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 270th amino acid is Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 271st amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 272nd amino acid is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Amino acid 273 is Phe or Ile; The 274th amino acid is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 275th amino acid is Leu or Trp, The 276th amino acid is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 278th amino acid is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The 279th amino acid is Ala. The 280th amino acid is Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr; The 281st amino acid is Asp, Lys, Pro, or Tyr; The 282nd amino acid is Glu, Gly, Lys, Pro, or Tyr; The 283rd amino acid is Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; The 284th amino acid is Asp, Glu, Leu, Asn, Thr, or Tyr; The 285th amino acid is Asp, Glu, Lys, Gln, Trp, or Tyr; The 286th amino acid is Glu, Gly, Pro, or Tyr; The 288th amino acid is Asn, Asp, Glu, or Tyr; The 290th amino acid is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr; The 291st amino acid is Asp, Glu, Gly, His, Ile, Gln, or Thr; The 292nd amino acid is Ala, Asp, Glu, Pro, Thr, or Tyr; The 293rd amino acid is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 294th amino acid is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 295th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 296th amino acid is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; The 297th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 298th amino acid is Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr; The 299th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The 300th amino acid is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The 301st amino acid is Asp, Glu, His, or Tyr; The 302nd amino acid is Ile; The 303rd amino acid is Asp, Gly, or Tyr; The 304th amino acid is Asp, His, Leu, Asn, or Thr; The 305th amino acid is Glu, Ile, Thr, or Tyr; The 311th amino acid is Ala, Asp, Asn, Thr, Val, or Tyr; The 313th amino acid is Phe. The 315th amino acid is Leu. The 317th amino acid is Glu or Gln; The amino acid at position 318 is His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; The amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; The 322nd amino acid is Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr; The 323rd amino acid is Ile, Leu, or Met; The 324th amino acid is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; The 325th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 326th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The 327th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; The 328th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 329th amino acid is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 330th amino acid is Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The 331st amino acid is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 332nd amino acid is Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 333rd amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, or Tyr; The 334th amino acid is Ala, Glu, Phe, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 335th amino acid is Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; The 336th amino acid is Glu, Lys, or Tyr; The 337th amino acid is Asp, Glu, His, or Asn; The 339th amino acid is Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr; The 376th amino acid is Ala or Val; The 377th amino acid is Gly or Lys; The 378th amino acid is Asp, The 379th amino acid is Asn, The 380th amino acid is Ala, Asn, or Ser; Amino acid 382 is Ala or Ile; The 385th amino acid is Glu; The 392nd amino acid is Thr; The 396th amino acid is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr; The 421st amino acid is Lys. The 427th amino acid is Asn, Amino acid 428 is Phe or Leu; The 429th amino acid is Met; The 434th amino acid is Trp, The 436th amino acid is Ile, and The amino acid at position 440 is Gly, His, Ile, Leu, or Tyr; The method according to any one of claims 1 to 8, wherein the modification is of at least one amino acid selected from the group consisting of:

13. The method according to any one of claims 1 to 12, wherein the human Fcγ receptor is FcγRIa, FcγRIIa, FcγRIIb, or FcγRIIIa.

14. The method according to any one of claims 1 to 8, wherein the amino acid modification in the IgG Fc region is Asp at amino acid position 238 and Gly at amino acid position 271 (EU numbering).

15. In the Fc region of IgG, the following amino acids are further divided into EU numbering 233, 234, 237, 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 264, 265, 266, 267, 268, 269, 270, 272, 279, 283, 285, 286, 288, 293, 296, 307, 308, 309, 311, 312, 314, 316, 317, 318, 326, 327, 330, 331, 332, 333, 15. The method of claim 14, wherein at least one amino acid selected from 339, 341, 343, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 389, 396, 423, 427, 428, 430, 431, 433, 434, 436, 438, 440 and 442 has been modified.

16. Amino acid modifications in the Fc region of IgG are shown in EU numbering; The 233rd amino acid is Asp, The 234th amino acid is Tyr. The 237th amino acid is Asp, The 264th amino acid is Ile; The 265th amino acid is Glu. Amino acid 266 is Phe, Met or Leu; The 267th amino acid is Ala, Glu, Gly, or Gln; The 268th amino acid is Asp or Glu; The 269th amino acid is Asp; The 272nd amino acid is Asp, Phe, Ile, Met, Asn, or Gln; The 296th amino acid is Asp, The 326th amino acid is Ala or Asp; The 327th amino acid is Gly; The 330th amino acid is Lys or Arg; The 331st amino acid is Ser; The 332nd amino acid is Thr; The 333rd amino acid is Thr, Lys, or Arg; The 396th amino acid is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr; The method according to claim 15, wherein the modification is of at least one amino acid selected from the group consisting of:

17. In the Fc region of IgG, the following residues are further divided into EU numbering 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 389, 423, 427, 17. The method according to any one of claims 14 to 16, wherein at least one amino acid selected from 428, 430, 431, 433, 434, 436, 438, 440 and 442 has been modified.

18. Amino acid modifications in the Fc region of IgG are shown in EU numbering; The 244th amino acid is Leu. The 245th amino acid is Arg; The 249th amino acid is Pro. The 250th amino acid is Gln or Glu, The 251st amino acid is Arg, Asp, Glu, or Leu; Amino acid 252 is Phe, Ser, Thr, or Tyr; The 254th amino acid is Ser or Thr; The 255th amino acid is Arg, Gly, Ile, or Leu; The 256th amino acid is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr; The 257th amino acid is Ala, Ile, Met, Asn, Ser, or Val; The 258th amino acid is Asp, The 260th amino acid is Ser; The 262nd amino acid is Leu. The 270th amino acid is Lys. The 272nd amino acid is Leu or Arg; The 279th amino acid is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; The 283rd amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; The 285th amino acid is Asn, The 286th amino acid is Phe. The 288th amino acid is Asn or Pro; The 293rd amino acid is Val; The 307th amino acid is Ala, Glu, Gln, or Met; Amino acid at position 308 is Ile, Pro or Thr; The 309th amino acid is Pro. The 311th amino acid is Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp; The 312th amino acid is Ala, Asp or Pro; The 314th amino acid is Ala or Leu; The 316th amino acid is Lys. The 317th amino acid is Pro. The 318th amino acid is Asn or Thr; Amino acid 332 is Phe, His, Lys, Leu, Met, Arg, Ser, or Trp; The 339th amino acid is Asn, Thr or Trp; The 341st amino acid is Pro. The 343rd amino acid is Glu, His, Lys, Gln, Arg, Thr, or Tyr; The 375th amino acid is Arg; The 376th amino acid is Gly, Ile, Met, Pro, Thr, or Val; The 377th amino acid is Lys. The 378th amino acid is Asp, Asn, or Val; The 380th amino acid is Ala, Asn, Ser, or Thr; Amino acid 382 is Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The 385th amino acid is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; The 386th amino acid is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; The 387th amino acid is Ala, Arg, His, Pro, Ser, or Thr; The 389th amino acid is Asn, Pro, or Ser; The 423rd amino acid is Asn, The 427th amino acid is Asn, Amino acid at position 428 is Leu, Met, Phe, Ser, or Thr; The 430th amino acid is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; The 431st amino acid is His or Asn, The amino acid at position 433 is Arg, Gln, His, Ile, Lys, Pro, or Ser; The 434th amino acid 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; Amino acid at position 438 is Lys, Leu, Thr or Trp; The 440th amino acid is Lys, and The 442nd amino acid is Lys. The method according to claim 17, wherein the modification is at least one amino acid selected from the group consisting of:

19. The method according to any one of claims 1 to 18, wherein the Fc region of IgG is an Fc region of IgG derived from a non-human animal.

20. The method according to any one of claims 1 to 18, wherein the Fc region of IgG is an Fc region of IgG of human origin.

21. 21. The method of any one of claims 1 to 20, wherein at least one amino acid in the antigen-binding domain is substituted with histidine and / or at least one histidine is inserted.

22. The method according to any one of claims 1 to 21, further comprising: having an antigen-binding activity that is lower under a low calcium ion concentration condition than under a high calcium ion concentration condition.

23. 23. The method of any one of claims 1 to 22, wherein the antigen-binding domain is obtained from a library.

24. The method of any one of claims 1 to 23, wherein the antigen-binding molecule is an antibody.

25. 25. The method of claim 24, wherein the antibody is either a chimeric antibody, a humanized antibody or a human antibody.

26. The method according to any one of claims 1 to 25, wherein the antigen-binding domain, whose antigen-binding activity changes depending on hydrogen ion concentration (pH) conditions, comprises amino acids selected from the following, in which at least one amino acid is histidine: Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102 (Kabat numbering), Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94 (Kabat numbering)

27. The method according to any one of claims 1 to 25, wherein the antigen-binding domain, whose antigen-binding activity changes depending on calcium concentration conditions, comprises at least one amino acid substitution in an amino acid selected from the following: Heavy chain: positions 95, 96, 100a and 101 (Kabat numbering); Light chain: positions 30, 31, 32, 50 and 92 (Kabat numbering)

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