Antigen-binding molecules that promote the elimination of antigens with multiple physiological activities.

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 an antigen-binding molecule capable of reducing the physiological activity in vivo of antigens that have multiple types of physiological activity and are difficult to inhibit in vitro, by promoting their elimination from the blood (serum or plasma), and to a pharmaceutical composition containing such an antigen-binding molecule as an active ingredient. [Background technology]

[0002] Many diseases are known to be caused by an excessive increase in the amount of bioactive substances (such as cytokines) present in the plasma compared to a healthy state, which disrupts the balance of bioactivity that is normally maintained. One effective means of treating such diseases is to inhibit the bioactivity of the excess bioactive substances. For example, antibodies that neutralize the bioactivity of a bioactive antigen by binding to it can be an effective therapeutic agent.

[0003] However, if an antigen has two or more types of biological activity, a single neutralizing antibody can usually inhibit only one type of biological activity, and it is expected that treating diseases caused by such biologically active substances with such antibodies will be difficult.

[0004] HMGB1 (High Mobility Group Box 1) is an example of a bioactive substance that possesses two or more types of biological activity. HMGB1 was identified as a member of the HMG family of nuclear proteins that contribute to the stability of the higher-order structure of DNA by binding to it. HMGB1 consists of 215 amino acids and is structurally composed of three main domains: the HMG A box, the HMG B box, and an acidic carboxyl terminus. Normally, it exists inside cells as a DNA-binding protein, but it is released extracellularly from inflammatory cells and necrotic cells through active or passive mechanisms. Released HMGB1 is known to induce diverse inflammatory responses by binding to various substances such as DNA, LPS (Lipopolysaccharide), and IL (Interleukin)-1β, thereby activating various cell surface receptors such as RAGE (Receptor for Advanced Glycation Endproducts), TLR4 (Toll-Like Receptor 4), and IL-1 receptor, and transmitting signals into the cell (Non-Patent Literature 1). Furthermore, in sepsis model mice administered LPS, elevated blood concentrations of HMGB1 were observed, and the mortality rate of the mice decreased after administration of a polyclonal antibody against HMGB1 (Non-Patent Literature 2), suggesting that HMGB1 plays an important role in the development of sepsis. Patent Literature 1 discloses the production of multiple high-affinity monoclonal antibodies against HMGB1, which inhibited the binding of HMGB1 to RAGE or TLR4, and reduced the mortality rate in sepsis model mice. However, there is no mention of obtaining an antibody that inhibits both the action of HMGB1 on RAGE and TLR4, suggesting that it is difficult to inhibit the multiple types of activity possessed by HMGB1 with a single antibody.

[0005] Antibodies (IgG) have long plasma retention due to their binding to FcRn (Neonatal Fc Receptor). IgG-FcRn binding is observed only under acidic conditions (pH 6.0), and hardly at all under neutral conditions (pH 7.4). Normally, IgG is taken up into cells nonspecifically via endocytosis, but under acidic conditions within endosomes, it binds to FcRn within the endosome, returning to the cell surface, and then dissociates from FcRn under neutral conditions in the plasma. IgG that does not bind to FcRn proceeds to lysosomes, where it is degraded. Introducing a mutation into the Fc region of IgG, thereby eliminating its binding to FcRn under acidic conditions, prevents recycling from endosomes into the plasma, significantly impairing IgG's plasma retention. Methods to improve IgG plasma retention by enhancing its binding to FcRn under acidic conditions have been reported. Introducing amino acid substitutions into the Fc region of IgG improves its binding to FcRn under acidic conditions, thereby increasing the recycling efficiency from endosomes to plasma, and consequently improving 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 acidic conditions within endosomes, it does not dissociate from FcRn in plasma under neutral conditions, resulting in no change in plasma retention or even a worsening of plasma retention (Non-Patent Literature 3-5).

[0006] Recently, an antibody that binds to an antigen in a pH-dependent manner has been reported (Patent Document 2). This antibody strongly binds to the antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in endosomes. After dissociating from the antigen, the antibody can be recycled into the plasma by FcRn and re-bind to the antigen, making it possible for a single antibody to repeatedly bind to multiple antigens. Since the retention period of the antigen in plasma is very short compared to antibodies with an FcRn-mediated recycling mechanism, the antigen's retention period in plasma is usually prolonged and the plasma antigen concentration increases when the antigen binds to an antibody. On the other hand, the above-mentioned pH-dependently binding antibody dissociates from the antigen in endosomes during the FcRn-mediated recycling process, and has been reported to promote the disappearance of the antigen from plasma compared to ordinary antibodies (Patent Document 2). However, no antibody engineering method is known to further improve this effect of promoting the disappearance of the antigen 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 has been made in view of such circumstances. An object of the present invention is to provide an antigen-binding molecule capable of reducing the physiological activity of an antigen with a single type of antigen-binding molecule in vivo by promoting the elimination of the antigen from blood (serum or plasma), wherein the antigen has two or more types of physiological activity, which makes it difficult to inhibit those physiological activities with a single type of antigen-binding molecule in vitro. The present invention also aims to provide a method for producing the antigen-binding molecule, and a pharmaceutical composition comprising the antigen-binding molecule as an active ingredient. Means for Solving the Problem

[0010] As a result of intensive research, the present inventors found that even for an antigen-binding molecule that inhibits some types of physiological activity but does not inhibit the remaining types of physiological activity in vitro against an antigen having two or more types of physiological activity, when the antigen-binding molecule is imparted with the following properties: (i) binds to human FcRn (Neonatal Fc Receptor) under acidic pH conditions, (ii) binds to human FcRn and / or human Fcγ receptor more strongly than native human IgG under neutral pH conditions, and (iii) the antigen-binding activity of the antigen-binding molecule changes depending on ion concentration conditions the present inventors newly found that imparting the above properties promotes the elimination of the antigen from blood (serum or plasma), and as a result can reduce the physiological activity in vivo.

[0011] This invention is based on such findings and specifically relates to the following inventions. [1] An antigen-binding molecule that reduces the concentration of an antigen in plasma and has the following characteristics: (1) to (6); (1) The antigen-binding molecule comprises an antigen-binding domain and at least one receptor-binding domain, (2) Under conditions in the pH range, the receptor-binding domain has the activity to bind to human FcRn (Neonatal Fc Receptor), (3) Under conditions of a neutral pH, the activity of the receptor-binding domain in binding to the human Fc receptor is higher than the activity of natural human IgG in binding to the human Fc receptor. (4) The activity of the antigen-binding domain in binding to the antigen changes depending on the ion concentration conditions. (5) The antigen has two or more types of physiological activity, (6) When an antigen-binding molecule binds to the antigen, one or more of the physiological activities of the antigen are inhibited, while at least one physiological activity is maintained. [2] The antigen-binding molecule according to [1], characterized in that, by binding to an antigen, it inhibits one or more of the target molecule binding activities of the antigen, while maintaining the binding activity of at least one target molecule. [3] The antigen-binding molecule according to [1] or [2], characterized in that the decrease in antigen concentration in plasma is due to the promotion of antigen uptake into cells. [4] An antigen-binding molecule according to any one of [1] to [3], characterized in that the physiological activity of the antigen in the body is reduced by a decrease in the antigen concentration in plasma. [5] An antigen-binding molecule as described in any one of items [1] to [4], wherein the antigen is HMGB1 (High Mobility Group Box 1). [6] The antigen-binding molecule described in [5] that inhibits the binding of HMGB1 to RAGE (Receptor for Advanced Glycation Endproducts). [7] An antigen-binding molecule as described in [5] or [6] that inhibits the binding of HMGB1 to TLR4 (Toll-Like Receptor 4). [8] An antigen-binding molecule described in any one of the items [1] to [4], wherein the antigen is CTGF (Connective Tissue Growth Factor). [9] An antigen-binding molecule according to any one of items [1] to [8], wherein the human Fc receptor is human FcRn.

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

[11] Amino acid modifications in the Fc region of IgG, EU numbering 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 described in

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

[12] Amino acid modifications in the Fc region of IgG are used for 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 either 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. The 252nd amino acid 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 either 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 either 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 either 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 either 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 328th amino acid is Arg, Asp, Glu, or Tyr. The 329th amino acid is either 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 either 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. 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 either 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 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, The 438th amino acid is Lys, Leu, Thr, or Trp. The 440th amino acid is Lys, and The 442nd amino acid is Lys, An antigen-binding molecule as described in

[11] , which is a modification of at least one amino acid selected from.

[13] An antigen-binding molecule according to any one of items

[10] to

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

[14] An antigen-binding molecule according to any one of items

[10] to

[12] , wherein the Fc region of IgG is the Fc region of human IgG.

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

[16] The antigen-binding molecule according to

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

[17] Amino acid modifications in the Fc region of IgG, EU numbering 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, An antigen-binding molecule as described in

[16] , which is a modification of at least one amino acid selected from positions 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] Amino acid modifications in the Fc region of IgG are used for EU numbering; The 221st amino acid is Lys or Tyr, The 222nd amino acid is Phe, Trp, Glu, or Tyr. The 223rd amino acid is Phe, Trp, Glu, or Lys. The 224th amino acid 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, The 254th amino acid 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 262nd amino acid 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. The 273rd amino acid 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 either 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 320th amino acid 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 either 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. The 428th amino acid is Phe or Leu, The 429th amino acid is Met, The 434th amino acid is Trp. The 436th amino acid is Ile, and The 440th amino acid is Gly, His, Ile, Leu, or Tyr. An antigen-binding molecule as described in

[17] , which is a modification of at least one amino acid selected from.

[19] An antigen-binding molecule according to any one of the items

[16] to

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

[20] The antigen-binding molecule described in

[17] , wherein the amino acid modification in the Fc region of IgG is such that the 238th amino acid in EU numbering is Asp and the 271st amino acid is Gly.

[21] In the Fc region of IgG, further 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, The antigen-binding molecule described in

[20] , wherein at least one amino acid selected from positions 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 is modified.

[22] Amino acid modifications in the Fc region of IgG are used for 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, The 266th amino acid is Phe, Met, or Leu. The 267th amino acid is Ala, Glu, Gly, or Gln. The 268th amino acid is either 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 either Ala or Asp. The 327th amino acid is Gly, The 330th amino acid is either 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. An antigen-binding molecule as described in

[21] , which is a modification of at least one amino acid selected from.

[23] In the Fc region of IgG, further 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, An antigen-binding molecule according to any one of the items

[20] to

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

[24] Amino acid modifications in the Fc region of IgG are used for 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. The 252nd amino acid 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 either 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 either Ala or Leu. The 316th amino acid is Lys, The 317th amino acid is Pro, The 318th amino acid is Asn or Thr. The 332nd amino acid 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. The 382nd amino acid 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 428th amino acid 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 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. The 438th amino acid is Lys, Leu, Thr, or Trp. The 440th amino acid is Lys, and The 442nd amino acid is Lys, An antigen-binding molecule as described in

[23] , which is a modification of at least one amino acid selected from.

[25] An antigen-binding molecule according to any one of items

[16] to

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

[26] An antigen-binding molecule according to any one of items

[16] to

[24] , wherein the Fc region of IgG is the Fc region of human-derived IgG.

[27] An antigen-binding molecule according to any one of items [1] to

[26] , wherein the ion concentration is the hydrogen ion concentration (pH), and the activity of binding to the antigen is lower under acidic pH conditions compared to under neutral pH conditions.

[28] The antigen-binding molecule described in

[27] , wherein the pH is acidic, which is the pH inside the endosome.

[29] The antigen-binding molecule described in

[27] or

[28] , wherein the pH is neutral, which is the pH of plasma.

[30] An antigen-binding molecule according to any one of items

[27] to

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

[31] An antigen-binding molecule according to any one of items

[27] to

[30] , wherein the ratio of activity to bind to an antigen under conditions in the acidic pH range and under conditions in the neutral pH range is 2 or more in terms of the KD(acidic pH) / KD(neutral pH) value.

[32] An antigen-binding molecule according to 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] Furthermore, an antigen-binding molecule according to any one of

[27] to

[32] , wherein the activity of binding to the antigen is lower under low calcium ion concentration conditions compared to high calcium ion concentration conditions.

[34] An antigen-binding molecule according to any one of items [1] to

[26] , wherein the ion concentration is the calcium ion concentration, and the activity of binding to the antigen is lower under low calcium ion concentration conditions compared to high calcium ion concentration conditions.

[35] The antigen-binding molecule described in

[33] or

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

[36] An antigen-binding molecule according to any one of items

[33] to

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

[37] An antigen-binding molecule according to any one of items

[33] to

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

[38] An antigen-binding molecule according to any one of the items

[33] to

[37] , wherein the ratio of activity binding to the antigen under low calcium ion concentration conditions to activity under high calcium ion concentration conditions is such that the value of KD(low calcium ion concentration) / KD(high calcium ion concentration) is 2 or more.

[39] An antigen-binding molecule described in any one of items [1] to

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

[40] An antigen-binding molecule as described in any one of items [1] to

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

[41] The antigen-binding molecule described in

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

[42] A pharmaceutical composition containing an antigen-binding molecule as described in any one of items [1] to

[41] as an active ingredient.

[43] The pharmaceutical composition according to

[42] for treating a disease in which the antigen is considered to be one of the causes.

[44] The pharmaceutical composition according to

[43] , wherein the antigen is HMGB1.

[45] The pharmaceutical composition according to

[43] or

[44] , wherein the disease is sepsis.

[46] The pharmaceutical composition according to

[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 described in [1], comprising the following steps; (a) A step of selecting antigens having two or more physiological activities, (b) Steps to obtain an antigen-binding domain, (c) A step of obtaining at least one receptor-binding domain, (d) A step of selecting from the antigen-binding domains obtained in step (b) a domain whose activity in binding to the antigen changes depending on the ion concentration conditions. (e) A step of selecting from among the receptor-binding domains obtained in step (c) a domain that has activity to bind to human FcRn under acidic pH conditions and whose activity to bind to human Fc receptors under neutral pH conditions is higher than the activity of natural human IgG to bind to human Fc receptors. (f) A step to produce 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. Furthermore (g) A step of selecting from the antigen-binding molecules produced in step (f) an antigen-binding molecule that, upon binding to an antigen, inhibits one or more of the 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 a domain that binds to an antigen and further modifying at least one amino acid in said domain.

[50] The method according to

[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 said domain.

[51] Process (f) (f) A polynucleotide is prepared by linking a polynucleotide encoding the antigen-binding domain selected in step (d) and a polynucleotide encoding the receptor-binding domain selected in step (e), and an antigen-binding molecule is prepared by linking the antigen-binding domain selected in step (d) and the receptor-binding domain selected in step (e) using the linked polynucleotide. The method described in any one of

[48] to

[50] .

[52] Step (g) is (g) A step of selecting from the antigen-binding molecules produced in step (f) an antigen-binding molecule in which, upon binding to the antigen, one or more of the binding activities of the antigen to target molecules are inhibited, while the binding activity to at least one target molecule is maintained. The method described in any one of the items

[48] to

[51] .

[53] The method according to any one of

[48] to

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

[0012] Furthermore, this invention relates to the following.

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

[41] or an antigen-binding molecule produced by a manufacturing method described in any one of items

[48] to

[53] .

[55] A method for promoting the uptake of an antigen into cells by administering an antigen-binding molecule described in any one of items [1] to

[41] or an antigen-binding molecule produced by a manufacturing method described in any one of items

[48] to

[53] .

[56] A method for reducing the physiological activity of an antigen in a living organism by administering an antigen-binding molecule described in any one of items [1] to

[41] or an antigen-binding molecule produced by a manufacturing method described in any one of items

[48] to

[53] .

[57] The method according to any one of the claims

[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 disease treatment agent containing as an active ingredient an antigen-binding molecule described in any one of items [1] to

[41] or an antigen-binding molecule manufactured by any one of items

[48] to

[53] .

[60] The therapeutic agent described in

[59] , wherein the antigen is HMGB1.

[61] The therapeutic agent described in

[59] or

[60] for a disease in which sepsis is present.

[62] The therapeutic agent described in

[59] , wherein the antigen is CTGF.

[63] The therapeutic agent described in

[59] or

[62] for a disease of fibrosis.

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

[54] to

[58] , comprising an antigen-binding molecule described in any one of [1] to

[41] or an antigen-binding molecule produced by the manufacturing method described in any one of

[48] to

[53] .

[0013] Furthermore, this invention relates to the following.

[101] A method for screening antibodies whose antigen-binding activity changes depending on conditions, comprising the following steps; (a) A step of preparing antibody-producing cells, (b) The step of bringing the antigen into contact with the cells of (a) under the first conditions, (c) A step of selecting cells from the cells of step (b) that have bound to a certain amount or more of antigen, (d) The step of placing the cells from step (c) under second conditions, and (e) A step in which cells from step (d) have a reduced antigen binding capacity compared to those in step (c).

[102] A method according to

[101] , comprising the following steps; (a) A step of preparing antibody-producing cells, (b) The step of bringing the antigen into contact with the cells of (a) under the first conditions, (c) A step of contacting the cells from step (b) with an anti-IgG antibody, (d) A step of selecting cells from the cells of step (c) that are bound to a certain amount or more of antigen and bound to a certain amount or more of anti-IgG antibody. (e) The step of placing the cells from step (d) under second conditions, and (f) A step in which cells from step (e) have a reduced antigen binding capacity compared to those in step (d).

[103] A method according to

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

[104] The method according to any one of

[101] to

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

[105] The method according to

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

[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 pH neutrality is pH 7.0 to 8.0 and the pH acidity 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 of less than or equal to that.

[110] The method according to any one of

[101] to

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

[111] The method according to any one of the claims

[101] to

[110] , wherein the antibody-producing cell is a B cell.

[112] The method according to

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

[113] The method according to any one of

[101] to

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

[114] The method according to any one of the items

[101] to

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

[115] The method according to any one of the items

[101] to

[114] , wherein the cell enrichment step is performed using MACS.

[0014] Furthermore, the present invention relates to the following inventions. [a] A method for treating a disease in which a physiologically active antigen is considered to be one of the causes, a method for reducing the concentration of an antigen in plasma, a method for promoting the uptake of an antigen into cells, or a method for reducing the physiological activity of an antigen in a living organism, comprising the step of administering an antigen-binding molecule of the present invention. [b] A therapeutic agent for diseases in which a physiologically active antigen is considered to be one of the causes, an agent for reducing antigen concentration in plasma, an agent for promoting the uptake of antigens into cells, or an agent for reducing the physiological activity of an antigen in the body, comprising the antigen-binding molecule of the present invention as an active ingredient. [c] The antigen-binding molecule of the present invention for use in methods for treating diseases in which a physiologically active antigen is considered to be one of the causes, methods for reducing the concentration of an antigen in plasma, methods for promoting the uptake of an antigen into cells, or methods for reducing the physiological activity of an antigen in a living organism. [d] Use of the antigen-binding molecule of the present invention in the manufacture of a therapeutic agent for a disease in which a physiologically active antigen is considered to be one of the causes, an agent for reducing the concentration of antigens in plasma, an agent for promoting the uptake of antigens into cells, or an agent for reducing the physiological activity of antigens in the body. (e) A process for producing a therapeutic agent for a disease in which a physiologically active antigen is considered to be one of the causes, an agent for reducing the concentration of an antigen in plasma, an agent for promoting the uptake of an antigen into cells, or an agent for reducing the physiological activity of an antigen in a living organism, comprising the step of using the antigen-binding molecule of the present invention. Examples of diseases include those in which HMGB1 is considered to be one of the causative agents, those in which CTGF is considered to be one of the causative agents, and those in which IgE is considered to be one of the causative agents. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the results of FACS sorting of B cells producing anti-HMGB1 antibodies. (A) shows the results of the first sorting under neutral pH and high calcium ion concentration conditions, and (B) shows the results of the second sorting under acidic pH and low calcium ion concentration conditions. The numbers (1, 2, 3) in the figure indicate the gate numbers. [Figure 2]This graph plots the HMGB1 binding activity of anti-HMGB1 antibodies. It shows the results of plotting 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 produced by B cells derived from gates 1, 2, and 3, specifically those whose antigen-binding ability is independent of pH and / or calcium ion concentration (gray area) and those whose antigen-binding ability is dependent on pH and / or calcium ion concentration (black area). [Figure 4-1] This figure shows the sensorgrams of Biacore anti-HMGB1 antibodies (HMG233-IgG1 and HMG236-IgG1) and human HMGB1. [Figure 4-2] This is a continuation of Figure 4-1. It shows sensorgrams of anti-HMGB1 antibodies (HMG481-IgG1 and HMG487-IgG1) and human HMG1. [Figure 5] This graph shows the results of RAGE ELISA for anti-HMGB1 antibodies (HMG233, HMG236, HMG481, HMG487, HMG446). The absorbance of the control under antibody-free conditions is set to 100, and the relative values ​​of each antibody are shown. [Figure 6] This graph shows the results of TLR4 / MD-2 ELISA for anti-HMGB1 antibodies (HMG233, HMG236, HMG481, HMG487, HMG446). The absorbance of the control under antibody-free conditions is set to 100, and the relative values ​​of each antibody are shown. [Figure 7] This figure shows the profile of human HMGB1 concentration in serum in normal mice. [Figure 8] This figure shows the serum concentration profile of anti-human HMGB1 antibody in normal mice. [Figure 9] This figure shows that antigens can be removed from plasma by using an antibody that binds to a target antigen in a pH-dependent manner and has FcRn binding activity in the neutral pH range. [Figure 10]This figure shows the sensorgrams of 6RKE02-IgG1 against hsIL-6R at pH 7.4 and pH 6.0. [Figure 11] This figure shows the results of the biological activity evaluation using human gp130-expressing BaF3 cells (BaF / gp130). [Figure 12] This figure shows the changes in plasma hsIL-6R concentration after antibody administration in an infusion study using normal mice. [Figure 13] This figure shows the protocol for the in vivo drug efficacy test (Test 1) using normal mice and the SAA inhibitory effect (mean ± SE) induced by antibody administration. [Figure 14] This figure shows the protocol for the in vivo drug efficacy test (Test 2) using normal mice and the SAA inhibitory effect (mean ± SE) induced by antibody administration. [Figure 15] This diagram illustrates a non-limited mechanism of action in which soluble antigens are eliminated from plasma upon administration of an ion-concentration-dependent antibody that has enhanced binding to the Fcγ receptor at neutral pH compared to existing neutralizing antibodies. [Figure 16] This figure shows the plasma concentration profile of human IL-6 receptor in human FcRn transgenic mice after administration of H54 / L28-IgG1 or Fv4-IgG1, which binds to the human IL-6 receptor in a pH-dependent manner. [Figure 17] This figure shows the plasma concentration profile of human IL-6 receptor in human FcRn transgenic mice after administration of Fv4-IgG1, which binds to the human IL-6 receptor in a pH-dependent manner; Fv4-IgG1-F760, a variant of Fv4-IgG1 lacking binding to mouse FcγR; Fv4-IgG1-F1022, a variant of Fv4-IgG1 with enhanced binding to mouse FcγR; or Fv4-IgG1-Fuc, a low-fucose antibody of Fv4-IgG1. [Figure 18]This figure shows the profile of human IL-6 receptor concentrations in the plasma of human FcRn transgenic mice after administration of antigen-binding molecules containing Fv4-IgG1, VH3-IgG1-F1022, and VH3-IgG1-F1093 (a modified version of VH3-IgG1-F1022 with improved binding to FcRn in the acidic pH range) as a heavy chain. [Figure 19] This figure shows the concentration profile of the administered antigen-binding molecules in the plasma of human FcRn transgenic mice when the antigen-binding molecules containing Fv4-IgG1, VH3-IgG1-F1022, and VH3-IgG1-F1093 (a modified version of VH3-IgG1-F1022 with improved binding to FcRn in the acidic pH range) as a heavy chain were administered to the mice. [Figure 20] This figure shows the plasma concentration profiles of human IL-6 receptors in human FcRn transgenic mice after administration of Fv4-IgG1, Fv4-IgG1-F1087 (a variant of Fv4-IgG1 with enhanced binding to mouse FcγR, particularly mouse FcγRIIb and mouse FcγRIII), and Fv4-IgG1-F1182 (a variant of Fv4-IgG1 with enhanced binding to mouse FcγR, particularly mouse FcγRI and mouse FcγRIV). [Figure 21] This figure shows the concentration profiles of administered antigen-binding molecules in the plasma of human FcRn transgenic mice after administration of Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified versions of Fv4-IgG1-F1087 with improved binding to FcRn in the acidic pH range. [Figure 22] This figure shows the concentration profiles of administered antigen-binding molecules in the plasma of human FcRn transgenic mice after administration of Fv4-IgG1, Fv4-IgG1-F1182, and Fv4-IgG1-F1181, a variant of Fv4-IgG1-F1182 with improved binding to FcRn in the acidic pH range. [Figure 23] This figure shows the changes in human IL-6 receptor concentration in the plasma of human FcRn transgenic mice after administration of Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified versions of Fv4-IgG1-F1087 with improved binding to FcRn in the acidic pH range. [Figure 24] This figure shows the changes in human IL-6 receptor concentration in the plasma of human FcRn transgenic mice after administration of Fv4-IgG1, Fv4-IgG1-F1182, and Fv4-IgG1-F1181, a variant of Fv4-IgG1-F1182 with improved binding to FcRn in the acidic pH range. [Figure 25] This figure shows the changes in human IL-6 receptor concentration in the plasma of normal mice after administration of Fv4-mIgG1, Fv4-mIgG1-mF44 (a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII), and Fv4-mIgG1-mF46 (another variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII). [Figure 26] This figure shows the changes in human IL-6 receptor concentration in the plasma of FcγRIII-deficient mice after administration of Fv4-mIgG1, Fv4-mIgG1-mF44 (a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII), and Fv4-mIgG1-mF46 (another variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII). [Figure 27]This figure shows the plasma concentration profiles of human IL-6 receptors in Fc receptor γ chain-deficient mice after administration of Fv4-mIgG1, Fv4-mIgG1-mF44 (a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII), and Fv4-mIgG1-mF46 (another variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII). [Figure 28] This figure shows the changes in human IL-6 receptor concentration in the plasma of FcγRIIb-deficient mice after administration of Fv4-mIgG1, Fv4-mIgG1-mF44 (a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII), and Fv4-mIgG1-mF46 (another variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII). [Figure 29] This figure shows the results of evaluating platelet aggregation ability by omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets derived from donors with FcγRIIa polymorphism (R / H). [Figure 30] This figure shows the results of evaluating platelet aggregation ability by omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets from donors with FcγRIIa polymorphism (H / H). [Figure 31] This figure shows the results of evaluating CD62p expression on the membrane surface of washed platelets. The graphs filled in black show the results when platelets were reacted with PBS and then stimulated with ADP, while the graphs that are not filled in 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 active integrins on the membrane surface of washed platelets. The graphs filled in black show the results when platelets were reacted with PBS and then stimulated with ADP, while the graphs that are not filled in 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 R-type. The binding amount of each PD variant to each FcγR was divided by the binding amount of the control antibody, IL6R-F652 (SEQ ID NO: 162) / IL6R-L (IL6R-F652 is an antibody heavy chain containing a modified Fc in which the Pro at position 238 in EU numbering has been replaced with Asp), and this value was multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The plot labeled F652 in the figure shows the value for IL6R-F652 / IL6R-L. [Figure 34] The vertical axis shows the relative binding activity to FcγRIIb of modified antibodies that have been introduced into GpH7-B3 (SEQ ID NO: 168) / GpL16-k0 (SEQ ID NO: 169) without the P238D modification. The horizontal axis shows the relative binding activity to FcγRIIb of modified antibodies that have been introduced into IL6R-F652 (SEQ ID NO: 162) / IL6R-L with the P238D modification. The relative binding activity was calculated by dividing the binding amount of each modified antibody to FcγRIIb by the binding amount of the antibody before modification, and then multiplying by 100. Here, modifications that exhibited a binding-enhancing effect on FcγRIIb when introduced into GpH7-B3 / GpL16-k0 which lacks P238D, and when introduced into IL6R-F652 / IL6R-L which has P238D, are included in region A. Modifications that exhibit a binding-enhancing effect on FcγRIIb when introduced into GpH7-B3 / GpL16-k0 which lacks P238D, but do not exhibit a binding-enhancing effect on FcγRIIb when introduced into IL6R-F652 / IL6R-L which has P238D, are included in region B. [Figure 35] This shows the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex. [Figure 36]This figure shows the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex and the model structure of the Fc (WT) / FcγRIIb extracellular domain complex superimposed on the FcγRIIb extracellular domain and Fc CH2 domain A using the least squares method based on the Cα interatomic distance. [Figure 37] This figure shows a comparison of the detailed structures near P238D by superimposing the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex and the model structure of the Fc (WT) / FcγRIIb extracellular domain complex using the least squares method based on the Cα interatomic distance for each of the Fc CH2 domains A and B individually. [Figure 38] This figure shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex, a hydrogen bond is observed between the Gly backbone at position 237, represented by the EU numbering of the Fc CH2 domain A, and the Tyr at position 160 of FcγRIIb. [Figure 39] This figure shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex, an electrostatic interaction is observed between the Asp at position 270, represented by the EU numbering of the Fc CH2 domain B, and the Arg at position 131 of FcγRIIb. [Figure 40] The horizontal axis represents the relative binding activity of each 2B variant to FcγRIIb, and the vertical axis represents the relative binding activity of each 2B variant to FcγRIIa R-type. The binding amount of each 2B variant to each FcγR was divided by the binding amount of the control antibody (a modified Fc antibody in which the Pro at position 238 in EU numbering was replaced with Asp) to each FcγR, and this value was then multiplied by 100 to obtain the relative binding activity of each 2B variant to each FcγR. [Figure 41] This diagram shows the Glu at position 233, represented by the EU numbering of Fc Chain A, and the surrounding residues in the FcγRIIb extracellular domain in the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain. [Figure 42]This diagram shows the Ala at position 330, represented by the EU numbering of Fc Chain A, and the surrounding residues in the FcγRIIb extracellular domain in the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain. [Figure 43] This figure shows the structure of Pro at position 271, represented by the EU numbering of Fc Chain B, by superimposing the crystal structures of the Fc (P238D) / FcγRIIb extracellular domain complex and the Fc (WT) / FcγRIIIa extracellular domain complex onto Fc Chain B using the least squares method based on the Cα interatomic distance. [Figure 44] This is a diagram of the Fc(P208) / FcγRIIb extracellular domain complex determined by X-ray crystallography. For the Fc portion, the left side is designated as domain A and the right side as domain B. [Figure 45] The structures of the Fc (P208) / FcγRIIb extracellular domain complex and the Fc (WT) / FcγRIIa extracellular domain complex (PDB code: 3RY6), determined by X-ray crystallography, were superimposed using the least squares method based on the Cα interatomic distance at the Fc CH2 domain A and compared. In the figure, the Fc (P208) / FcγRIIb extracellular domain complex is depicted with a thick line, and the Fc (WT) / FcγRIIa extracellular domain complex is depicted with a thin line. Note that in the structure of the Fc (WT) / FcγRIIa extracellular domain complex, only the Fc CH2 domain A is depicted. [Figure 46] This image shows the detailed structure of the X-ray crystal structure of the Fc(P208) / FcγRIIb extracellular domain complex, specifically the area around position 237 of the Fc-subordinate CH2 domain A, which forms a hydrogen bond with position 160 of FcγRIIb in the main chain, represented by EU numbering. [Figure 47]This figure shows the structure of the amino acid residues surrounding the Asp side chain at position 237, represented by EU numbering, of the Fc (P208) / FcγRIIb extracellular domain complex in the X-ray crystal structure. [Figure 48] This figure compares the X-ray crystal structures of the Fc (P238D) / FcγRIIb extracellular domain complex and the Fc (P208) / FcγRIIb extracellular domain complex shown in Reference Example 15, superimposed using the least squares method based on the Cα interatomic distance in the Fc CH2 domain B, around the loop from position 266 to 271, as represented by EU numbering. Within this loop, Fc (P208) has a modification of H268D at position 268, as represented by EU numbering, and a modification of P271G at position 271, as represented by EU numbering, compared to Fc (P238D). [Figure 49] This figure shows the structure around Ser239 of the Fc CH2 domain B in the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular domain complex, along with the electron density obtained by X-ray crystallography, with 2Fo-Fc as the coefficient. [Figure 50] This figure compares the three-dimensional structures of the Fc(P208) / FcγRIIaR extracellular domain complex and the Fc(P208) / FcγRIIb extracellular domain complex, determined by X-ray crystallography, superimposed using the least squares method based on Cα interatomic distances. [Figure 51] This figure compares the X-ray crystal structures of the Fc (P208) / FcγRIIaR extracellular domain complex and the Fc (P208) / FcγRIIb extracellular domain complex, along with the electron density obtained by X-ray crystallography, using 2Fo-Fc as a coefficient, near Asp position 237, which is represented by the EU numbering of the Fc-part CH2 domain A. [Figure 52]This figure compares the X-ray crystal structures of the Fc (P208) / FcγRIIaR extracellular domain complex and the Fc (P208) / FcγRIIb extracellular domain complex, along with the electron density obtained by X-ray crystallography, using 2Fo-Fc as a coefficient, near Asp position 237, which is represented by the EU numbering of the Fc CH2 domain B. [Figure 53] This figure compares the sequences of the constant regions of G1d and G4d. In the figure, the amino acids enclosed in thick borders indicate the sites where the amino acid residues differ between G1d and G4d. [Figure 54] This figure shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody as determined by X-ray crystallography. (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] This figure shows the changes in plasma concentrations of H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody in normal mice that were administered these antibodies. [Figure 56] This figure shows the concentration profiles of soluble human IL-6 receptor (hsIL-6R) in the plasma of normal mice administered with H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody. [Figure 57] This figure shows ion-exchange chromatograms of an antibody containing the human Vk5-2 sequence and an antibody containing the hVk5-2_L65 sequence, which is a modified version of the human Vk5-2 sequence with a modified glycosylation sequence. The solid line represents the chromatogram of the antibody containing 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 containing the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 67), light chain: hVk5-2_L65 (SEQ ID NO: 70)). [Figure 58A]These are ion-exchange chromatograms of antibodies containing the LfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55), light chain: LfVk1_Ca (SEQ ID NO: 83)) and antibodies containing a sequence in which the Asp(D) residue in the LfVk1_Ca sequence is modified to an Ala(A) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in the ion-exchange chromatogram after 5°C storage is used as the main peak, and the y-axis is normalized at the main peak. The figure also shows the chromatogram of the antibody containing LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 58B] This figure shows the chromatogram of an antibody containing LfVk1_Ca1 (SEQ ID NO: 85) as its light chain. [Figure 58C] This figure shows the chromatogram of an antibody containing LfVk1_Ca2 (SEQ ID NO: 86) as its light chain. [Figure 58D] This figure shows the chromatogram of an antibody containing LfVk1_Ca3 (SEQ ID NO: 87) as its light chain. [Figure 59A] These are ion exchange chromatograms of antibodies containing the LfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55), light chain: LfVk1_Ca (SEQ ID NO: 83)) and antibodies containing 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) of the LfVk1_Ca sequence is modified to a Ser(S) residue. These chromatograms were obtained after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in the ion exchange chromatogram after storage at 5°C was used as the main peak, and the y-axis was normalized at the main peak. The figure also shows the chromatogram of the antibody containing LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 59B] This figure shows a chromatogram of an antibody containing LfVk1_Ca6 (SEQ ID NO: 88) as its light chain. [Figure 60]This figure shows the relationship between the amino acid distribution (labeled "Library") of sequence information from 290 clones isolated from E. coli that had been introduced with an antibody gene library that binds to an antigen in a Ca-dependent manner, and the designed amino acid distribution (labeled "Design"). The horizontal axis represents the amino acid location, expressed in Kabat numbering. The vertical axis represents the ratio of the amino acid distribution. [Figure 61] This figure shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 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 anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 6RC1IgG_019 antibody under low calcium ion concentration conditions (3 μM). The horizontal axis represents time, and the vertical axis represents the RU value. [Figure 63] This figure shows the relationship between the amino acid distribution (labeled "Library") of sequence information for 132 clones isolated from E. coli into which an antibody gene library that binds to an antigen in a pH-dependent manner was introduced, and the designed amino acid distribution (labeled "Design"). The horizontal axis represents the amino acid location represented by Kabat numbering. The vertical axis represents the ratio of the amino acid distribution. [Figure 64] This figure 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 the RU value. [Figure 65] This figure 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 the RU value. [Modes for carrying out the invention]

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

[0017] In this invention, physiological activity is a general term for activity that brings about quantitative and / or qualitative changes or effects on living organisms, tissues, cells, proteins, DNA, RNA, etc., and includes activities that regulate biological functions such as metabolism, growth, reproduction, homeostasis, mental activity, and biological defense. More specifically, it includes activities that regulate cell proliferation and maturation, metabolism via the endocrine system, information transmission in the nervous system, blood circulation, wound healing, immune response, and cell migration. Physiological activity can also be rephrased as biological activity. A physiologically active substance is a substance that possesses such physiological activity. A physiologically active substance exerts its physiological activity by acting on specific biological molecules (target molecules) and causing some kind of change or effect. In this invention, a physiologically active substance is also expressed as a physiologically active antigen. In this invention, a physiologically active substance may be any substance that possesses physiological activity, but preferably it is a physiologically active polypeptide and its modifications (physiologically active peptides). Preferred target molecules for a physiologically active substance to exert its physiological activity are receptors present on the cell surface or inside cells, and the physiologically active substance transmits a signal to the cell by binding to a specific receptor, thereby exerting its physiological activity. When a physiologically active substance is converted from a precursor lacking physiological activity to a mature form possessing 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 an artificially synthesized substance. In the present invention, physiologically active substances that are considered to be one of the causes of disease in organisms (preferably humans) are preferred.

[0018] Examples of bioactive peptides include cell growth factors such as fibroblast growth factor (FGF), transforming growth factor (TGF), bone morphogenetic factor (BMP), epidermal growth factor (EGF), 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 factor (BMP); cytokines such as interferon (IFN), interleukin (IL), colony-stimulating factor (CSF), erythropoietin, and tumor necrosis factor (TNF); enzymes such as various hormones like insulin and paratyloid hormone (PTH), or proteases such as matrix metalloproteinases (MMP); and enzyme inhibitors such as TIMP (Tissue Inhibitor of Metalloprotease).

[0019] The physiologically active substance in this invention is preferably a physiologically active substance derived from mammals, and particularly preferably a physiologically active substance derived from humans.

[0020] Bioactive substances can be obtained, for example, by purification from within a living organism. They can also be produced by chemical synthesis. If the bioactive substance is a bioactive peptide, it is also possible to produce recombinant peptides using genetic engineering techniques. That is, from the amino acid sequence of the bioactive peptide or the nucleic acid sequence encoding it, a nucleic acid encoding the bioactive peptide can be synthesized by gene cloning methods or nucleic acid synthesis methods known to those skilled in the art. After inserting the nucleic acid into a known expression vector and transforming a suitable host cell, the target bioactive peptide can be purified from the host cell or culture supernatant by known methods. Purification can be performed by using multiple chromatography methods, such as conventional ion chromatography or affinity chromatography, once, multiple times in combination, or individually. Furthermore, if it possesses the original bioactivity, it is also possible to produce a partial peptide containing a part of the bioactive peptide, or a fusion peptide fused with a different polypeptide such as a peptide tag or Fc fragment. A fusion peptide can be produced by in-frame fusion of genes encoding two or more desired polypeptide fragments and inserting the fusion gene into an expression vector as described above (Sambrook J et al., Molecular Cloning 2 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] Methods for measuring the physiological activity of a bioactive substance include, for in vitro physiological activity, preparing the bioactive substance and its target molecule and detecting their binding using methods such as ELISA, FACS, or Biacore. Alternatively, it can be measured by reacting the bioactive substance with cells expressing the receptor and detecting the changes that occur in the cells (e.g., changes in cell proliferation or morphology, or changes in gene or protein expression). Furthermore, for in vivo physiological activity, it can be measured by administering the bioactive substance to an animal and observing the 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 it may be a substance other than the target molecule that binds to a substance that promotes binding to the target molecule, and then indirectly bind to the target molecule. The physiologically active substance has domains for binding to the target molecule, and it is preferable that there are multiple different binding domains on the physiologically active substance corresponding to two or more different target molecules. In particular, it is preferable that the above-mentioned binding domains are located in positions that are structurally distant from each other to the extent that the binding of the antigen-binding molecule of the present invention does not simultaneously inhibit the binding of the physiologically active substance to all target molecules.

[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 measuring a negative control). For example, "having binding activity" means that in a system in which binding activity can be measured, such as ELISA, FACS, or Biacore, the measured value is higher than the background value. In the present invention, the height of the measured value relative to the background value is preferably 2 times or more, more preferably 3 times or more, even more preferably 5 times or more, and particularly preferably 10 times or more.

[0024] In the present invention, "inhibiting activity" means that, in a system capable of measuring activity, the measured value after adding a certain substance becomes lower than the measured value before adding the 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 adding a certain substance becomes lower than the measured value before adding the substance. In the present invention, the decrease in the measured value after adding a certain substance compared to the measured value before adding the 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 activity, the measured value after adding a certain substance is 80% or more of the measured value before adding the 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 adding a certain substance is 80% or more of the measured value before adding the substance. In the present invention, the measured value after adding a certain substance is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, compared to the measured value before adding the substance (or when a negative control is added).

[0026] In the present invention, "reducing the antigen concentration in plasma" means that when the antigen-binding molecule of the present invention is administered to a target, the concentration of the antigen present in the plasma decreases more in the former than in the latter when administered to a negative control. 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 the antigen from the plasma," "reducing the residence time of the antigen in the plasma," or "shortening the plasma half-life of the antigen." Plasma may also refer to serum. Administration of the antigen-binding molecule provided by the present invention to a target (living organism) can be carried out by, for example, intradermal injection, intravenous injection, intravitreous injection, subcutaneous injection, intraperitoneal injection, parenteral injection, intramuscular injection, etc. The target to which the antigen-binding molecule of the present invention is administered is preferably an animal, more preferably a mammal, and even more preferably a human.

[0027] A decrease in antigen concentration may be achieved by promoting the uptake of the antigen into cells. Furthermore, it is preferable that the physiological activity of the antigen in the body is reduced by the decrease in antigen concentration, and it is particularly preferable that all types of physiological activity of the antigen are reduced.

[0028] The concentration of an antigen can be measured by appropriately using methods known to those skilled in the art. If the antigen is a bioactive peptide, the concentration of the bioactive peptide in the sample of unknown concentration can be measured by preparing a standard of known concentration and a system capable of quantitatively measuring it (e.g., ELISA or Biacore), and creating a standard curve representing the relationship between each concentration and the measured value. Examples of samples include biological plasma, cell culture medium, or cell extracts.

[0029] In this invention, the uptake of an antigen into cells means that the antigen is taken into cells by endocytosis. Whether the uptake of an antigen into cells has been promoted can be determined, for example, by measuring whether the concentration of the antigen in the cell culture medium has decreased compared to the control, or whether the concentration of the antigen in the cells has increased compared to the control, after adding the antigen to the cell culture medium. Promoted uptake of an antigen into cells means that the elimination of the antigen from the plasma in the body is promoted. Therefore, whether the uptake of an antigen into cells has been promoted can also be determined, for example, by measuring whether the concentration of the antigen in the plasma has decreased compared to the control, after administering the antigen to the body.

[0030] The antigen-binding molecule provided by the present invention is not particularly limited as long as it has the characteristics shown in (1) to (6) above, but is preferably a polypeptide having the property of specifically binding to an antigen, human FcRn, and human Fc receptor, more preferably an antibody, and particularly preferably IgG. The antibody may be a chimeric antibody, a humanized antibody, a human antibody, etc. It may also be a bispecific antibody, an antibody modified by attaching various molecules, or a polypeptide containing an antibody fragment. The antigen-binding molecule provided by the present invention includes an antigen-binding domain and a receptor-binding domain. Here, a domain means a constituent unit that can be divided and isolated, but its size is not particularly limited. Each domain contains a polypeptide. The antigen-binding domain in the present invention is not particularly limited as long as it has the property of specifically binding to an antigen, but preferred examples include antibodies and their fragments (variable region, Fab, F(ab')2, Fv, CDR, etc.), antibody-like molecules called scaffolds (DARPins (WO2002 / 020565), Affibody (WO1995 / 001937), Avimer (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925), etc.), or target molecules (receptors) or their fragments (soluble receptors) that bind to physiologically active substances. A particularly preferred example is the variable region of an antibody. Furthermore, the receptor-binding domain in 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 their fragments (constant region, Fc, etc.), albumin and its fragments (domain 3), anti-FcRn antibodies and their fragments (variable region, Fab, F(ab')2, Fv, CDR, etc.), anti-FcRn antibody-like molecules (DARPins (WO2002 / 020565), Affibody (WO1995 / 001937), Avimer (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925), etc.), and anti-FcRn peptides. A more preferred example is the Fc region of IgG.The human Fc receptor in this invention is not particularly limited as long as it is a receptor to which the Fc region of human IgG binds, but is preferably a human FcRn or human Fcγ receptor. The IgG may be derived from a non-human animal or from humans, but is preferably human IgG, and particularly preferably human IgG1. It is preferable that the amino acids in the Fc region of IgG are modified as described below.

[0031] The antigen-binding molecule of the present invention only needs to contain at least one receptor-binding domain. For example, one 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 one antigen-binding molecule contains multiple receptor-binding domains, these receptor-binding domains may all bind to the same type of human Fc receptor, or they may each bind to different types of human Fc receptors. On the other hand, having activity to bind to human FcRn is one of the requirements that the antigen-binding molecule of the present invention must satisfy, so it is preferable that at least one receptor-binding domain included in the antigen-binding molecule of the present invention is a domain that binds to human FcRn. While not particularly limited, examples of cases in which one antigen-binding molecule contains two receptor-binding domains include an antigen-binding molecule in which both are domains that bind to human FcRn, and an antigen-binding molecule in which one is a domain that binds to human FcRn and the other is a domain that binds to human Fcγ receptors. Furthermore, if a single antigen-binding molecule contains a single receptor-binding domain, that receptor-binding domain only needs to be a domain that binds to at least 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] In this specification, "natural human IgG" refers to naturally occurring human IgG, and it is desirable that a fucose-containing glycan is bound to the EU numbering position 297 of the Fc region. Naturally occurring human IgG1, IgG2, IgG3, or IgG4 may be used as the natural human IgG, but naturally occurring human IgG1 is preferred. Whether or not the bound glycan is a fucose-containing glycan can be determined by, for example, the following method: The glycan 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 concentrated dry product of the reaction solution from which proteins have been removed by reacting with ethanol (Schenk et al., J. Clin. Investigation (2001) 108 (11) 1687-1695) is fluorescently labeled with 2-aminopyridine (Bigge et al., Anal. Biochem. (1995) 230 (2) 229-238). The fluorescently labeled 2-AB-conjugated glycans, which have been removed by solid-phase extraction using a cellulose cartridge, are analyzed by normal-phase chromatography. By observing the peaks in the detected chromatogram, it is possible to determine whether the glycans bound to the Fc region of the human IgG sample are fucose-containing glycans.

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

[0034] Humanized antibodies, also called reshaped human antibodies, are antibodies derived from non-human mammals, such as mouse antibodies, in which the complementarity determining region (CDR) of a human antibody has been transplanted. Methods for identifying CDRs are well 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 transplanting CDRs are also well 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 within the same antibody molecule that recognize different epitopes. A bispecific antibody may recognize two or more different antigens, or it may recognize 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 (dAb) (WO2004 / 058821, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, and Fc fusion proteins. Any molecule containing an Fc region can use that Fc region as a receptor-binding domain. The Fc region refers to the heavy chain constant region in an antibody molecule, from the N-terminus of the hinge region of the papain cleavage site, including the hinge, CH2, and CH3 domains. In IgG, the Fc region, in EU numbering, means, for example, from cysteine ​​position 226 to the C-terminus, or from proline position 230 to the C-terminus, but is not limited to these. While not particularly limited, examples of the Fc region of IgG 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). Preferably, it is the Fc region of human IgG1.

[0037] Antibody-like molecules (scaffold molecules) are a general term for molecules that have a common skeletal structure and the property of specifically binding 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 include DARPins (WO2002 / 020565), Affibody (WO1995 / 001937), Avimer (WO2004 / 044011, WO2005 / 040229), and Adnectin (WO2002 / 032925).

[0038] Antibodies may contain modified sugar chains. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (e.g., WO99 / 54342), antibodies lacking fucose to be attached to the sugar chain (e.g., WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913), and antibodies having sugar chains with bisecting GlcNAc (e.g., WO02 / 79255).

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

[0040] The present invention provides an antigen-binding molecule characterized by inhibiting one or more of the binding activities of an antigen to target molecules while maintaining the binding activity of at least one of those target molecules. Specifically, it is preferable that when the antigen-binding molecule of the present invention binds to an antigen having two or more types of physiological activity, one or more of the physiological activities of the antigen are inhibited. Inhibiting one or more types of physiological activity (or having physiological activity inhibited) means inhibiting the binding activity of one or more of the multiple types of target molecules that the antigen has binding activity to. Furthermore, even when the antigen-binding molecule of the present invention binds to an antigen having two or more types of physiological activity, it is preferable that at least one of the physiological activities of the antigen is maintained (or has its physiological activity maintained). Maintaining at least one type of physiological activity (or having its physiological activity maintained) means maintaining the binding activity of at least one of the multiple types of target molecules that the antigen has binding activity to.

[0041] When an excess of a physiologically active antigen in the body causes certain diseases, molecules that inhibit the physiological activity of the antigen by binding to it, such as neutralizing antibodies, are considered useful in treating those diseases. However, if an antigen has two or more physiological activities, neutralizing antibodies can only inhibit one type of physiological activity. On the other hand, the antigen-binding molecule of the present invention can reduce in vivo physiological activity by promoting the elimination of the antigen from the blood (serum or plasma), even if at least one type of physiological activity is maintained. Therefore, it is far more useful than general neutralizing antibodies.

[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 an Fc region modifier in which at least one amino acid in the Fc region of IgG has been modified. IgG may be derived from non-human animals or from humans, but is preferably human IgG (IgG1, IgG2, IgG3, IgG4), and particularly preferably human IgG1. Examples of amino acid modification include amino acid substitution, insertion, deletion, etc., but amino acid substitution is preferred. The number of amino acids to be modified is not particularly limited; only one amino acid may be modified, or two or more amino acids may be modified. Amino acid modification is permitted as long as the modified Fc region modifier has activity to bind to human FcRn under acidic and neutral pH conditions, and its activity to bind to human FcRn under neutral pH conditions is stronger than that of human IgG. Generally, it is known that in living organisms, the pH outside cells (e.g., in plasma) is neutral, and the pH inside cells (e.g., inside endosomes) is acidic. Furthermore, it is known that the binding of IgG and FcRn is observed only under acidic (intracellular) conditions and is hardly observed under neutral (extracellular) conditions. The antigen-binding molecule of the present invention preferably has an acidic pH that corresponds to the pH inside endosomes and a neutral pH that corresponds to the pH in plasma.

[0043] If the receptor-binding domain of the antigen-binding molecule provided by the present invention can be conferred with properties such as having activity to bind to human FcRn under intracellular and extracellular pH conditions, and having stronger activity to bind to human FcRn than human IgG under extracellular pH conditions, then the antigen-binding molecule of the present invention, which has bound to the antigen extracellularly, will further bind to FcRn on the cell surface and move into the cell, thereby promoting the uptake of the antigen from extracellular to intracellular. By administering such an antigen-binding molecule to a living organism, it is possible to reduce the concentration of the antigen present in the plasma and reduce the physiological activity of the antigen in vivo, making the antigen-binding molecule provided by the present invention useful.

[0044] Human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides and has 22 to 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) of β2 microglobulin and a transmembrane α-chain (or heavy chain). The α-chain of FcRn consists of three extracellular domains (α1, α2, 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 are registered in GenBank under accession numbers NM_001136019 (sequence number: 16) and NP_001129491 (sequence number: 17), respectively. For non-human organisms, the gene sequence and amino acid sequence of mouse FcRn are registered in GenBank under accession numbers NM_010189 (sequence number: 18) and NP_034319 (sequence number: 19), and the gene sequence and amino acid sequence of rat FcRn are registered in GenBank under accession numbers NM_033351 (sequence number: 20) and NP_203502 (sequence number: 21), respectively.

[0046] Human FcRn (SEQ ID NO: 17) forms a complex with human β2-microglobulin (SEQ ID NO: 38) in vivo. The complex of soluble human FcRn and β2-microglobulin can be produced using a conventional recombinant expression method. The binding activity of the receptor-binding domain of the present invention can be evaluated using such a soluble human FcRn / β2-microglobulin complex. In the present invention, unless otherwise specified, human FcRn refers to a form that can bind to the receptor-binding domain of the present invention, and an example is the complex of human FcRn and human β2-microglobulin.

[0047] A receptor-binding domain that binds more strongly to FcRn than natural human IgG under neutral pH conditions can be created 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, an antigen-binding domain characterized by binding to FcRn can also be used as the receptor-binding domain. Whether the binding activity of the receptor-binding domain to FcRn is higher than that of the Fc region of natural human IgG can be appropriately determined using the method described above.

[0048] In the present invention, the binding activity to human FcRn under acidic pH conditions means the human FcRn binding activity at pH 4.0 to pH 6.5. Preferably, it means the human FcRn binding activity at pH 5.0 to pH 6.5, and more preferably, it means the human FcRn 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, or 6.5, and particularly preferably, it means the human FcRn binding activity at pH 5.8 to pH 6.0, which is close to the pH in early endosomes in living organisms. Furthermore, in the present invention, the binding activity to human FcRn under neutral pH conditions means the human FcRn binding activity at pH 6.7 to pH 10.0. Preferably, this refers to human FcRn binding activity at pH 7.0 to pH 9.0, more preferably at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, and particularly preferably at human FcRn binding activity at pH 7.4, which is close to the pH of plasma in vivo.

[0049] If the binding affinity between the receptor-binding domain and human FcRn is very low at pH 7.4, making it difficult to accurately measure the affinity, pH 7.0 can be used instead. The binding affinity between the receptor-binding domain and human FcRn may be measured at any temperature between 10°C and 50°C as the temperature used for the measurement conditions. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between the receptor-binding domain and human FcRn. More preferably, any of the following temperatures are used: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C. While not particularly limited, 25°C is one preferred temperature.

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

[0051] [Table 1]

[0052] Table 2 also shows examples of modifications that can strengthen binding to human FcRn under acidic pH conditions compared to natural human IgG. Of these modifications, those that can strengthen binding to human FcRn even under neutral pH conditions can be appropriately selected and used in the present invention. When modifying the Fc region of IgG, particularly preferred modification locations include, for example, EU numbering 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, 332nd, 334th, 338th, 339th, 340th, 341st, 343rd, 345th, 360th, 361st, 362nd, 375th, 376th, 377th, 378th, 380th, 382nd, 384th, 385th, 386th, The amino acids at positions 387, 389, 390, 391, 413, 422, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 437, 438, 440 and 442 can be cited. In addition, preferred positions other than those listed above include the EU numbering positions 252, 254, 256, 309, 311, 315, as described in WO1997 / 034631.The 433rd and / or 434th amino acids, as well as the 253rd, 310th, 435th and / or 426th amino acids combined with these amino acids, EU numbering as described in WO2000 / 042072: 238th, 252nd, 253rd, 254th, 255th, 256th, 265th, 272nd, 286th, 288th, 303rd, 305th, 307th, 309th, 311th, 312th, 317th, 340th, 356th, 360th, 362nd, 376th, 378th, 380th, 382nd, 386th, 388th, 400th, 413th, 415th, Amino acids 424, 433, 434, 435, 436, 439 and / or 447, EU numbering 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and / or 436, EU numbering 250, 314 and 428, EU numbering 238, 244, 245, 249, as described in WO2006 / 020114 252nd, 256th, 257th, 258th, 260th, 262nd, 270th, 272nd, 279th, 283rd, 285th, 286th, 288th, 293rd, 307th, 311th, 312th, 316th, 317th, 318th, 332nd, 339th, 341st, 343rd, 375th, 376th, 377th, 378th, 380th, 382nd, 423rd, 427th, 430th, 431st, 434th, 436th, 438th, The 440th and / or 442nd amino acids, EU numbering as described in WO2010 / 045193, 251st, 252nd, 307th, 308th, 378th,Examples include amino acids at positions 428, 430, 434, and / or 436. By modifying at least one amino acid selected from these, human FcRn binding activity under neutral pH conditions can be enhanced. The number of amino acids modified is not particularly limited; only one amino acid may be modified, or two or more amino acids may be modified.

[0053] [Table 2]

[0054] Furthermore, a receptor-binding domain that already possesses human FcRn binding activity under conditions in the acidic and neutral pH ranges is, for example, the Fc region of IgG, specifically 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 either 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. The 252nd amino acid 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 either 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 either 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 either 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 either 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 328th amino acid is Arg, Asp, Glu, or Tyr. The 329th amino acid is either 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 either 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. The 389th amino acid is Ala, Asn, Pro, or Ser. The 390th amino acid is Ala, the amino acid at position 391 is Ala, the amino acid at position 413 is Ala, the amino acid at position 423 is Asn, the amino acid at position 424 is Ala or Glu, the amino acid at position 427 is Asn, the amino acid at position 428 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 amino acid at position 431 is His or Asn, the amino acid at position 433 is Arg, Gln, His, Ile, Pro, Ser or Lys, the amino acid at position 434 is Ala, Phe, Gly, Met, His, Ser, Trp or Tyr, the amino acid at position 435 is Lys, Arg or Asn, the amino acid at position 436 is Ala, His, Ile, Leu, Glu, Phe, Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp or Val, the amino acid at position 437 is Arg, the amino acid at position 438 is Lys, Leu, Thr or Trp, the amino acid at position 440 is Lys, and the amino acid at position 442 is Lys, an Fc region in which amino acids are selected from the above is mentioned. The position of the selected amino acid may be only one position, or may be two or more positions. Examples of combinations of two or more amino acids include combinations of amino acids described 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] In this specification, "higher activity to bind to human FcRn than natural human IgG" means, for example, that the activity to bind to human FcRn is 105% or more of natural human IgG, 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.

[0062] These amino acid modifications can be carried out as appropriate using known techniques, for example, as seen 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 Modifications to the Fc region of human IgG1 were made in 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.

[0063] According to Yeung et al. (The Journal of Immunology, 2009 182: 7663-7671), the human FcRn binding activity of human IgG1 under acidic pH conditions (pH 6.0) is KD 1.7 μM, and the human FcRn binding activity of human IgG1 under neutral pH conditions is almost undetectable. Therefore, a preferred embodiment of the antigen-binding molecule provided by the present invention is an antigen-binding molecule in which the human FcRn binding activity under acidic pH conditions is KD 20 μM or stronger, and the human FcRn binding activity under neutral pH conditions is the same as or stronger than that of human IgG. A more preferred embodiment is an antigen-binding molecule in which the human FcRn binding activity under acidic pH conditions is KD 2.0 μM or stronger, and the human FcRn binding activity under neutral pH conditions is KD 40 μM or stronger. A more preferred embodiment is an antigen-binding molecule that exhibits human FcRn binding activity with a KD of 0.5 μM or stronger under acidic pH conditions and human FcRn binding activity with a KD of 15 μM or stronger under neutral pH conditions. The KD values ​​shown here are those measured using the method described in The Journal of Immunology, 2009 182: 7663-7671 (immobilizing the antigen-binding molecule on a chip and flowing human FcRn as an analyte).

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

[0065] While it is possible to use the KD (dissociation constant) as a value for human FcRn binding activity, it is difficult to calculate it as KD because the human FcRn binding activity of human IgG is hardly observed under neutral pH conditions (pH 7.4). One method to determine whether the human FcRn binding activity at pH 7.4 is higher than that of human IgG is to judge by the magnitude of the binding response in Biacore when analytes are flowed at the same concentration. That is, if the response when human FcRn is flowed through a chip immobilized with the antigen-binding molecule provided by the present invention is greater than the response when human FcRn is flowed through a chip immobilized with human IgG, then it can be determined that the human FcRn binding activity of that antigen-binding molecule is higher than that of human IgG.

[0066] An Fcγ receptor (FcγR) is a receptor that can bind to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), and essentially refers to any member of the Fcγ receptor family. In humans, this family includes, but is not limited to, FcγRI(CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; FcγRIII(CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR isoform or allotype. The organisms from which FcγR originates include, but are not limited to, humans, mice, rats, rabbits, and monkeys, and may originate from any organism. Mouse FcγR receptors include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(FcγRIV,CD16-2), as well as any undiscovered mouse FcγR 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 assigned to 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 assigned to SEQ ID NOs: 41 (BC020823.1) and 42 (AAH20823.1), respectively (allotype R131 is the sequence in which the 166th amino acid of SEQ ID NO: 42 is substituted with Arg), and the polynucleotide sequence of FcγRIIb is assigned to 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 assigned to SEQ ID NOs: 41 (BC020823.1) and 42 (AAH20823.1) (allotype R131 is the sequence in which the 166th amino acid of SEQ ID NO: 42 is substituted with Arg), and the polynucleotide sequence of FcγRIIb is assigned to 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 assigned to SEQ ID NOs: 41 (BC020823.1) and 42 (AAH20823.1), respectively (allotype R131 is the sequence in which the 166th amino acid of SEQ ID NOs: 42 is substituted with Arg), and the polynucleotide sequence of FcγRIIb is assigned to FcγRIIb The amino acid sequences of FcγRIIIa are described in SEQ ID NOs: 43 (BC146678.1) and 44 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIa are described in SEQ ID NOs: 45 (BC033678.1) and 46 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIb are described in SEQ ID NOs: 47 (BC128562.1) and 48 (AAI28563.1), respectively (the numbers in parentheses indicate the RefSeq registration numbers). Whether or not the Fcγ receptor has binding activity to the Fc region of IgG can be confirmed by FACS, ELISA, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), and BIACORE, which utilizes the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0067] Furthermore, the term "Fc ligand" or "effector ligand" refers to a molecule derived from any organism, preferably a polypeptide, that binds to the Fc region of an antibody to form a complex. Binding of an Fc ligand to the Fc region preferably induces one or more effector functions. 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. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors homologous to FcγR (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands also include undiscovered molecules that bind to Fc.

[0068] FcγRI (CD64) comprising FcγRIa, FcγRIb and FcγRIc, and FcγRIII (CD16) comprising isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) are each composed of two subunits: an α chain that binds to the Fc region of IgG, and a common γ chain having an ITAM that transduces activation signals into cells. On the other hand, the intracellular domains of FcγRII (CD32) comprising isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc contain ITAMs. These receptors are expressed on many immune cells including macrophages, mast cells, and antigen-presenting cells. Activation signals transduced by the binding of these receptors to the Fc region of IgG promote the phagocytic capacity of macrophages, production of inflammatory cytokines, degranulation of mast cells, and activation of antigen-presenting cells. Fcγ receptors having the ability to transduce 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 transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses the activation signal from the BCR, resulting in suppression of antibody production by B cells. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic activity 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 this invention.

[0070] Receptor-binding domains that bind more strongly to the Fcγ receptor than natural human IgG under neutral pH conditions can be created 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, an antigen-binding domain characterized by binding to the Fcγ receptor can also be used as a receptor-binding domain. Examples of such receptor-binding domains include Fab fragments that bind to FcγRIIIa, camel-derived single-domain antibodies and single-chain Fv, 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, as well as FcγRI-binding cyclic peptides described in FASEB J. 2009 Feb;23(2):575-85. Whether the binding activity of the receptor-binding domain to the Fcγ receptor is higher than the binding activity of the Fc region of natural human IgG to the Fcγ receptor can be appropriately determined using the method described above.

[0071] In the present invention, binding activity to human Fcγ receptors under acidic pH conditions means human Fcγ receptor binding activity at pH 4.0 to pH 6.5. Preferably, it means human Fcγ receptor binding activity at pH 5.0 to pH 6.5, and more preferably, it means 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, or 6.5, and particularly preferably, it means human Fcγ receptor binding activity at pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Furthermore, in the present invention, binding activity to human Fcγ receptors under neutral pH conditions means human Fcγ receptor binding activity at pH 6.7 to pH 10.0. Preferably, this refers to human Fcγ receptor binding activity at pH 7.0 to pH 9.0, more preferably at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, and most preferably at human Fcγ receptor binding activity at pH 7.4, which is close to the pH of plasma in vivo.

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

[0073] A suitable example of a receptor-binding domain in the present invention is the Fc region of human IgG. The origin of the Fc region is not limited, but it can be obtained from any non-human animal or from humans. 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 may 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 specific class of IgG. That is, the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the receptor-binding domain. Examples of naturally occurring or artificially modified IgG variants are described in, but are not limited to, the publicly available 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).

[0074] Any amino acid can be modified as long as the receptor-binding domain binds more strongly to the Fcγ receptor than natural human IgG under neutral pH conditions. When the receptor-binding domain is created by modifying the Fc region of human IgG1, amino acid modifications to enhance binding activity to the Fcγ receptor 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 modifying the Fc region of IgG to form a receptor-binding domain that binds to the human Fcγ receptor, preferred amino acids are, for example, the 221st, 222nd, 223rd, 224th, 225th, 227th, 228th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 247th, 249th, 250th, 251st, 252nd, 254th, 255th, 256th, 257th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 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,These are at least one amino acid selected from groups 440 and 442. Modifications of these amino acids enhance the binding activity of the IgG Fc region to the Fcγ receptor under neutral pH conditions.

[0076] A particularly preferred modification for enhancing binding to the Fcγ receptor under neutral pH conditions is, for example, the EU numbering of the Fc region; The 221st amino acid is Lys or Tyr, The 222nd amino acid is Phe, Trp, Glu, or Tyr. The 223rd amino acid is Phe, Trp, Glu, or Lys. The 224th amino acid 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, The 254th amino acid 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 262nd amino acid 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. The 273rd amino acid 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 either 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 320th amino acid 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 either 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. The 428th amino acid is Phe or Leu, The 429th amino acid is Met, The 434th amino acid is Trp. The 436th amino acid is Ile, and The 440th amino acid is Gly, His, Ile, Leu, or Tyr. This includes the modification of at least one amino acid selected from the group. Furthermore, the number of amino acids to be modified is not particularly limited; only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of modifications of two or more amino acids are those listed 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] In this specification, "higher activity to bind to human Fcγ receptor than native human IgG" means that the activity of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb to bind to human Fcγ receptor is higher than that of native human IgG. For example, this means that the activity binding to the human Fcγ receptor is 105% or more of that of natural human IgG, 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.

[0081] The receptor-binding domain in the present invention may have the property of having higher binding activity to a specific Fcγ receptor than to other Fcγ receptors (selectively binding to a specific Fcγ receptor). An example of such a receptor-binding domain is one in which binding activity to inhibitory Fcγ receptors is higher than binding activity to active Fcγ receptors. Preferably, the receptor-binding domain has higher binding activity to the inhibitory Fcγ receptor FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) than the binding activity to an active Fcγ receptor selected from any of the following: FcγRI(CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc. Particularly preferred are receptor-binding domains that exhibit higher binding activity to FcγRIIb-1 and / or FcγRIIb-2 than 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, the value obtained by dividing the KD value of the receptor-binding domain for the active Fcγ receptor by the KD value for the inhibited Fcγ receptor is 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 3 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, 35 or higher, 40 or higher, 45 or higher, 50 or higher, 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher. 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 or higher, 420 or higher, 430 or higher, 440 or higher, 450 or higher, 460 or higher, 470 or higher, 480 or higher, 490 or higher, 500 or higher, 520 or higher, 540 or higher, 560 or higher, 580 or higher, 600 or higher, 620 or higher, 640 or higher, 660 or higher, 680 or higher, 700 or higher, 720 or higher, 740 or higher, 760 or higher, 780 or higher, 800 or higher, 820 or higher, 840 or higher, 860 or higher, 880 or higher, 900 or higher, 920 or higher, 940 or higher, 960 or higher, 980 or higher If the values ​​are 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 100000 or more, the receptor-binding domain can be judged to selectively bind to inhibitory Fcγ receptors rather than active Fcγ receptors.

[0083] While not particularly limited, examples of receptor-binding domains that exhibit higher binding activity to inhibitory Fcγ receptors than to active Fcγ receptors (selectively binding to inhibitory Fcγ receptors) include Fc regions in which the 238th and / or 328th amino acids, as represented by EU numbering, are modified to different amino acids, and more preferably, Fc regions in which the 238th amino acid is modified to Asp and / or the 328th amino acid is modified to Glu. Suitable examples include IgG Fc region variants described in WO2012 / 115241. Furthermore, IgG Fc region variants described in US2009 / 0136485 can also be appropriately selected.

[0084] In addition to the above modification of the Fc region of IgG, at least one further modification may be made, preferably resulting in enhanced binding activity to FcγRIIb and maintenance or reduction of binding activity to FcγRIIa (allotype H131) and FcγRIIa (allotype R131). Such modifications improve binding selectivity to FcγRIIb compared to FcγRIIa. Modifications that improve binding selectivity to FcγRIIb compared to FcγRIIa (allotype R131) are preferred, and modifications that improve binding selectivity to FcγRIIb compared to both FcγRIIa (allotype R131) and FcγRIIa (allotype H131) are even more preferred. Examples of such modifications, though not particularly limited, include EU numbering; The 233rd amino acid is Asp, The 234th amino acid is Trp or Tyr, The 235th amino acid 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, The 238th amino acid 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 either 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. The 331st amino acid 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. This includes modifications of at least one amino acid selected from the group.

[0085] Furthermore, among these, a desirable example of modification is 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. This includes modifications of at least one amino acid selected from the group.

[0086] The above modifications may be made in one place or in combination of two or more places. Preferred examples of such modifications include those listed in Tables 24-25, 27-34, and 36-37.

[0087] As one embodiment of the receptor-binding domain contained in the antigen-binding molecule of the present invention, although not particularly limited, examples include modified 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). An example of such a modified Fc region is the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) where the 238th amino acid represented by the EU numbering is Asp and the 271st amino acid is Gly. The Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) where the 238th amino acid represented by the EU numbering is Asp and the 271st amino acid is Gly, and the antigen-binding molecule containing said Fc region, have higher binding activity to inhibitory Fcγ receptors than to active Fcγ receptors.

[0088] The present invention may also involve at least one further modification to the Fc region, in which the 238th amino acid, represented by EU numbering, is Asp and the 271st amino acid is Gly, preferably resulting in enhanced binding activity to FcγRIIb-1 and / or FcγRIIb-2, and maintenance or reduction of binding activity to FcγRIIa (allotype H131) and FcγRIIa (allotype R131). 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 active 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)). By making such modifications, the binding selectivity to FcγRIIb is improved compared to FcγRIIa.

[0089] As an example of a selective receptor-binding domain, though not particularly limited, the 238th amino acid in the EU numbering of the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) is modified to Asp, and the 271st amino acid is modified to Gly, and the 233rd, 234th, 237th, 244th, 245th, 249th, 250th, 251st, 252nd, 254th, 255th, 256th, 257th, 258th, 260th, 262nd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 272nd, 279th, 283rd, 285th, 286th, 288th, 293rd, 296th, 307th, 308th, 309th, 311th, 312th, 314th, 316th, 317th, 318th, 326th, 327th, 330th, 331st, 332nd, 333rd, 339th, 341st, 343rd, 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 position 440 and 442 are modified.

[0090] Furthermore, as an example of a selective receptor-binding domain, although not particularly limited, it is possible to modify the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4) by changing the 238th amino acid to Asp and the 271st amino acid to Gly, and the 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, The 266th amino acid is Phe, Met, or Leu. The 267th amino acid is Ala, Glu, Gly, or Gln. The 268th amino acid is either 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 either Ala or Asp. The 327th amino acid is Gly, The 330th amino acid is either 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. An example is an Fc region in which one or more amino acids selected from the group have been modified.

[0091] While not particularly limited, examples of the above-mentioned Fc region include the Fc regions described 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] Preferably, the receptor-binding domain that binds to the human Fcγ receptor described above includes further amino acid modifications that enhance binding to FcRn under acidic pH conditions. Such modifiable amino acids include, for example, the 252nd, 254th, 256th, 309th, 311th, 315th, 433rd and / or 434th amino acids with EU numbering as described in WO1997 / 034631, as well as the 253rd, 310th, 435th and / or 426th amino acids to be combined with these, and the 238th, 252nd, 253rd, 254th, 255th, 256th, 265th, 272nd, 286th, 288th, 303rd, 305th, 307th, 309th, 311th, 312th, 317th, 340th, 356th, 360th amino acids with EU numbering as described in WO2000 / 042072. The 362nd, 376th, 378th, 380th, 382nd, 386th, 388th, 400th, 413th, 415th, 424th, 433rd, 434th, 435th, 436th, 439th and / or 447th amino acids, as listed in EU numbering WO2002 / 060919, the 251st, 252nd, 254th, 255th, 256th, 308th, 309th, 311th, 312th, 385th, 386th, 387th, 389th, 428th, 433rd, The 434th and / or 436th amino acid, the 250th, 314th and 428th amino acids with EU numbering as described in WO2004 / 092219, the 238th, 244th, 245th, 249th, 252nd, 256th, 257th, 258th, 260th, 262nd, 270th, 272nd, 279th, 283rd, 285th, 286th, 288th, 293rd, 307th, 311th, 312th, 316th, 317th, 318th, 332nd, 339th, 341st, 343rd,Examples include amino acids 375, 376, 377, 378, 380, 382, ​​423, 427, 430, 431, 434, 436, 438, 440 and / or 442, and amino acids 251, 252, 307, 308, 378, 428, 430, 434 and / or 436, as described in WO2010 / 045193. Modifications of these amino acids enhance the binding of the Fc region of IgG to FcRn under acidic pH conditions.

[0099] More specifically, an example of such a modification is 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. The 252nd amino acid 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 either 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 either Ala or Leu. The 316th amino acid is Lys, The 317th amino acid is Pro, The 318th amino acid is Asn or Thr. The 332nd amino acid 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. The 382nd amino acid 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 428th amino acid 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 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. The 438th amino acid is Lys, Leu, Thr, or Trp. The 440th amino acid is Lys, and The 442nd amino acid is Lys, This includes modifications of at least one amino acid selected from the group.

[0100] An example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modified formulations containing Ile at amino acid position 308, Pro at amino acid position 309, and / or Glu at amino acid position 311. Modified versions containing 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: amino acid 308 is Ile or Thr, amino acid 309 is Pro, amino acid 311 is Glu, Leu or Ser, amino acid 312 is Ala, and / or amino acid 314 is Ala or Leu, Modified versions 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. This includes at least one modification selected from the group.

[0101] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modified versions containing Leu at the 251st amino acid, Tyr at the 252nd amino acid, Ser or Thr at the 254th amino acid, Arg at the 255th amino acid, and / or Glu at the 256th amino acid. These are some examples.

[0102] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modifications including: amino acid 428 being Leu, Met, Phe, Ser or Thr; amino acid 433 being Arg, Gln, His, Ile, Lys, Pro or Ser; amino acid 434 being His, Phe or Tyr; and / or amino acid 436 being Arg, Asn, His, Lys, Met or Thr; Modifications in which the 428th amino acid is His or Met, and / or the 434th amino acid is His or Met, This includes at least one modification selected from the group.

[0103] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modifications including one in which the 385th amino acid is Arg, the 386th amino acid is Thr, the 387th amino acid is Arg, and / or the 389th amino acid is Pro, Modified versions containing Asp at amino acid 385, Pro at amino acid 386, and / or Ser at amino acid 389. This includes at least one modification selected from the group.

[0104] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modifications including Gln or Glu at the 250th amino acid, and Modified version containing Leu or Phe at amino acid position 428. This includes at least one modification selected from the group.

[0105] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modifications including Gln at amino acid 250 and / or Leu or Phe at amino acid 428, and Modified versions containing Glu at the 250th amino acid and / or Leu or Phe at the 428th amino acid. This includes at least one modification selected from the group.

[0106] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modified versions containing Gln at amino acid position 307 and Ala or Ser at amino acid position 434. Modified form containing Pro at amino acid 308 and Ala at amino acid 434. Modified version containing Tyr at amino acid 252 and Ala at amino acid 434. Modified version containing Val at amino acid 378 and Ala at amino acid 434. Modified form containing Leu at amino acid position 428 and Ala at amino acid position 434. Modified version containing Ala at amino acid position 434 and Ile at amino acid position 436. Modifications include Pro at amino acid 308 and Tyr at amino acid 434, and Modified form containing Gln at amino acid 307 and Ile at amino acid 436. This includes at least one modification selected from the group.

[0107] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modified form containing Gln at amino acid position 307, Ala at amino acid position 380, and Ser at amino acid position 434. Modified form containing Gln at amino acid position 307, Ala at amino acid position 380, and Ala at amino acid position 434. Modifications including Tyr at amino acid 252, Pro at amino acid 308, and Tyr at amino acid 434, and Modified version containing Asp at amino acid 251, Gln at amino acid 307, and His at amino acid 434. This includes at least one modification selected from the group.

[0108] In addition to the above, an example of a modification that can enhance binding to human FcRn under acidic pH conditions compared to human IgG is EU numbering; Modified versions containing Ile at amino acid position 257 and Ile at amino acid position 311. Modifications include one in which amino acid 257 is Ile and amino acid 434 is His, and Modified version containing Val at amino acid 376 and His at amino acid 434. This includes at least one modification selected from the group.

[0109] If the antigen-binding molecule of the present invention is prepared 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 the same subclass of IgG as a control. As appropriate, the Fc regions of human IgG can be used as control molecules: human IgG1 (SEQ ID NO: 49, RefSeq registration number AAC82527.1 with an A added to the N-terminus), human IgG2 (SEQ ID NO: 50, RefSeq registration number AAB59393.1 with an A added to the N-terminus), human IgG3 (SEQ ID NO: 51, RefSeq registration number CAA27268.1), and human IgG4 (SEQ ID NO: 52, RefSeq registration number AAB59394.1 with an A added to the N-terminus).

[0110] In mice, four types of Fcγ receptors have been identified to date: FcγRI(CD64), FcγRIIb(CD32), FcγRIII(CD16), and FcγRIV(CD16-2, or FcγRIII-2). Similar to humans, FcγRIIb is considered to be the only inhibitory Fcγ receptor. 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] To date, it has been reported that FcγRIIb dysfunction and decreased expression are correlated with the development of autoimmune diseases in humans. For example, in some SLE patients, there are reports of decreased FcγRIIb expression due to weakened binding of transcription activators caused by gene polymorphisms in the promoter region of FcγRIIb expression (Hum. Genet. (2005) 117, 220-227, J. Immunol. (2004) 172, 7192-7199, J. Immunol. (2004) 172, 7186-7191). In addition, two types of gene polymorphisms have been reported in SLE patients, where the 233rd amino acid of FcγRIIb is either Ile or Thr. This region 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 be present in the lipid draft compared to when it is Ile, resulting in a decrease in FcγRIIb signaling function (Nat. Med. (2005) 11, 1056-1058, Hum. Mol. Genet., (2005) 14, 2881-2892). In mice, knockout mice in which the FcγRIIb gene of C57BL / 6 mice is disrupted have been reported to exhibit SLE-like symptoms such as the production of autoantibodies and glomerulonephritis (Immunity 13 (2000) 277-285, J. Exp. Med. (2002) 195, 1167-1174). Furthermore, decreased FcγRIIb expression has been reported in mice, which have been considered a spontaneous model of SLE (Immunogenetics (2000) 51, 429-435, Int. Immunol. (1999) 11, 1685-1691, Curr. Biol. (2000) 10, 227-230, J. Immunol. (2002) 169, 4340-4346). From these findings, it is thought that FcγRIIb regulates humoral immunity in mice, just as it does in humans.

[0112] When the antibody having the Fc region of the present invention eliminates an antigen via FcγRIIb, the endocytotic function of FcγRIIb is considered to be the most important contributing factor among the functions of FcγRIIb. As mentioned above, there are two splicing variants of FcγRIIb, FcγRIIb1 and FcγRIIb2, but it has been reported that the latter is mainly involved in the endocytosis of the antibody-antigen immune complex (J. Immunol. (1994), 152 574-585, Science (1992) 256, 1808-1812, Cell (1989) 58, 317-327). To date, it has 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 a 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 also suggests that FcγRIIb2 possesses endocytic activity in humans, just as it does in mice.

[0113] On the other hand, unlike FcγRIIb2, FcγRIIb1 has been reported not to induce endocytosis. FcγRIIb1 has an insertion sequence in its intracellular domain that is not found in FcγRIIb2. It is thought that this sequence inhibits the uptake of FcγRIIb1 into clathrin-coated pits, and as a result, endocytosis is inhibited (J. Cell. Biol. (1992) 116, 875-888, J. Cell. Biol. (1989) 109, 3291-3302). In humans, as in mice, FcγRIIb1 also has an insertion sequence, so it is expected that a similar mechanism causes the difference in endocytic ability between FcγRIIb1 and FcγRIIb2. Furthermore, it has been reported that in both humans and mice, approximately 40% of immune complexes on the cell surface are taken into the cell within 20 minutes (Mol. Immunol. (2011) 49, 329-337, Science (1992) 256, 1808-1812). From this, it is expected that FcγRIIb2 takes up immune complexes into cells in humans at a similar rate to that in mice.

[0114] Among the Fcγ receptor family, FcγRIIb is the only one that possesses an intracellular ITIM in both humans and mice, and the distribution of cells expressing it is identical, suggesting that its function in regulating immunity is also similar. Furthermore, considering that immune complexes are taken up into cells at a similar rate in both humans and mice, it is thought that using mice can predict the effect of antibody-mediated antigen elimination via FcγRIIb in humans. In fact, in the examples described later, 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 when mIgG1 was administered.

[0115] Furthermore, in the examples described later, similar experiments were conducted using Fc receptor γ chain-deficient mice. In mice, it has been reported that FcγRs other than FcγRIIb are only expressed in the presence of the γ chain; therefore, only FcγRIIb is expressed in Fc receptor γ chain-deficient mice. By administering mF44 and mF46 to Fc receptor γ chain-deficient mice, it becomes possible to investigate the effect on antigen elimination when selective binding activity of FcγRIIb is enhanced. The results from 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. In addition, the results from the examples revealed that mF44 and mF46 eliminated the antigen to almost the same extent when administered to Fc receptor γ chain-deficient mice as when administered to normal mice.

[0116] Furthermore, in the examples described later, similar experiments were conducted using FcγRIII-deficient mice. Since mIgG1, mF44, and mF46 bind only to FcγRIIb and FcγRIII among the mouse FcγRs, administering these antibodies to FcγRIII-deficient mice allows for consideration of the effect on antigen clearance when selective binding activity to FcγRIIb is enhanced. The results from the examples showed that mF44 and mF46 administered to FcγRIII-deficient mice increased antigen clearance compared to mIgG1 administered to the same mice. In addition, the results from the examples revealed that mF44 and mF46, when administered to FcγRIII-deficient mice, caused antigen clearance to almost the same extent as when administered to normal mice and Fc receptor γ chain-deficient mice.

[0117] These results revealed that while binding activity to the active Fcγ receptor is not enhanced, selective binding activity to FcγRIIb is selectively enhanced, thereby accelerating antigen elimination.

[0118] In addition to the literature reports discussed above, the results of the mouse-based verification described above suggest that, in human vivo, the uptake of immune complexes via FcγRIIb occurs in the same way as in mice. As a result, antibodies with an Fc region that selectively enhances binding activity to human FcγRIIb can accelerate antigen elimination. Furthermore, as discussed earlier, since the uptake of immune complexes via FcγRIIb occurs at a similar rate in mice and humans, it is thought that an effect comparable to that of antibodies with an Fc region that enhances affinity to mouse FcγRIIb in accelerating antigen elimination can be achieved by antibodies with an Fc region that enhances affinity to human FcγRIIb.

[0119] The Kabat numbering system is generally used when referring to residues in the variable region of an antibody (approximately residues 1-107 of the light chain and residues 1-113 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 constant region of the heavy chain of an antibody (e.g., Kabat et al., the EU index reported above). "Kabat's EU index" means the residue numbering of 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, for example, WO2006 / 073941).

[0120] The antigen-binding domain in the present invention is characterized by having different antigen-binding activity under intracellular and extracellular conditions. Intracellular and extracellular conditions refer to conditions that differ inside and outside the cell within a living organism. Examples of condition categories include ion concentration, more specifically hydrogen ion concentration (pH) and calcium ion concentration. Intracellular conditions are preferably the environment characteristic of the inside of an endosome, and extracellular conditions are preferably the environment characteristic of plasma.

[0121] Antigen-binding domains that exhibit changes in antigen-binding activity depending on ion concentration conditions can be obtained by screening for such domains from a large number of antigen-binding domains. For example, if the antigen-binding molecule of the present invention is an antibody, antibodies with the above-mentioned properties can be obtained by producing a large number of antibodies with different sequences using hybridoma or antibody library methods, and measuring their antigen-binding activity under different ion concentration conditions. The B cell cloning method exemplified in Example 1 of this specification is particularly suitable as a method for screening such antibodies. Furthermore, as will be described later, by identifying at least one characteristic amino acid residue that can confer the property of changing antigen-binding activity depending on ion concentration to an antigen-binding domain, and by producing a library of a large number of antigen-binding domains with different sequences that share such a characteristic amino acid residue as a common structure, antigen-binding domains with the above-mentioned properties can be efficiently screened from such a library.

[0122] In one aspect of the present invention, the ion concentration condition refers to the hydrogen ion concentration condition or the pH condition. In the present invention, the condition of the concentration of protons, i.e., the nuclei of hydrogen atoms, is treated as synonymous with the condition of the hydrogen ion concentration index (pH). If the activity of hydrogen ions in an aqueous solution is represented by aH+, then pH is defined as -log10aH+. The ionic strength in the aqueous solution is (for example, 10 -3If the value is lower, aH+ is approximately equal to the hydrogen ion 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, pH=7 is neutral, aqueous solutions with a pH less than 7 are acidic, and aqueous solutions with a pH greater than 7 are alkaline. In the present invention, when pH is used as the ion concentration condition, it is desirable that the binding activity to the antigen under acidic pH conditions (i.e., high hydrogen ion concentration or low pH) is lower than the binding activity to the antigen under neutral pH conditions (i.e., low hydrogen ion concentration or high pH).

[0123] The pH inside a cell is more acidic than outside the cell, and conversely, the pH outside a cell is more neutral than inside the cell. The present invention provides an antigen-binding molecule in which the extracellular conditions are in the pH neutral range and the intracellular conditions are in the pH acidic range. In the present invention, the preferred pH acidic range is pH 4.0 to pH 6.5, more preferably pH 5.0 to pH 6.5, and 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, or 6.5, and particularly preferably pH 5.8 to pH 6.0, which is close to the pH in early endosomes in living organisms. Furthermore, in the present invention, the preferred pH neutral range is pH 6.7 to pH 10.0, more preferably pH 7.0 to pH 9.0, and 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, or 8.0, with a particularly preferred pH of 7.4, which is close to the pH in plasma (blood).

[0124] When comparing the strength of the antigen-binding activity of the antigen-binding domain provided by the present invention under conditions in the acidic pH range and the neutral pH range, it is preferable that binding is stronger under the neutral pH range than under the acidic pH range. When the strength of binding activity is expressed by 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. The upper limit of the value of KD(acidic pH) / KD(neutral pH) is not particularly limited, and any value such as 100, 400, 1000, 10000, etc., is acceptable as long as it can be produced by 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 by the magnitude of the binding response when analytes are flowed at the same concentration in Biacore. When an antigen is passed through a chip on which the antigen-binding molecule provided by the present invention is immobilized, the binding response under acidic pH 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 neutral pH conditions.

[0125] Generally, it is known that in living organisms, the pH outside cells (e.g., in plasma) is neutral, while the pH inside cells (e.g., inside endosomes) is acidic. If the antigen-binding domain of the antigen-binding molecule provided by the present invention can be conferred with properties such that its activity to bind to antigens is weaker under intracellular pH conditions compared to extracellular pH conditions, then antigens bound to the antigen-binding molecule of the present invention outside cells will dissociate from the antigen-binding molecule of the present invention inside cells, and as a result, the uptake of antigens from outside cells into cells will be promoted. By administering such an antigen-binding molecule to a living organism, it becomes possible to reduce the concentration of antigens present in plasma and reduce the physiological activity of the antigens in vivo, making the antigen-binding molecule provided by the present invention useful.

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

[0127] In alanine scanning methods known to those skilled in the art, histidine can be randomly substituted and / or inserted into the antigen-binding domains of antigen-binding molecules provided by the present invention by methods such as histidine scanning, in which alanine is replaced with histidine. From these, antigen-binding domains that bind weakly to the antigen under acidic pH conditions compared to neutral pH conditions can be selected.

[0128] The number of histidine substitutions and / or insertions can be appropriately determined by those skilled in the art. Histidine may be substituted at only one location, or inserted at only one location. Alternatively, histidine may be substituted at two or more locations, or inserted at two or more locations. Furthermore, a combination of histidine substitution and insertion at two or more locations may be performed.

[0129] The antigen-binding domain preferably has equivalent antigen-binding activity before and after histidine substitution and / or insertion. Equivalent activity here means 10% or more, preferably 30% or more, more preferably 50% or more, more preferably 80% or more, and particularly preferably 90% or more of the original activity.

[0130] If the antigen-binding molecule provided by the present invention includes an antibody constant region, one method for conferring to the antigen binding property provided by the present invention such that it binds weakly to the antigen under acidic pH conditions compared to neutral pH conditions is to modify the antibody constant region. For example, one method for modifying the antibody constant region is to compare the isotypes of the constant region (IgG1, IgG2, IgG3, IgG4) and select the isotype that has lower binding activity to the antigen under acidic pH conditions (faster dissociation rate under acidic pH conditions). Another method is to introduce amino acid modifications into the amino acid sequences of the isotypes (IgG1, IgG2, IgG3, IgG4) to reduce binding activity to the antigen under acidic pH conditions (faster dissociation rate under acidic pH conditions). The hinge region of the antibody constant region differs significantly depending on the isotype (IgG1, IgG2, IgG3, IgG4). Differences in the amino acid sequence of the hinge region greatly affect antigen binding activity. Therefore, by selecting the appropriate isotype based on the type of antigen to bind, it is possible to reduce antigen binding activity under acidic pH conditions. Furthermore, when introducing amino acid modifications into the isotype's amino acid sequence, the hinge region is considered a desirable location for these modifications.

[0131] By creating a library of numerous antigen-binding domains that have different sequences but share a common structure containing amino acid residues that change antigen-binding activity depending on the hydrogen ion concentration conditions, and then screening from this library, it is possible to efficiently obtain antigen-binding domains that have activity to bind to a desired antigen and whose antigen-binding activity changes depending on the hydrogen ion concentration conditions.

[0132] For example, by combining a light chain variable region that includes at least one amino acid residue in its framework sequence that changes antigen-binding activity depending on hydrogen ion concentration conditions with a heavy chain variable region having a random sequence, a library can be created that contains multiple antigen-binding domains with different sequences, while all having an amino acid residue that changes antigen-binding activity depending on hydrogen ion concentration conditions as a common structure. In a preferred embodiment, when such amino acid residues are introduced into a light chain modification region, they may be included in CDR1 of the light chain modification region, and more preferably, they may be included in positions 24, 27, 28, 31, 32 and / or 34 as represented by the Kabat numbering of CDR1 of the light chain variable region. In another preferred embodiment, they may be included in CDR2 of the light chain variable region, and more preferably, they may be included in positions 50, 51, 52, 53, 54, 55 and / or 56 as represented by the Kabat numbering of 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, and more preferably in positions 89, 90, 91, 92, 93, 94 and / or 95A as represented by the Kabat numbering of the CDR3 of the light chain variable region. Such amino acid residues may be contained individually or in combination of two or more, as long as the antigen-binding activity is altered by the hydrogen ion concentration conditions.

[0133] When constructing 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 the hydrogen ion concentration conditions with a heavy chain variable region having a random sequence, it is also possible to design the light chain variable region to include flexible residues. The number and position 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 the hydrogen ion concentration conditions. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the light chain. While not particularly limited, suitable examples of flexible residues introduced into the light chain variable region sequence include the amino acid residues listed in Table 7 or Table 8. Furthermore, while not particularly limited, 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) can be suitably used as sequences of the light chain variable region other than amino acid residues and flexible residues whose antigen-binding activity changes depending on the hydrogen ion concentration conditions.

[0134] [Table 7] (The position represents the Kabat numbering.)

[0135] [Table 8] (The position represents the Kabat numbering.)

[0136] Any amino acid residue can be suitably used as an amino acid residue that changes antigen-binding activity depending on the hydrogen ion concentration conditions, but specifically, amino acid residues with a side chain pKa of 4.0-8.0 are examples of such electron-donating amino acids, including natural amino acids such as histidine or glutamic acid, as well as unnatural amino acids such as histidine analogs (US20090035836) or 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). Furthermore, particularly suitable examples of such amino acid residues include those with a side chain pKa of 6.0-7.0. Histidine is a suitable example of such an electron-donating amino acid.

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

[0138] Furthermore, the calcium ion concentration is lower inside cells than outside cells, and conversely, the calcium ion concentration is higher outside cells than inside cells. In the present invention, preferred low calcium ion concentrations 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. Also in the present invention, preferred high calcium ion concentrations 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, it is preferable that the low calcium ion concentration is the calcium ion concentration in endosomes, and the high calcium ion concentration is the calcium ion concentration in plasma.

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

[0140] When the strength of binding activity is expressed by KD (Dissociation constant), the value of KD(low calcium ion concentration) / KD(high calcium ion concentration) is greater than 1, preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD(low calcium ion concentration) / KD(high calcium ion concentration) is not particularly limited and any value such as 100, 400, 1000, 10000, etc., is acceptable as long as it can be produced by 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 by the magnitude of the binding response when analytes are flowed at the same concentration in Biacore. When an antigen is flowed through a chip immobilized with the antigen-binding molecule provided by the present invention, 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] Generally, it is known that in living organisms, the calcium ion concentration is high extracellularly (e.g., in plasma) and low intracellularly (e.g., within endosomes). Therefore, in the present invention, it is preferable that the extracellular conditions are under high calcium ion concentration conditions and the intracellular conditions are under low calcium ion concentration conditions.

[0142] If the antigen-binding domain of the antigen-binding molecule provided by the present invention can be conferred with properties such that its activity to bind to the antigen is weaker under intracellular calcium ion concentration conditions compared to extracellular calcium ion concentration conditions, then the antigen bound to the antigen-binding molecule of the present invention outside the cell will dissociate from the antigen-binding molecule of the present invention inside the cell, and as a result, the uptake of the antigen from outside the cell into the cell will be promoted. By administering such an antigen-binding molecule to a living organism, it becomes possible to reduce the concentration of the antigen present in the plasma and reduce the physiological activity of the antigen in vivo, making the antigen-binding molecule provided by the present invention useful.

[0143] As a method for screening antigen-binding domains or antigen-binding molecules that exhibit lower antigen-binding activity under low calcium ion concentration conditions compared to high calcium ion concentration conditions, the method described in WO2012 / 073992, etc. (e.g., paragraphs 0200-0213) is exemplified.

[0144] The method for conferring the property of weaker binding to the antigen under low calcium ion concentration conditions compared to high calcium ion concentration conditions to the antigen-binding domain provided by the present invention is not particularly limited and may be carried out by any method. Specifically, as described in Japanese Patent Application No. 2011-218006, for example, one method is to replace at least one amino acid residue in the antigen-binding domain with an amino acid residue having metal-chelating activity, and / or to insert at least one amino acid residue having metal-chelating activity. An antigen-binding molecule provided by the present invention in which at least one amino acid residue in the antigen-binding domain is replaced with an amino acid residue having metal-chelating activity, and / or to insert at least one amino acid residue having metal-chelating activity is one preferred embodiment of the antigen-binding molecule provided by the present invention. Suitable examples of amino acid residues having metal-chelating activity include serine, threonine, asparagine, glutamine, aspartic acid, or glutamic acid.

[0145] Furthermore, as examples of amino acid residues that alter the antigen-binding activity of the antigen-binding domain depending on the calcium ion concentration, amino acid residues that form calcium-binding motifs can be preferably cited. 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, troponin C, calmodulin, parvalbumin, the EF domain contained in myosin light chains, the C2 domain contained in Protein kinase C, the Gla domain contained in the blood coagulation protein FactorIX, asialoglycoprotein receptors and mannose-binding receptors, ASGPR, CD23, C-type lectins contained in DC-SIGN, the A domain, annexin domain, cadherin domain, thrombospondin type 3 domain and EGF-like domain contained in LDL receptors can be suitably used as calcium-binding motifs. In addition to the above, the calcium-binding motif contained in the antigen-binding domain described in Sequence ID No. 57 can also be suitably used.

[0146] The antigen-binding domain of the present invention may include amino acid residues that change their antigen-binding activity depending on the calcium ion concentration, such as amino acid residues having metal chelating activity or amino acid residues forming calcium-binding motifs. The position of the antigen-binding domain containing such amino acid residues is not particularly limited and may be any position as long as it changes the antigen-binding activity depending on the calcium ion concentration. Furthermore, as long as it changes the antigen-binding activity depending on the calcium ion concentration, such amino acid residues may be included individually or in combination of two or more. Suitable examples of such amino acid residues include serine, threonine, asparagine, glutamine, aspartic acid, or glutamic acid. If the antigen-binding domain is a variable region of the antibody, these amino acid residues may be included in the heavy chain variable region and / or the light chain variable region. In a preferred embodiment, these amino acid residues may be included in the CDR3 of the heavy chain variable region, and more preferably in positions 95, 96, 100a, and / or 101 as represented by the Kabat numbering of the 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, and more preferably in positions 30, 31 and / or 32 as represented by 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, and more preferably in position 50 as represented by 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, and more preferably in position 92 as represented by the Kabat numbering of CDR3 of the light chain variable region.

[0148] Furthermore, 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, and more preferably, it may be contained in one or more of the 30th, 31st, 32nd, 50th, and / or 92nd positions represented by the Kabat numbering of the light chain variable region.

[0149] By creating a library of numerous antigen-binding domains that have different sequences but share a common structure containing amino acid residues that change antigen-binding activity depending on the calcium ion concentration conditions, and then screening from this library, it is possible to efficiently obtain antigen-binding domains that have activity to bind to a desired antigen and whose antigen-binding activity changes depending on the calcium ion concentration conditions.

[0150] When the antigen-binding domain is the variable region of an antibody, a particularly preferred embodiment is one in which the framework sequences of the light chain and / or heavy chain variable regions have a framework sequence of human germline. Therefore, if the framework sequence is entirely human in one embodiment of the present invention, it is considered that the antigen-binding domain of the present invention will cause little to no immunogenic reaction when administered to a human (e.g., for the treatment of a disease). In this sense, in the present invention, "having a germline sequence" means that a part of the framework sequence of the present invention is identical to a part of the framework sequence of any human germline. For example, even if the antigen-binding domain of the present invention has a sequence that combines framework sequences of multiple different human germlines, it is still considered an antigen-binding domain of the present invention that "has a germline sequence".

[0151] While this invention is not bound by any particular theory, one reason why the use of germline sequences is expected to eliminate harmful immune responses in most individuals is thought to be as follows: As a result of affinity maturation steps that occur during normal immune responses, somatic mutations frequently occur in the variable regions of immunoglobulins. These mutations mainly occur around CDRs, whose sequences are hypervariable, but also affect residues in framework regions. Since these framework region mutations are not present in germline sequences, they may exhibit immunogenicity 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 potential for immune tolerance, the genes encoding the variable regions can be selected from a set of functional (normally expressed) germline genes.

[0152] Examples of frameworks include sequences of currently known fully human-type framework regions, such as those 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 included 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 is classified into 7 subgroups, Vλ, which is classified into 10 subgroups, and VH, which is classified into 7 subgroups.

[0153] Fully human-type VH sequences are not limited to those listed below, but include, for example, 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), and the VH3 subgroup (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-3) VH sequences such as 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), VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61), VH5 subgroup (VH5-51), VH6 subgroup (VH6-1), and VH7 subgroup (VH7-4, VH7-81) are preferably mentioned. These are also described in publicly available literature (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)), and those skilled in the art can appropriately design the antigen-binding domain of the present invention based on this sequence information. Other fully human-type framework regions or framework sub-regions can also be suitably used.

[0154] Completely human-type VK sequences are not limited to those listed below, but for example, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, O18 are classified in the Vk1 subgroup, and A1, A2, A3, A5, A7, A17, A are classified in the Vk2 subgroup. Preferred examples include 18, A19, A23, O1, O11, A11, A27, L2, L6, L10, L16, L20, L25 classified in Vk3 subgroup, B3 classified in Vk4 subgroup, B2 classified in Vk5 subgroup (also referred to as Vk5-2 in this specification)), A10, A14, A26 classified in VK6 subgroup, etc. (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] Fully human-type VL sequences are not limited to those listed below, but for example, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, V1-22, which are classified as VL1 subgroups, and V2-1, V2-6, V2-7, V2- Suitable examples include V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V3-2, V3-3, V3-4 (classified in the VL3 subgroup), V4-1, V4-2, V4-3, V4-4, V4-6 (classified in the VL4 subgroup), and V5-1, V5-2, V5-4, V5-6 (classified in the VL5 subgroup) (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).

[0156] Typically, these framework sequences differ from one or more amino acid residues. These framework sequences can be used in conjunction with at least one amino acid residue that alters antigen-binding activity under the aforementioned ion concentration conditions. Examples of frameworks, though not limited to these, include 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 includes at least one amino acid residue whose antigen-binding activity changes depending on the calcium ion concentration, with a heavy chain variable region having a random sequence, a library can be created containing multiple antigen-binding domains that have different sequences but share a common structure of amino acid residues whose antigen-binding activity changes depending on the calcium ion concentration. While not particularly limited, a library of antigen-binding domains combining a light chain variable region belonging to the Vk5-2 family, represented by SEQ ID NO: 57 (Vk5-2), with a heavy chain variable region having a random sequence is a suitable example. Alternatively, a library of antigen-binding domains combining a light chain variable region sequence in which specific amino acid residues in 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) are replaced with at least one amino acid residue whose antigen-binding activity changes depending on the calcium ion concentration, with a heavy chain variable region having a random sequence is also a suitable example.

[0158] Furthermore, it is possible to design the light chain variable region, which includes at least one amino acid residue in its framework sequence that alters antigen-binding activity depending on calcium ion concentration conditions, to also include flexible residues. The number and position 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 conditions. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the heavy chain variable region and / or light chain variable region. Examples of flexible residues introduced into the light chain variable region described in Sequence ID No. 57 (Vk5-2) include the amino acid residues listed in Table 9 or Table 10.

[0159] [Table 9]

[0160] [Table 10]

[0161] In this specification, a flexible residue refers to an amino acid residue located in a position 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 antibody or antigen-binding domains. These highly diverse positions are generally located in the CDR region. When determining these highly diverse positions in known and / or natural antibodies, data provided by, for example, Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful. Additionally, several databases on the internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html) provide sequences of numerous collected human light and heavy chains, and this sequence information is useful for determining these highly diverse positions in the present invention. According to the present invention, if a certain position contains 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, and preferably 10 to 12 different amino acids, then that position can be said to be very diverse. Furthermore, a certain position can 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, and preferably at least about 12 different amino acids.

[0162] When constructing 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 the 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 include flexible residues. The number and position of flexible residues are not particularly limited, as long as the antigen-binding activity changes depending on the calcium ion concentration. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the light chain. While not particularly limited, preferred examples of flexible residues introduced into the light chain variable region include the amino acid residues listed in Table 9 or Table 10.

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

[0164] In another embodiment, a synthetic library in which any CDR sequence in the V gene in genomic DNA or in a reconstructed functional V gene is replaced with a set of synthetic oligonucleotides encoding a codon set of appropriate length is suitably 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 introduce diversity in the amino acid residues at positions exposed on the surface of the antigen-binding molecule. Positions exposed on the surface refer to positions that are judged to be possible to be exposed on the surface and / or to be able to contact the antigen, based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and in the variable region, these are generally CDRs. Positions exposed on the surface 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). Furthermore, the exposed positions on the surface can be determined using algorithms known in the art (e.g., Lee and Richards (J.Mol.Biol. (1971) 55, 379-400), Connolly (J.Appl.Cryst. (1983) 16, 548-558)). Additionally, the exposed positions on the surface can be determined using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. SYBYL Biomolecular Module Software (Tripos Associates) is a suitable example of software for this purpose. If the algorithm requires a user input size parameter, the "size" of the probe used in the calculation is generally set to a radius of approximately 1.4 angstroms or less. Furthermore, methods for determining exposed areas and regions on a surface using personal computer software are described in Pacios (Comput.Chem. (1994) 18 (4), 377-386 and J.Mol.Model. (1995) 1, 46-53).

[0165] Furthermore, in another embodiment, naive libraries constructed from antibody genes derived from lymphocytes of healthy individuals are also particularly suitable as randomized variable region libraries (Gejima et al. (Human Antibodies (2002) 11, 121-129), and Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)). Since the repertoire of antibody sequences derived from lymphocytes of healthy individuals does not contain bias, high diversity can be expected. In this invention, an amino acid sequence containing a naive sequence refers to an amino acid sequence obtained from such a naive library.

[0166] In one aspect of the present invention, the antigen-binding domain of the present invention can be obtained from a library containing multiple antigen-binding domains with different sequences, prepared by combining a heavy chain variable region containing at least one amino acid residue that changes antigen-binding activity depending on calcium ion concentration conditions with a light chain variable region having a random sequence. While not particularly limited, a library of antigen-binding domains combining the heavy chain variable region described 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 is a preferred example. Alternatively, a light chain variable region having a germline sequence may be appropriately selected and used instead of a light chain variable region having a random sequence. While not particularly limited, a library of antigen-binding domains combining the heavy chain variable region described 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 is a preferred example.

[0167] Furthermore, it is possible to design the heavy chain variable region, which includes at least one amino acid residue that changes antigen-binding activity depending on the calcium ion concentration conditions, to also include flexible residues. The number and position of such 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 the calcium ion concentration conditions. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. Although not particularly limited, examples of flexible residues introduced into the heavy chain variable region described in SEQ ID NO: 117 (6RL#9-IgG1) include all amino acid residues of heavy chain CDR1 and heavy chain CDR2, as well as amino acid residues other than positions 95, 96 and / or 100a of heavy chain CDR3. Also, examples of flexible residues introduced into the heavy chain variable region described in SEQ ID NO: 119 (6KC4-1#85-IgG1) include all amino acid residues of heavy chain CDR1 and heavy chain CDR2, as well as amino acid residues other than positions 95 and / or 101 of heavy chain CDR3.

[0168] Furthermore, a library containing multiple antigen-binding domains with different sequences can also be prepared by combining a heavy chain variable region into which at least one amino acid residue that changes antigen-binding activity depending on the calcium ion concentration conditions is introduced, with a light chain variable region having a random sequence or a light chain variable region having a germline sequence. As an example, a library of antigen-binding domains is preferably provided in which a heavy chain variable region in which a specific amino acid residue of the heavy chain variable region is replaced with at least one amino acid residue that changes antigen-binding activity depending on the calcium ion concentration conditions is combined with a light chain variable region having a random sequence or a light chain variable region having a germline sequence. Examples of such amino acid residues, though not particularly limited, include amino acid residues contained in heavy chain CDR1, amino acid residues contained in heavy chain CDR2, and amino acids at positions 95, 96, 100a and / or 101 of heavy chain CDR3. These amino acid residues may be included individually or in combination of two or more, as long as they form a calcium-binding motif and / or their antigen-binding activity changes depending on the calcium ion concentration conditions.

[0169] Even when combining a heavy chain variable region into which at least one amino acid residue that alters antigen-binding activity depending on the calcium ion concentration conditions is introduced, with a light chain variable region having a random sequence or a light chain variable region having a germline sequence, it is also possible to design the heavy chain variable region to include flexible residues. As long as the antigen-binding activity of the antigen-binding domain of the present invention changes depending on the calcium ion concentration conditions, the number and position of flexible residues are not particularly limited. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the heavy chain. Furthermore, the amino acid sequences of CDR1, CDR2 and / or CDR3 of the heavy chain variable region other than the amino acid residue that alters antigen-binding activity depending on the calcium ion concentration conditions may be randomized sequences as in the synthetic library described above. When a germline sequence is used as the light chain variable region, there are no particular limitations, but 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) are preferred examples.

[0170] For the modification of amino acids 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 employed. In addition, several known methods can be employed for modifying 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 tRNA to which a non-natural amino acid is bound as a complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, 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, and flow cytometers. Specifically, in the case of Biacore, the antigen-binding molecule containing the antigen-binding domain provided by the present invention can be immobilized on a chip, and the KD value can be measured by flowing the antigen through it as an analyte. By performing the measurement under conditions in the acidic pH range and the neutral pH range, it is possible to calculate the KD (acidic pH) / KD (neutral pH) value, and by performing the measurement under conditions in the low calcium ion concentration range and the high calcium ion concentration range, it is possible to calculate the KD (low calcium ion concentration) / KD (high calcium ion concentration) value.

[0172] In the antigen-binding domain provided by the present invention, the property that the activity of binding to an antigen changes depending on the ion concentration conditions may be simultaneously met under multiple conditions. For example, the antigen-binding domain provided by the present invention may have the property that the activity of binding to an antigen is lower under acidic pH conditions compared to neutral pH conditions, and that the activity of binding to an antigen is lower under low calcium ion concentration conditions compared to high calcium ion concentration conditions.

[0173] Specific examples of antigens having two or more physiological activities 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 β, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, anti-Id, antithrombin III, anthrax, apoA1, apo-serum amyloid A, apo-SAA, β-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 TissueGrowth 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, hapten (NP-cap or NIP-cap), HB-EGF, HCMV gB coat glycoprotein, Hep B gp120, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, hepcidin, Herpes simplex virus (HSV) gB glycoprotein, HIV coat proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HMGB1 (High Mobility Group Box 1), HSP47, Hsp90, HSV gD glycoprotein, human cytomegalovirus (HCMV), human serum albumin, human tissue plasminogen activator (t-PA), IFN-α, IFN-β, IFN-γ, IgE, IGF, immunoglobulin immune complex, immunoglobulin, influenza, inhibin, inhibin α, inhibin β, laminin 5, incubation-related 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, non-structural protein type 3 derived from hepatitis C virus(NS3), Oncostatin M, Osteopontin, Oxidized LDL, Polyglycol chains of different sizes (e.g., PEG-20, PEG-30, PEG-40), Prekallikrein, Prion protein, Procalcitonin, Proinsulin, Prolactin, Proprotein convertase PC9, Prorelaxin, Polynuclear Respiratory 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 Panspecific (TGF-β Pan Examples of specific 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 (TGF) 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 sequence and amino acid sequence of human HMGB1 are registered in GenBank under accession numbers NM_002128 (sequence number: 22) and NP_002119 (sequence number: 23), respectively. For non-human organisms, the gene sequence and amino acid sequence of mouse HMGB1 are registered in GenBank under accession numbers NM_010439 (sequence number: 24) and NP_034569 (sequence number: 25), and the gene sequence and amino acid sequence of rat HMGB1 are registered in GenBank under accession numbers NM_012963 (sequence number: 26) and NP_037095 (sequence number: 27), respectively.

[0175] The gene sequence and amino acid sequence of human CTGF are registered in GenBank under accession numbers NM_001901 (sequence number: 28) and NP_001892 (sequence number: 29), respectively. For non-human organisms, the gene sequence and amino acid sequence of mouse CTGF are registered in GenBank under accession numbers NM_010217 (sequence number: 30) and NP_034347 (sequence number: 31), and the gene sequence and amino acid sequence of rat CTGF are registered in GenBank under accession numbers NM_022266 (sequence number: 32) and NP_071602 (sequence number: 33), respectively.

[0176] The gene sequence for the human IgE constant region is registered under GenBank accession number L00022 (sequence number: 34), and the gene sequence for the mouse IgE constant region is registered under GenBank accession number X01857 (sequence number: 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 have been reported to include DNA, RNA, LPS (Lipopolysaccharide), IL-1β (Interleukin-1β), CXCL12 (chemokine (CXC motif) Ligand 12), and nucleosomes. Examples of preferred 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 and FcεRII have been reported as target molecules to which IgE binds. These are also preferred as examples of target molecules in the present invention.

[0180] This invention provides polynucleotides that encode antigen-binding molecules. Polynucleotides are mainly composed of DNA, RNA, and other nucleic acid analogs.

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

[0182] The present invention provides host cells that hold the vector provided by the present invention. The host cells that can be used are not particularly limited, and for example, Escherichia coli and various animal cells can be suitably used. The host cells can be used, for example, as a production system for the production and expression of the antigen-binding molecule 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] Examples of eukaryotic cells that can be used as host cells include 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, Vero, etc., amphibian cells such as African clawed frog oocytes (Valle et al., Nature (1981) 291: 338-340), and insect cells such as Sf9, Sf21, and Tn5. Preferably, CHO-DG44, CHO-DX11B, COS7, HEK293, and BHK are used. CHO is particularly preferred when the goal is high-level expression. For introducing the vector into host cells, methods known to those skilled in the art can be used, such as the calcium phosphate method, the DEAE dextran method, the cationic ribosome DOTAP method (manufactured by Boehringer Mannheim), electroporation, lipofection, and microinjection. Furthermore, the Free Style 293 Expression System (manufactured by Invitrogen) can be used to perform gene transfer and polypeptide expression.

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

[0185] When using prokaryotic cells, there are production systems that utilize bacterial cells. Examples of bacterial cells known to be used in protein production systems include Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis.

[0186] On the other hand, examples of systems for producing polypeptides in vivo include production systems using animals and production systems using plants. The target polynucleotide is introduced into these animals or plants, causing them to produce polypeptides within their bodies, which are then recovered. In this invention, the term "host" encompasses these animals and plants.

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

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

[0189] Furthermore, when using plants to produce the antigen-binding molecules provided by the present invention, for example, tobacco can be used. When using tobacco, a polynucleotide encoding the target antigen-binding molecule 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 obtained from the leaves of this tobacco plant (Ma et al., Eur. J. Immunol. (1994) 24: 131-8). Alternatively, a similar bacterium can be used to infect duckweed (Lemna minor), and after cloning, the desired antigen-binding molecule can be obtained 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 host cells or extracellularly (e.g., culture medium, milk) and purified as substantially pure and homogeneous molecules. The separation and purification of the antigen-binding molecules provided by the present invention can be performed using any separation and purification method commonly used for the purification of polypeptides, and is not limited in any way. For example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc., can be appropriately selected and combined for separation and purification.

[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 chromatography methods 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 (manufactured by Pharmacia).

[0192] If necessary, the antigen-binding molecule provided by the present invention can be modified or partially removed by acting on it with an appropriate protein-modifying enzyme. Examples of protein-modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

[0193] The present invention also provides a pharmaceutical composition containing the antigen-binding molecule of the present invention as an active ingredient. The pharmaceutical composition can be used to treat a disease, but the pharmaceutical composition provided by the present invention is preferably used to treat a disease in which a physiologically active antigen is considered to be one of the causes. The antigen is preferably two or more physiologically active antigens whose physiological activity is reduced in vivo by the antigen-binding molecule provided by the present invention. In this specification, “treatment” means obtaining a pharmacological and / or physiological effect. The effect can be preventive in that it completely or partially prevents the symptoms of the disease, and can be therapeutic in that it completely or partially treats the symptoms of the disease. In this specification, “treatment” includes all treatments of diseases in mammals, especially humans. Furthermore, “treatment” also includes preventing the onset of the disease in subjects who have not yet been diagnosed with the disease, and suppressing or mitigating the progression of the symptoms of the disease.

[0194] The pharmaceutical compositions provided by the present invention can be formulated using methods known to those skilled in the art (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). The antigen-binding molecules provided by the present invention can also be formulated in combination with other pharmaceutical components as needed. For example, they may include pharmaceutically acceptable carriers or additives. Furthermore, the pharmaceutical compositions provided by the present invention can be used parenterally, for example, as sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. Dosage forms for oral and parenteral administration and their manufacturing methods are well known to those skilled in the art, and the pharmaceutical compositions provided by the present invention can be manufactured according to conventional methods by mixing them with pharmaceutically acceptable carriers, etc. Examples of carriers used in the present invention include, but are not limited to, sterile water, physiological saline, vegetable oil, emulsifiers, surfactants, excipients, vehicles, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, and taste enhancers. Other commonly used carriers can be used as appropriate. Specifically, 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 triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. It is conceivable to formulate the pharmaceutical compositions provided by the present invention by appropriately combining these and mixing them in a unit dose form generally accepted for pharmaceutical production. The amount of active ingredient in these formulations is set so as to obtain an appropriate dose within the indicated range.

[0195] The pharmaceutical composition provided by the present invention can be administered orally or parenterally, but parenteral administration is preferred, specifically including injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection administration include intravenous administration, intramuscular administration, intraperitoneal administration, and subcutaneous administration. The dosage can be appropriately selected from 0.0001 mg to 1000 mg per kg of patient body weight or from 0.001 mg to 10000 mg per patient, but is not limited to these ranges. The target of administration is mammals, preferably humans.

[0196] The present invention also provides a kit containing an antigen-binding molecule or pharmaceutical composition provided by the present invention, and a kit for use in various methods provided by the present invention. The kits provided by the present invention may also optionally include instructions describing how to use them. Furthermore, the kits of the present invention can be suitably used for (i) a method to reduce the concentration of an antigen in plasma, (ii) a method to promote the uptake of an antigen into cells, or (iii) a method to reduce the physiological activity of an antigen in a living organism.

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

[0198] Furthermore, the present invention provides a method for producing the antigen-binding molecule of the present invention, comprising the following steps: (a) A step of selecting antigens having two or more physiological activities, (b) Steps to obtain an antigen-binding domain, (c) A step of obtaining at least one receptor-binding domain, (d) A step of selecting from the antigen-binding domains obtained in step (b) a domain whose activity in binding to the antigen changes depending on the ion concentration conditions. (e) A step of selecting from among the receptor-binding domains obtained in step (c) a domain that has activity to bind to human FcRn under acidic pH conditions and whose activity to bind to human Fc receptors under neutral pH conditions is higher than the activity of natural human IgG to bind to human Fc receptors. (f) A step to produce 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. Furthermore (g) A step of selecting from the antigen-binding molecules produced in step (f) an antigen-binding molecule that, upon binding to an antigen, inhibits one or more of the physiological activities of the antigen while maintaining at least one physiological activity.

[0199] The antigen-binding domain in this invention may be obtained by any method. For example, if the antigen-binding domain is an antibody or a fragment thereof (variable region, Fab, F(ab')2, Fv, CDR, etc.), they 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 way may be modified by at least one amino acid.

[0200] Many methods for preparing antibody libraries are already known. For example, phage display libraries can be prepared by those skilled in the art by referring 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 Japanese Patent Publication No. 10-504970. If a phage display library is used in which the variable region of an antibody is expressed as a single-chain antibody (scFv) on the surface of a phage, then 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 that antigen can be determined by analyzing the genes of the selected phages. Once the DNA sequence of the antibody's variable region is known, it can be ligated with the DNA encoding the desired constant region, inserted into a suitable expression vector, and introduced into host cells for expression to produce a recombinant antibody. 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 found.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 known antibody libraries such as Throughput Screen (1991) 4, 185-192).

[0201] The hybridoma method for obtaining antibodies basically uses known techniques, employing a desired antigen or cells expressing the desired antigen as a sensitizing antigen. These are immunized according to standard immunization methods, the resulting immune cells are fused with known parent cells using standard cell fusion methods, and monoclonal antibody-producing cells (hybridoms) are screened using standard screening methods. Furthermore, cDNA encoding the variable region of the antibody can be obtained from the mRNA of the obtained hybridomas using reverse transcriptase. This cDNA is then ligated with DNA encoding the desired constant region, inserted into a suitable expression vector, and introduced into host cells for expression, thereby producing a recombinant antibody.

[0202] More specifically, although not limited to the following examples, sensitizing antigens can include both immunogenic complete antigens and incomplete antigens, such as haptens that do not exhibit immunogenicity. For example, the full-length protein or partial peptide of the target protein can be used. Other substances composed of polysaccharides, nucleic acids, lipids, etc., are known to be able to act as antigens and are not particularly limited. Antigen preparation can be carried out by methods known to those skilled in the art, for example, by following methods using baculoviruses (e.g., WO98 / 46777). Hybridoma preparation can be carried out by following methods such as those of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). If the immunogenicity of the antigen is low, it can be bound to an immunogenic macromolecule such as albumin and immunization can be performed. The antigen can also be bound to other molecules as needed.

[0203] Hybridomas can be obtained by immunizing animals with the appropriate sensitizing antigens described above. Alternatively, antibody-producing lymphocytes can be immortalized in vitro to become antibody-producing cells. Various mammals can be used as animals for immunization, but rodents, lagomorphs, and primates are commonly used. Examples include rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as cynomolgus macaques, rhesus macaques, baboons, and chimpanzees. In addition, transgenic animals with a repertoire of human antibody genes are known, and human antibodies can also 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). Alternatively, instead of using such transgenic animals, it is also possible to obtain a desired human antibody with antigen-binding activity by, for example, sensitizing human lymphocytes with a desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells, such as U266 (see Japanese Patent Publication No. 1-59878).

[0204] Immunization of animals is carried out, for example, by appropriately diluting and suspending the sensitizing antigen in phosphate-buffered saline (PBS) or physiological saline, emulsifying it with an adjuvant if necessary, and then injecting it intraperitoneally or subcutaneously into the animal. Subsequently, preferably, the sensitizing antigen mixed with Freund's incomplete adjuvant is administered several times every 4 to 21 days. Confirmation of antibody production can be performed by measuring the titer of the target antibody in the animal's serum using conventional methods.

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

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

[0207] The receptor-binding domain in this invention may be obtained by any method. For example, if 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 the region containing it (the constant region of an antibody or a full-length antibody). If the receptor-binding domain is the Fc region of IgG, at least one amino acid may be modified.

[0208] Methods for adding, deleting, and / or substituting arbitrary amino acids into polypeptides can be carried out 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 well known. For example, in the case of human-mouse chimeric antibodies, a chimeric antibody can be obtained by ligating DNA encoding the variable region of a mouse antibody with DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host to induce production.

[0210] Humanized antibodies, also called reshaped human antibodies, are created by transplanting the complementarity determining region (CDR) of an antibody derived from a non-human mammal, such as a mouse antibody, into the CDR of a human antibody. Methods for identifying CDRs are well 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 transplanting CDRs are also well known (see European Patent Application Publication No. EP 125023, WO 96 / 02576). Humanized antibodies can be produced using a system with a standard expression vector by, for example, determining the CDR of a mouse antibody using a known method, obtaining DNA encoding an antibody in which the CDR and the framework region (FR) of a human antibody are linked, and then producing the humanized antibody. Such DNA can be synthesized by assembly PCR using several oligonucleotides as primers, each having overlapping portions at both the CDR and FR terminal regions (see the method described in publication WO98 / 13388). The FR of the human antibody ligated via the CDR is selected so that the CDR forms a good antigen-binding site. If necessary, the amino acids of the FR in the variable region of the antibody may be modified so that the CDR of the reconstituted human antibody forms an appropriate antigen-binding site (Sato et al., Cancer Res. (1993) 53: 10.01-6). The amino acid residues in the FR that can be modified include parts that bind directly to the antigen via non-covalent bonds (Amit et al., Science (1986) 233: 747-53), parts that affect or act on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and parts related to VH-VL interactions (Patent Publication No. EP239400).

[0211] In addition to the humanization described above, modifications may also be made to improve the biological properties of the antibody, such as its ability to bind to antigens. Modifications in this invention can be carried out by methods such as site-directed mutation (see, for example, Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutation, or cassette mutation. Generally, antibody variants with improved biological properties have 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.) of amino acid sequence homology and / or similarity with respect to the amino acid sequence of the variable region of the original antibody. In this specification, sequence homology and / or similarity is defined as the percentage of amino acid residues that are homologous (the same residue) and / or similar (residues classified into the same group based on the general side-chain characteristics of amino acids) with respect to the amino acid residues of the original sequence, after the sequence has been aligned and gaps introduced as necessary to maximize sequence homology. Typically, natural amino acid residues are 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 affect chain orientation: glycine and proline; and (6) Aromaticity: Tyrosine, tryptophan, and phenylalanine It is classified into the following group.

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

[0213] In the method for producing an antigen-binding molecule provided by the present invention, whether the antigen-binding molecule or its antigen-binding domain inhibits one or more of the physiological activities of the antigen, or maintains at least one of the physiological activities, can also be confirmed by measuring whether it inhibits one or more of the target molecule-binding activities of the antigen, or maintains at least one target molecule-binding activity.

[0214] In the method for producing an antigen-binding molecule provided by the present invention, multiple receptor-binding domains may be obtained, and from among them, a domain that has activity to bind to human FcRn under acidic pH conditions and activity to bind to human Fc receptors under neutral pH conditions that is higher than that of natural human IgG may be selected. Alternatively, multiple domains may be created by modifying any receptor-binding domain by adding, deleting, and / or substituting at least one amino acid, and from among them, a domain that has activity to bind to human FcRn under acidic pH conditions and activity to bind to human Fc receptors under neutral pH conditions that is higher than that of natural human IgG may be selected.

[0215] In the method for producing antigen-binding molecules provided by the present invention, an antigen-binding molecule in which an antigen-binding domain and a 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, linking them in-frame, introducing them into an expression vector, and expressing this vector in host cells. The antigen-binding domain and the receptor-binding domain may be directly linked, or they may be indirectly linked using any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (for example, the linker disclosed in Protein Engineering (1996) 9, 299-305). The length and amino acid sequence of the peptide linker are not particularly limited, but usually a peptide 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 is used.

[0216] The present invention also provides a method for screening antibodies whose antigen-binding activity changes depending on conditions, comprising the following steps: (a) A step of preparing antibody-producing cells, (b) The step of bringing the antigen into contact with the cells of (a) under the first conditions, (c) A step of selecting cells from the cells of step (b) that have bound to a certain amount or more of antigen, (d) The step of placing the cells from step (c) under second conditions, and (e) A step in which cells from step (d) have a reduced antigen binding capacity compared to those in step (c).

[0217] A more preferred embodiment of the above method includes the following steps: (a) A step of preparing antibody-producing cells, (b) The step of bringing the antigen into contact with the cells of (a) under the first conditions, (c) A step of contacting the cells from step (b) with an anti-IgG antibody, (d) A step of selecting cells from the cells of step (c) that are bound to a certain amount or more of antigen and bound to a certain amount or more of anti-IgG antibody. (e) The step of placing the cells from step (d) under second conditions, and (f) A step in which cells from step (e) have a reduced antigen binding capacity compared to those in step (d).

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

[0219] In the present invention, "antibody-producing cells" are not particularly limited as long as they contain antibody genes and express antibody proteins, but are preferably cells that naturally exist in the animal body and produce antibodies, more preferably lymphocytes, and even more preferably B cells. As the animal, various mammals (mouse, rat, hamster, rabbit, cynomolgus monkey, rhesus monkey, hamadryas baboon, chimpanzee, human, etc.) can be appropriately selected, but rabbits are particularly preferred in the present invention. It is also preferable to use animals immunized with the desired antigen. In addition to naturally occurring cells, antibody-producing cells may also be artificially produced cells such as hybridomas or genetically modified cells. It is preferable that the antibody-producing cells used in the present invention have the property of secreting antibodies outside the cell (secretory antibodies) and / or the property of presenting antibodies on the cell membrane (membrane antibodies). Antibody-producing cells that naturally exist in the animal body can be suitably recovered from, for example, the spleen, lymph nodes, blood (peripheral blood mononuclear cells), and such methods are known to those skilled in the art and are also described in the examples below.

[0220] In the present invention, the "first condition" and the "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 category of conditions is not particularly limited as long as it is the condition to which antibody-producing cells are exposed, but examples include temperature, pH, and the composition contained in the culture medium and its concentration. Preferably, it is the ion concentration, and particularly preferably, it is the hydrogen ion concentration (pH) or calcium ion concentration. Intracellular conditions preferably refer to conditions characteristic of the environment inside endosomes, 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, while the pH inside the cell is acidic compared to the pH outside the cell. In the present invention, the preferred neutral pH range is pH 6.7 to pH 10.0, more preferably pH 7.0 to pH 9.0, and 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, or 8.0, with a particularly preferred pH of 7.4, which is close to the pH in plasma (blood). Also in the present invention, the preferred acidic pH range is 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, or 6.5, with a particularly preferred pH of 5.8 to pH 6.0, which is close to the pH in early endosomes in living organisms.

[0222] Furthermore, the calcium ion concentration is higher extracellularly than intracellularly, and conversely, lower intracellularly than extracellularly. 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). Also, preferred low calcium ion concentration conditions in the present invention 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 the "second condition" may include multiple conditions simultaneously. For example, the "first condition" may be a neutral pH condition with a high calcium ion concentration condition, while the "second condition" may be an acidic pH condition with a low calcium ion concentration condition.

[0224] The antigen can be any substance against which an antibody can be produced, and is not particularly limited in type, but it is preferably an antigen containing a polypeptide. Furthermore, in the 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 bound to the antigen, or it may be indirectly bound to the antigen using an antigen-antibody reaction or a biotin-avidin reaction, etc. Examples of labeling substances include radioactive isotopes, chemiluminescent compounds, fluorescent compounds, phosphorescent compounds, magnetic particles, enzymes, etc., but fluorescent compounds are 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, 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 the anti-IgG antibody is bound from among antibody-producing cells, the proportion of cells expressing IgG as a subclass can be increased. The expression of IgG in B cells means that a class switch to IgG has occurred, and enriching cells that produce such mature antibodies is considered advantageous in screening antibodies with high binding activity. The labeling substance may be directly bound to the antigen, or it may be indirectly bound to the antigen using an antigen-antibody reaction or a biotin-avidin reaction, etc. 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 to which they are labeled are different from each other and that the detection methods are different (so that both can be detected individually). Methods for labeling the antigen and the anti-IgG antibody can be carried out by referring to methods known to those skilled in the art (e.g., US5057313, US5156840, etc.).

[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 performed by detecting the labeling substance. For example, if the antigen and / or anti-IgG antibody are each labeled with different types of fluorescent compounds, whether a certain amount or more of antigen and / or anti-IgG antibody is bound to a cell can be evaluated by whether the fluorescence emitted by each fluorescent compound is detected at a certain intensity or higher. The certain amount can be arbitrarily set by those skilled in the art depending on the purpose. In the present invention, the selection step is preferably performed using FACS (Fluorescence Activated Cell Sorting).

[0227] In the antibody screening method provided by the present invention, the step of concentrating cells bound to an antigen is preferably carried out by detecting the labeling substance. For example, if the antigen is labeled with magnetic particles, cells bound to the antigen and cells not bound to the antigen can be separated using a magnetic device, and cells bound to the antigen can be concentrated by removing the cells not bound to the antigen. In the present invention, the concentration step is preferably carried out using MACS (Magnetic Activated Cell Sorting, registered trademark).

[0228] By using the antibody screening method provided by the present invention, it becomes possible to easily and efficiently screen for antibodies whose antigen-binding activity changes depending on the conditions from among a large number of antibody-producing cells. Compared to conventional methods, the number of cells that can be screened can be greatly increased, so the probability of finding rare antibodies that could not be found before is greatly increased, making the antibody screening method provided by the present invention useful.

[0229] Furthermore, the present invention provides a method for reducing the concentration of an antigen in plasma, a method for promoting the uptake of an antigen into cells, or a method for reducing the physiological activity of an antigen in a living organism by administering the antigen-binding molecule of the present invention.

[0230] Furthermore, the present invention provides a disease treatment agent containing the antigen-binding molecule of the present invention as an active ingredient. Examples of diseases include diseases in which HMGB1 is considered to be one of the causative agents, diseases in which CTGF is considered to be one of the causative agents, or diseases in which IgE is considered to be one of the causative agents. The present invention also provides a kit containing the antigen-binding molecule of the present invention for use in methods for reducing the concentration of an antigen in plasma, for promoting the uptake of an antigen into cells, or for reducing the physiological activity of an antigen in a living organism. Furthermore, the present invention provides a method for treating a disease in which a physiologically active antigen is considered to be one of the causes, a method for reducing the concentration of an antigen in plasma, a method for promoting the uptake of an antigen into cells, or a method for reducing the physiological activity of an antigen in a living organism, comprising the step of administering the antigen-binding molecule of the present invention. Furthermore, the present invention provides a therapeutic agent for diseases in which a physiologically active antigen is considered to be one of the causes, an agent for reducing antigen concentration in plasma, an agent for promoting the uptake of antigens into cells, or an agent for reducing the physiological activity of an antigen in the body, all of which contain the antigen-binding molecule of the present invention as an active ingredient. Furthermore, the present invention provides an antigen-binding molecule for use in methods for treating diseases in which a physiologically active antigen is considered to be one of the causes, methods for reducing the concentration of antigens in plasma, methods for promoting the uptake of antigens into cells, or methods for reducing the physiological activity of antigens in living organisms. Furthermore, the present invention provides for the use of the antigen-binding molecule in the production of therapeutic agents for diseases in which a physiologically active antigen is considered to be one of the causes, agents for reducing antigen concentration in plasma, agents for promoting the uptake of antigens into cells, or agents for reducing the physiological activity of antigens in living organisms. The present invention also provides a process for producing a therapeutic agent for diseases in which a physiologically active antigen is considered to be one of the causes, an agent for reducing antigen concentration in plasma, an agent for promoting the uptake of antigens into cells, or an agent for reducing the physiological activity of an antigen in a living organism, which includes a step of using the antigen-binding molecule of the present invention. Examples of diseases include diseases in which HMGB1 is considered to be one of the etiologies, diseases in which CTGF is considered to be one of the etiologies, or diseases in which IgE is considered to be one of the etiologies.

[0231] It should be noted that the amino acids included in the amino acid sequence described in this invention may undergo post-translational modifications (for example, modification of the N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art), but even when amino acids are modified post-translation in this way, they are naturally still included in the amino acid sequence described in this invention.

[0232] All prior art documents cited herein are incorporated herein by reference. [Examples]

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

[0234] [Example 1] Production of anti-HMGB1 antibody by rabbit B cell cloning method Preparation of HMGB1 The antigen HMGB1 was prepared as follows: An animal cell expression vector was created by inserting the 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). The HMGB1 protein was purified from the obtained culture supernatant using cation exchange column chromatography, anion exchange column chromatography, and gel filtration column chromatography.

[0235] Antigen-induced immunization of animals Rabbits were immunized with HMGB1. For the initial immunization, 100 μg of HMGB1 protein contained in complete Freund's adjuvant (CFA) was injected intradermally. Subsequently, two or more booster immunizations were performed, each containing 50 μg of HMGB1 protein contained in incomplete Freund's adjuvant (IFA), at intervals of more than one week. Antibody titers were measured to confirm that antibodies were being produced within the animals.

[0236] Tissue collection from immunized animals and preparation of single-cell suspensions Individuals showing antibody production 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 the tubes were 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 removal of the supernatant. The same washing procedure was repeated to prepare PBMCs. The PBMCs were stained with trypan blue, and the cell density was determined on a hemocytometer.

[0237] After harvesting, single-cell suspensions were prepared by processing the spleen and lymph nodes through a 70 μm cell strainer (BD Falcon) using a 5 mL syringe plunger. The cells were collected in sterile 50 mL tubes in RPMI medium containing 2% FBS. The cells were washed by centrifugation at 1000 × g for 5 minutes and removal of the supernatant. The washing procedure was repeated by adding 50 mL of RPMI-1640 containing 2% FBS. After the final wash, trypan blue staining was performed and the cell density was determined on a hemocytometer.

[0238] Recovery of antigen-binding B cells Single-cell suspensions of blood, spleen, and lymph nodes prepared by the method described above were centrifuged at 1000 × g for 5 minutes, and the cells were 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). A solution of biotinylated human HMGB1 diluted to 500 nM with HBSS was prepared and added to the cells to a density of 1E08 cells / 100 μL or less, and the cells were suspended. 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, and then dialyzing it with TBS 300mM NaCl (10mM Tris-HCl, 300mM NaCl) using a microdialyzer (TOMY). The cell suspension was incubated on ice for 30 minutes. Then, it was washed with 50 mL of HBSS to remove biotinylated HMGB1 that was not bound to the cells. A solution of MACS® streptavidin beads (Miltenyi Biotech) diluted 10-fold with HBSS was prepared and added to the cells to a density of 1E08 cells / 500 μL or less, and the cells were suspended. The cell suspension was incubated on ice for 30 minutes. After washing with 50 mL of HBSS, HBSS was added to the cells to a density of 1E08 cells / 500 μL or less, and the cells were suspended. The cell suspension was collected using the autoMACS Pro Separator to obtain the positive cell fraction to which MACS streptavidin beads were bound.

[0239] For the harvested cells, a solution of biotinylated HMGB1 diluted with HBSS was prepared again, and the cells were suspended by adding it to the cells at a density of 1E08 cells / 100μL or less. The cell suspension was incubated on ice for 30 minutes. Then, it was washed with 50 mL of HBSS. This re-incubation with biotinylated human HMGB1 may be omitted. A solution of Streptavidin-FITC (BD) and Mouse anti-rabbit IgG-PE (Southern Biotech) diluted with HBSS was prepared, and the cells were suspended by adding it to the cells at a density of 1E08 cells / 100μL or less. The cell suspension was incubated on ice for 30 minutes. Then, it was washed with 50 mL of HBSS. Then, HBSS was added to the cells at a density of 1E07 cells / 100μL or less, and the cells were suspended. Cells from the cell suspension were collected using FACSAria (BD) to separate the fractions with high fluorescence values ​​for both FITC and PE.

[0240] pH or Ca 2+ Recovery of antigen-binding antibody-expressing B cells whose dissociation ability changes with changes in ion concentration. pH or Ca 2+A method was used to concentrate and recover B cells expressing antibodies whose dissociation ability changes with changes in ion concentration. Single cell suspensions from blood, spleen, and lymph nodes were prepared and then 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). A solution of biotinylated HMGB1 diluted to 500 nM in HBSS was prepared and added to the cells to a density of 1E08 cells / 100 μL or less to suspend the cells. The cell suspension was incubated on ice for 30 minutes. Subsequently, it was washed with 50 mL of HBSS to remove biotinylated HMGB1 that was not bound to the cells. A solution of MACS streptavidin beads (Miltenyi Biotech) was prepared by diluting it 10-fold with HBSS. This solution was added to cells at a density of 1E08 cells / 500 μL or less and suspended. The cell suspension was incubated on ice for 30 minutes. After washing with 50 mL of HBSS, HBSS was added to the cells at a density of 1E08 cells / 500 μL or less and suspended. The cell suspension was collected using an autoMACS Pro Separator to obtain the positive cell fraction to which MACS streptavidin beads were bound.

[0241] For the harvested cells, a solution of biotinylated HMGB1 diluted to 500 nM with HBSS was prepared again, and the cells were suspended by adding this solution to the cells at a density of less than 1E08 cells / 100 μL. The cell suspension was incubated on ice for 30 minutes. Then, it was washed with 50 mL of HBSS. A solution of Streptavidin-FITC (BD) and Mouse anti-rabbit IgG-PE (Southern Biotech) diluted with HBSS was prepared, and the cells were suspended by adding this solution to the cells at a density of less than 1E08 cells / 100 μL. The cell suspension was incubated on ice for 30 minutes. Then, it was washed with 50 mL of HBSS. Then, HBSS was added to the cells at a density of less than 1E07 cells / 100 μL, and the cells were suspended. The cell suspension was gated using FACSAria (BD) to fractions with high fluorescence values ​​for both FITC and PE, and the cells were harvested. During recovery, MBSS (20mM MES, 5.3mM KCl, 0.4mM KH2PO4, 4.2mM NaHCO3, 0.3mM Na2HPO4, 0.1% BSA, 2mM EDTA, 5mM Glucose, 138mM NaCl, pH 5.8) was placed in the recovery tube. The tube was then left in the MBSS solution for 30 minutes. pH decrease or Ca 2+ For antibodies that readily dissociate due to decreased ion concentration, leaving the MBSS at a low pH and in the presence of EDTA causes the antigen to dissociate and the FITC fluorescence value to decrease. After 30 minutes, FACSAria was used again to gate the cell population where the PE fluorescence value was the same as the first sorting, but the FITC fluorescence value was lower than the first sorting value, and the cells were collected. The results are shown in Figure 1. (A) shows the dot plots of the first sorting. Cells were collected from the gate indicated by 1. (B) shows the dot plots of the second sorting. In the second sorting, cells were collected separately from gates 1, 2, and 3.

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

[0243] B cell culture The recovered cells were seeded into a 96-well microtiter plate at a density of 1 cell or less per well. Activated rabbit T cell-conditioned medium was added to a final concentration of 5%, and EL4 cells (European Collection of Cell Cultures) were added at approximately 25,000 cells per well. The activated rabbit T cell-conditioned medium was prepared by: treating thymus collected from rabbits through a 70 μm cell strainer (BD Falcon) using the plunger of a 5 mL syringe to obtain cells, culturing the cells in RPMI-1640 containing PHA (Roche), phorbol 12-myristate 13-acetate (Sigma) and 2% FBS, and freezing the culture supernatant at -70°C or lower. For use as feeder cells, EL4 cells are treated by adding Mytomycin C (Sigma) to a concentration of 10 μg / mL to stop cell proliferation, and culturing the cells at 37°C under 5% CO₂ for 2 hours or more before use. After culturing these cultures at 37°C under 5% CO₂ for 5 to 7 days, a portion of the supernatant containing secreted antibodies was collected. Using the collected supernatant, the HMGB1-binding ability of the antibodies was evaluated by the method described below. The cells were maintained under conditions of 37°C and 5% CO₂ until evaluation of the HMGB1-binding ability of the antibodies.

[0244] Screening of culture supernatants for monoclonal antibodies with desired specificity Antibody antigen recognition was screened using the ELISA method. A 384-well plate coated with streptavidin was prepared, and biotinylated HMGB1 was captured. The supernatant containing secreted antibodies was applied to the plate. After standing 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. Then, a 40,000-fold dilution of Goat anti-rabbit IgG Fc HRP conjugate (BETHYL) in TBS containing 2 mM CaCl2 was dispensed and stood 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 / well of chromogenic substrate (ABTS peroxidase substrate (KPL)) was added. After incubation for 1 hour, the absorbance at 405 nm was measured using a SpectraMax from Molecular Devices. By analyzing the absorbance measurements at 405 nm, we identified the 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 An ELISA was performed using the culture supernatant to evaluate whether pH / Ca-dependent dissociation occurred. Goat anti-rabbit IgG-Fc (BETHYL), diluted to 1 μg / mL with PBS(-), was added to a 384-well MAXISorp (Nunc) plate and left at room temperature for at least 1 hour. Then, the PBS(-) diluted Goat anti-rabbit IgG-Fc was removed from the plate, and TBS (pH 7.4) containing 1% BSA and 2 mM CaCl2 was added and left for at least 1 hour. The TBS (pH 7.4) containing 1% BSA and 2 mM CaCl2 was removed from the plate, and the culture supernatant was added. At this time, pH or Ca 2+In evaluating antibodies whose dissociation ability changes with ion concentration changes, two wells of culture supernatant were added to each type of B cell culture supernatant in an ELISA plate. The supernatant was left at room temperature for at least one hour, or at 4°C overnight, to trap the antibodies in the culture supernatant with Goat anti-rabbit IgG-Fc. Subsequently, the supernatant was washed three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20, and biotinylated HMGB1 was added and left at room temperature for at least one hour. This resulted in the binding of the rabbit antibody trapped by Goat anti-rabbit IgG-Fc to the biotinylated HMGB1. The biotinylated HMGB1 that did not bind to the rabbit antibody was washed away three times with 80 μL of TBS (pH 7.4) containing 2 mM CaCl2 and 0.05% Tween-20. 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 as described above, and 20 mM MES, 150 mM NaCl, 2 mM EDTA, pH 5.8 (Buffer B) was added to the other well, and the mixture was left to stand at 37°C or below for at least one hour. While Buffer A and Buffer B were being added, biotinylated human HMGB1 dissociated from the rabbit antibody. However, depending on the antibody's properties, the antigen may dissociate more easily at lower pH, or CaCl2 may be used to maintain the binding of the antigen to the antibody. 2+When ions were required, antigens detached more easily from the antibody in wells with Buffer B than in wells with 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 of Streptavidine-HRP (Genscript) prepared in TBS containing 2 mM CaCl2 was added and left at room temperature for 1 hour. Streptavidine-HRP bound to biotinylated HMGB1 that remained undissociated 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 chromogenic substrate (ABTS peroxidase substrate) was added. After incubation for 1 hour, the absorbance at 405 nm was measured using a SpectraMax from Molecular Devices. By analyzing the absorbance measurements at 405 nm, the antibodies in the culture supernatant where the wells with Buffer A developed a stronger color than the wells with Buffer B were found to be at 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 characteristics. First, for each type of B cell culture supernatant, two wells of culture supernatant are prepared in the ELISA plate, and after the antigen and antibody are bound, one well is subjected to a high pH of around 7.0 or higher, and Ca 2+ Set the conditions so that ions are present in a range of approximately 1 mM or more, and the other side at a low pH of around 6.0 or lower, and Ca 2+ The key is to incubate the dissociation of the antigen and antibody under conditions that reduce the ion concentration. By including this incubation step, pH or Ca can be efficiently controlled. 2+This method allows for screening 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 the antigen is bound to them. When rabbit antibodies are reacted after the antigen has been bound to the plate, a strong binding reaction occurs because both arms of the antibody bind to the antigen immobilized on the plate. Even antibodies that exhibit pH / Ca-dependent dissociation become difficult to dissociate, and only those exhibiting strong dependence can be obtained. However, by trapping rabbit antibodies in the culture supernatant with Goat anti-rabbit IgG-Fc and then binding the antigen to them, the binding of the antigen and antibody becomes more likely to occur in a monovalent state. Therefore, pH or Ca 2+ Even when the change in dissociation ability due to changes in ion concentration is weak, it becomes possible to determine whether or not there is pH / Ca dependence.

[0247] The results are shown in Figures 2 and 3. Figure 2 shows pH or Ca 2+ This is a dot plot of the results of screening antibodies whose dissociation ability changes with changes in ion concentration. The Y axis represents the OD (405nm) value under incubation conditions of 20mM MES, 150mM NaCl, 2mM CaCl2, pH 7.4. The X axis represents the OD (405nm) value under incubation conditions of 20mM MES, 150mM NaCl, 2mM EDTA, pH 5.8. (A) shows the results of culturing B cells derived from gate 1 in Figure 1(B), and testing under conditions of pH change or Ca 2+ This is the result of screening antibodies whose dissociation ability changes with changes in ion concentration. (B) shows the results of culturing B cells derived from gate 2 in Figure 1(B), and testing for changes in pH or Ca 2+ This is the result of screening antibodies whose dissociation ability changes with changes in ion concentration. (C) shows the results of culturing B cells derived from gate 3 in Figure 1(B), and testing for changes in pH or Ca 2+ This is the result of screening antibodies whose dissociation ability changes with changes in ion concentration. In incubation at pH 5.8 in the presence of 2 mM EDTA, the results were obtained for antibodies at pH 7.4 in the presence of 2 mM Ca 2+Antibodies that dissociate more easily from antigens compared to incubation in the presence of EDTA are indicated with a circle (○). The conditions are pH 5.8, with 2mM EDTA and pH 7.4, with 2mM Ca. 2+ Conditions that did not show a significant change in value under the conditions including are indicated by a square (□). Figure 3 is a graph of the number of clones in Figures 2(A), (B), and (C). The gray areas indicate pH changes or Ca 2+ The number of clones (indicated by □ in (A), (B), and (C)) in which no change is observed in the dissociation of antibody and antigen even with changes in ion concentration, is shown in black areas when pH decreases or Ca 2+ This represents the number of clones (indicated by ○ in (A), (B), and (C)) in which antibody-antigen dissociation is more likely to occur when the ion concentration decreases. In the recovery of antigen-binding B cells, similar to the result in (A), the pH or Ca of the antigen-binding antibody is used. 2+ The percentage of antigen-binding antibodies whose dissociation ability changes with changes in ion concentration is at most a few percent. On the other hand, pH or Ca 2+ By concentrating and recovering B cells that express antibodies whose dissociation ability changes with changes in ion concentration, the pH or Ca of the antigen-binding antibody can be determined as shown in (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. By using this recovery method, pH or Ca 2+ This method allows for the efficient acquisition of extremely rare antibodies whose dissociation ability changes with changes in ion concentration and which also possess physiological activity.

[0248] Identification of L and H chain sequences in the variable region of B cells and expression of recombinant antibodies Screening results for monoclonal antibodies with desired specificity from cell culture plates incubated at 37°C and 5% CO2, or pH or Ca 2+Using the screening results for antibodies whose dissociation ability changes with changes in ion concentration as an indicator, cells and culture supernatant were collected in new 96-well plates using MS2000 (J-Tek). From the plates containing the collected cells and culture supernatant, only the culture supernatant was transferred to another 96-well plate. Meanwhile, the plates containing the cells collected by MS2000 were frozen and stored at -70°C or below. Antibody cDNA was obtained from the cells stored at -70°C, and an antibody expression vector was constructed. The primers for the PCR reaction used to obtain the cDNA were designed to anneal to the 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). Reverse transcription and first PCR were performed using the purified RNA with the OneStep RT-PCR kit (TAKARA). The primer sequences used are shown in Table 11. Subsequently, nested PCR was performed on the first PCR product using PrimeSTAR HS (TAKARA). The primer sequences used for PCR are shown in Table 11. In the table, R represents a mixed base of A and G, V represents a mixed base of A, C, and G, W represents a mixed base of A and T, and Y represents a mixed base of C and T.

[0249] [Table 11]

[0250] Cassette vectors were created by introducing the antibody constant region sequence into animal cell expression vectors, and antibody expression vectors were then produced using these cassette vectors. Two types of cassette vectors were created: one containing the rabbit antibody H chain constant region sequence, and another containing the rabbit antibody L chain constant region sequence. The vector containing the H chain constant region sequence had an ampicillin resistance gene inserted into it. The vector containing the L chain constant region sequence had a kanamycin resistance gene inserted into it. These two types of vectors contained sequences that partially overlapped with the primer sequences of nested PCR. By using the In-Fusion PCR cloning kit (Clontech), the nested PCR product was incorporated into the cassette vector containing the antibody constant region sequence, and an expression vector containing the full-length rabbit antibody gene was produced. The nested PCR product was inserted into the vector using the Clontech In-Fusion PCR cloning kit. Subsequently, the cells were transformed into bacteria for plasmid propagation and production. The transformed bacteria were cultured in LB medium containing ampicillin or kanamycin. Plasmids were purified from the grown bacteria using a 96-well EndoFree ezFilter Plasmid Miniprep Kit (Biomiga), and antibodies were obtained according to Reference Example 1.

[0251] [Example 2] Measurement of affinity and pH / Ca dependence of anti-HMGB1 antibody Production of MedG4-IgG1 antibody MedG4H-IgG1 (SEQ ID NO: 36) and MedG4L-CK (SEQ ID NO: 37) were designed by ligating the human IgG1 constant region and the human Igκ constant region to the VH region (WO2007 / 084253 SEQ ID NO: 19) and VL region (WO2007 / 084253 SEQ ID NO: 17) of the G4 antibody described in WO2007 / 084253, respectively. The DNA encoding these antibodies was prepared using genetic engineering techniques, and MedG4-IgG1, an anti-HMGB1 antibody, was produced by expressing it 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. To determine whether the acquired antibodies possessed pH and pH / Ca-dependent binding ability, they were evaluated using Biacore T100 and T200 (GE Healthcare). Plasma conditions were set to pH 7.4 and calcium ion concentration 1.2 mM. Two intraendosomal conditions were set: pH 5.8 and calcium ion concentration 1.2 mM, and pH 5.8 and calcium ion concentration 3 μM. An appropriate amount of protein A (Invitrogen) was immobilized on a Sensor chip CM4 (GE Healthcare) using amine coupling, and the target antibody was captured onto it. Human HMGB1 was used as the antigen. Measurements were performed using three types of running buffers: (1; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween20, 2 mmol / L CaCl2, pH 7.4; 2; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween20, 2 mmol / L CaCl2, pH 5.8; 3; 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween20, 3 μmol / L CaCl2, pH 5.8). Each running buffer was used to dilute human HMGB1.

[0253] HMG233-IgG1, HMG236-IgG1, HMG481-IgG1, HMG487-IgG1 The antibody, diluted in running buffer, was injected at a flow rate of 10 μL / min for 1 minute and captured on the sensor tip. Then, human HMGB1 dilution (500 nM) and running buffer (as a reference solution) were injected at a flow rate of 10 μL / min for 1 minute to interact with the captured antibody, and the dissociation of human HMGB1 was observed by further flowing running buffer at a flow rate of 10 μL / min for 1 minute. 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 tip.

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

[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 to capture on the sensor tip. Then, human HMGB1 dilution and running buffer (as a reference solution) were injected at a flow rate of 10 μL / min for 1 minute to interact with the captured antibodies. Subsequently, running buffer was flowed at a flow rate of 10 μL / min for 2 minutes to observe the 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 tip.

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

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

[0258] [Table 12]

[0259] [Example 3] Evaluation of the binding of HMGB1 to cell surface receptors ELISA-based binding of HMGB1 to RAGE-Fc A 5 μg / ml PBS solution of recombinant human RAGE-Fc fusion protein (R&D SYSTEMS) was added to each well of an 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 of HMGB1 and 100 μg / mL of anti-HMGB1 antibody or buffer were pre-incubated at room temperature in the presence of 2.5% skim milk for 1 hour, and then transferred to a plate coated with the blocked RAGE. 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. Next, the plate was washed five times, 20 μl of TMB chromogenic agent was added, and the absorbance of the plate at 450 nm was measured.

[0260] We confirmed that the anti-HMGB1 antibody does not inhibit the binding of the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody used for detection to HMGB1 by pitting the anti-HMGB1 antibody against the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody on a plate immobilized with HMGB1.

[0261] ELISA-based binding of HMGB1 to TLR4 / MD-2 A 5 μg / ml PBS solution of recombinant human TLR4 / MD-2 protein (R&D SYSTEMS) was added to each well of an 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 of HMGB1 and 100 μg / mL of anti-HMGB1 antibody or buffer were pre-incubated at room temperature in the presence of 2.5% skim milk for 1 hour, and then transferred to a plate coated with the blocked TLR4 / MD-2. 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. Next, the plate was washed five times with PBS / Tween, 20 μl of TMB chromogenic agent was added, and the absorbance of the plate at 450 nm was measured.

[0262] We confirmed that the anti-HMGB1 antibody does not inhibit the binding of the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody used for detection to HMGB1 by pitting the anti-HMGB1 antibody against the peroxidase-labeled mouse anti-HMGB1 monoclonal antibody on a plate immobilized with HMGB1.

[0263] result Both RAGE and TLR4 have been identified as putative receptors for HMGB1. Several anti-HMGB1 antibodies were evaluated by ELISA assays for their ability to inhibit the binding of RAGE-Fc fusions or TLR4 / MD-2 fusions to HMGB1. For both RAGE and TLR4, the measured value under conditions without anti-HMGB1 antibody was set to 100, and the measured values ​​under each anti-HMGB1 antibody-added condition were calculated as relative values ​​and shown in Figures 5 and 6. Antibodies showing values ​​lower than 100 were judged to have the ability to inhibit binding to each receptor. Among the antibodies subjected to RAGE ELISA, HMG233-IgG1 and HMG236-IgG1 inhibited the binding of HMGB1 to RAGE. The inhibition rates were 53.1% for HMG233-IgG1 and 64.1% for HMG236-IgG1. Among the antibodies subjected to TLR4 / MD-2 ELISA, HMG481-IgG1, HMG487-IgG1, and HMG446-IgG1 inhibited the binding of HMGB1 to TLR4 / MD-2. The inhibition 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, one antibody was demonstrated to inhibit the binding of HMGB1 to RAGE but not to TLR4. Another antibody was demonstrated to inhibit the binding of HMGB1 to TLR4 / MD-2 but not to RAGE.

[0265] [Example 4] Investigation of the effect of pH-dependent anti-human HMGB1 antibody on accelerating human HMGB1 elimination. In vivo testing 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 to normal mice (C57BL / 6J mouse, Charles River Japan). A mixed solution of human HMGB1 (0.1 mg / mL) and anti-human HMGB1 antibodies (MedG4-IgG1 1 mg / mL, HMG446 2.05 mg / mL) was administered as a single dose of 10 mL / kg via tail vein. The antibody concentration in this mixed solution was set to a concentration at which 99.0% or more of the human HMGB1 contained in the mixed solution bound to the antibody. Blood samples were collected at 5 minutes, 10 minutes, 15 minutes, 1 hour, 4 hours, 2 days, and 7 days after administration. The collected blood was allowed to stand for 2 hours and then centrifuged at 4°C and 12,000 rpm for 5 minutes to obtain serum. The separated serum was stored in a freezer set to -20°C or below until measurement was performed. In this specification, MedG4-IgG1 may also be referred to as med G4. Similarly, HMG446-IgG1 may also be referred to as HMG446-G1.

[0266] Measurement of serum anti-human HMGB1 antibody concentration by ELISA method The concentration of anti-human HMGB1 antibody in mouse serum was 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 to stand overnight at 4°C to prepare an Anti-Human IgG immobilized plate. Calibration curve samples at serum concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 μg / mL, as well as mouse serum measurement samples diluted more than 100-fold, were prepared. 150 μL of 2000 ng / mL human HMGB1 was added to 150 μL of these calibration curve samples and serum measurement samples, and the mixture was left to stand at room temperature for 1 hour. Then, the mixture was dispensed into the Anti-Human IgG immobilized plate and left to stand at room temperature for another 1 hour. Subsequently, Goat Anti-Human IgG (γ chain specific) Biotin (BIOT) Conjugate (Southern Biotech Association) was reacted at room temperature for 1 hour, followed by Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) being reacted at room temperature for 1 hour. A color reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate, and after stopping the reaction with 1N-Sulfuric acid (Showa Chemical), the absorbance at 450 nm was measured using a microplate reader. The concentration of anti-human HMGB1 antibody in mouse serum was calculated from the absorbance of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). Figure 8 shows the changes in serum anti-human HMGB1 antibody concentration in mice after intravenous administration, as measured by this method.

[0267] Measurement of human HMGB1 concentration in serum using ELISA method Human HMGB1 concentrations in mouse serum were measured using the HMGB1 ELISA kit II (shino-test). Calibration curve samples at serum concentrations of 12800, 6400, 3200, 1600, 800, 400, and 200 μg / mL were prepared, along with mouse serum measurement samples diluted more than 100-fold. These samples were then mixed in equal volumes with either 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. Subsequently, the samples were dispensed onto the provided solid-phase plates and incubated at 37°C for 20-24 hours. The provided labeled antibody solution was then reacted at 25°C for 2 hours, followed by a 30-minute reaction with the chromogenic reagent, after which the reaction was stopped with the stop solution. The absorbance at 450 nm was then measured using a microplate reader. Human HMGB1 concentrations in mouse serum were calculated from the absorbance of the calibration cur...

Claims

1. A pharmaceutical composition for treating a condition caused by a soluble antigen, comprising an 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) Under conditions in the acidic pH range, the receptor-binding domain has the activity to bind to human FcRn (Neonatal Fc Receptor), (3) Under conditions of a neutral pH, the receptor-binding domain has higher activity in binding to human FcRn than in native human IgG has higher activity in binding to human FcRn, wherein the receptor-binding domain is the receptor-binding domain shown in (a) or (b) below: (a) The receptor is a human FcRn, and the receptor-binding domain includes an Fc region in which the amino acids in the Fc region of IgG have been modified, and further, The amino acid modification corresponds to EU numbering 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, 437, 438, The modification includes at least one amino acid selected from positions 440 and 442; (b) The receptor is a human Fcγ receptor, and the receptor-binding domain includes an Fc region in which the amino acids in the Fc region of IgG have been modified, and further, The modifications of the amino acids are EU numbered 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, 329, 330, Includes modifications of at least one amino acid selected from positions 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 activity of binding to the antigen under conditions in the acidic pH range is lower than the activity of binding to the antigen under conditions in the neutral pH range. Here, the antigen-binding domain, whose antigen-binding activity is lower under acidic pH conditions than under neutral pH conditions, comprises at least one histidine at a position selected from the following amino acids: Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102 (Kabat numbering), Light chains: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94 (Kabat numbering) (5) The antigen has two or more types of physiological activity, (6) When an antigen-binding molecule binds to an antigen, one or more of the physiological activities of the antigen are inhibited, while at least one physiological activity is maintained.

2. The pharmaceutical composition according to claim 1, characterized in that, by binding to an antigen, it inhibits one or more of the target molecule binding activities of the antigen, while maintaining the binding activity of at least one target molecule.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antigen-binding molecule reduces the plasma concentration of the antigen.

4. Modification of amino acids in the Fc region of IgG is used for 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 either 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. The 252nd amino acid 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 either 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 either 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 either 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 either 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 328th amino acid is Arg, Asp, Glu, or Tyr. The 329th amino acid is either 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 either 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. 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 either 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 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, The 438th amino acid is Lys, Leu, Thr, or Trp. The 440th amino acid is Lys, and The 442nd amino acid is Lys, A pharmaceutical composition according to any one of claims 1 to 3, wherein the modification is of at least one amino acid selected from.

5. Modification of amino acids in the Fc region of IgG is used for EU numbering; The 221st amino acid is Lys or Tyr, The 222nd amino acid is Phe, Trp, Glu, or Tyr. The 223rd amino acid is Phe, Trp, Glu, or Lys. The 224th amino acid 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, The 254th amino acid 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 262nd amino acid 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. The 273rd amino acid 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 either 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 320th amino acid 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 either 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 either 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. The 428th amino acid is Phe or Leu, The 429th amino acid is Met, The 434th amino acid is Trp. The 436th amino acid is Ile, and The 440th amino acid is Gly, His, Ile, Leu, or Tyr. A pharmaceutical composition according to any one of claims 1 to 4, wherein the modification is of at least one amino acid selected from.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human Fcγ receptor is FcγRIa, FcγRIIa, FcγRIIb, or FcγRIIIa.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the amino acid modification in the Fc region of IgG is such that the 238th amino acid in EU numbering is Asp and the 271st amino acid is Gly.

8. In the Fc region of IgG, further 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, The pharmaceutical composition according to claim 7, wherein at least one amino acid selected from positions 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 is modified.

9. Modification of amino acids in the Fc region of IgG is used for 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, The 266th amino acid is Phe, Met, or Leu. The 267th amino acid is Ala, Glu, Gly, or Gln. The 268th amino acid is either 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 either Ala or Asp. The 327th amino acid is Gly, The 330th amino acid is either 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 pharmaceutical composition according to claim 8, wherein the modification is of at least one amino acid selected from the following.

10. In the Fc region of IgG, further EU numbering is applied to 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, A pharmaceutical composition according to any one of claims 7 to 9, wherein at least one amino acid selected from positions 428, 430, 431, 433, 434, 436, 438, 440, and 442 is modified.

11. Modification of amino acids in the Fc region of IgG is used for 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. The 252nd amino acid 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 either 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 either Ala or Leu. The 316th amino acid is Lys, The 317th amino acid is Pro, The 318th amino acid is Asn or Thr. The 332nd amino acid 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. The 382nd amino acid 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 428th amino acid 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 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. The 438th amino acid is Lys, Leu, Thr, or Trp. The 440th amino acid is Lys, and The 442nd amino acid is Lys, The pharmaceutical composition according to claim 10, wherein the modification is of at least one amino acid selected from.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the antigen is selected from the group consisting of cytokines, interferons, and cell growth factors.

13. The pharmaceutical composition according to claim 12, wherein the antigen is selected from the group consisting of fibroblast growth factor (FGF), transforming growth factor (TGF), bone morphogenetic factor (BMP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), bone morphogenetic factor (BMP), and interferon (IFN), interleukin (IL), colony-stimulating factor (CSF), erythropoietin, and tumor necrosis factor (TNF).

Images