Antigen binding molecules that accelerate disappearance of antigens

Abstract

To provide an antigen binding molecule, a pharmaceutical composition comprising the antigen binding molecule and a production method thereof, where the binding molecule has such functions that when it binds to an antigen, it enhances uptake of the antigen into a cell, it can bind a larger number of antigen per molecule, it has improved pharmacokinetics, and it accelerates intracellular dissociation of the antigen to which it has bound outside the cell, it is released in a free state from the cell, thereby reducing the total or free antigen concentration in the serum.SOLUTION: A pharmaceutical composition comprises an antigen binding molecule comprising an antigen binding domain and an Fcγ receptor binding domain, where the antigen binding domain has a binding activity to a human FcRn under acidic pH conditions and whose antigen binding activity changes according to the ionic concentration, and the Fcγ receptor binding domain has a higher Fcγ receptor binding activity under neutral pH conditions, compared with the Fcγ receptor binding domain of a naturally occurring human IgG Fc region in which the sugar chain at position 297 by EU numbering is a fucose-containing sugar chain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides antigen-binding molecules that enhance intracellular uptake of bound antigens, antigen-binding molecules that can bind an increased number of antigens per molecule, antigen-binding molecules with improved pharmacokinetics, antigen-binding molecules that enhance intracellular dissociation of extracellularly bound antigens, antigen-binding molecules that enhance extracellular release in an unbound state, antigen-binding molecules that have the function of reducing the total or free antigen concentration in plasma, pharmaceutical compositions containing the antigen-binding molecules, and methods for producing them. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Document 1 and Non-Patent Document 2). On the other hand, various technologies applicable to second-generation antibody drugs have been developed, and technologies for improving effector function, antigen binding ability, pharmacokinetics, and stability, or reducing the risk of immunogenicity, etc. have been reported (Non-Patent Document 3). Since antibody drugs generally require a very high dose, problems include the difficulty of preparing a subcutaneous formulation and the high manufacturing cost. Methods for reducing the dose of antibody drugs include a method for improving the pharmacokinetics of the antibody and a method for improving the affinity between the antibody and the antigen.

[0003] Artificial amino acid substitution in the constant region has been reported as a method for improving the pharmacokinetics of antibodies (Non-Patent Documents 4 and 5). Affinity maturation technology (Non-Patent Document 6) has been reported as a technique for enhancing antigen-binding ability and antigen-neutralizing ability, and it is possible to enhance binding activity to an antigen by introducing mutations into amino acids in the CDR region of the variable region, etc. By enhancing antigen-binding ability, it is possible to improve in vitro biological activity or reduce the dosage, and further to improve the drug efficacy in vivo (in a living organism) (Non-Patent Document 7).

[0004] On the other hand, the amount of antigen that can be neutralized by one antibody molecule depends on the affinity, and by strengthening the affinity, it is possible to neutralize the antigen with a small amount of antibody, and it is possible to strengthen the affinity of an antibody by various methods (Non-Patent Document 6). Furthermore, if it is possible to covalently bind to the antigen and make the affinity infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of bivalent antibodies) with one molecule of antibody. However, in the previous methods, the stoichiometric neutralization reaction of one molecule of antigen (two antigens in the case of bivalent antibodies) with one molecule of antibody was the limit, and it was impossible to completely neutralize the antigen with an antibody amount less than the amount of antigen. In other words, there was a limit to the effect of strengthening affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain its neutralizing effect for a certain period of time, it is necessary to administer an antibody amount equal to or greater than the amount of antigen produced in the body during that period, and there was a limit to reducing the required antibody dosage only by the above-mentioned improvement of the pharmacokinetics of the antibody or affinity maturation technology. Therefore, in order to maintain the neutralizing effect of an antigen for a desired period of time with an antibody amount equal to or less than the antigen amount, it is necessary to neutralize multiple antigens with one antibody. As a new method for achieving this, an antibody that binds to an antigen in a pH-dependent manner has recently been reported (Patent Document 1). A pH-dependent antigen-binding antibody that strongly binds to an antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in endosomes can dissociate from the antigen in endosomes. A pH-dependent antigen-binding antibody can bind to an antigen again when the antibody is recycled into plasma by FcRn after dissociating from the antigen, so that one pH-dependent antigen-binding antibody can repeatedly bind to multiple antigens.

[0005] In addition, the plasma retention of antigens is very short compared to antibodies that bind to FcRn and are recycled. When an antibody with such a long plasma retention binds to the antigen, the plasma retention of the antibody-antigen complex becomes as long as that of the antibody. Therefore, when an antigen binds to an antibody, the plasma retention of the antigen becomes longer, and the plasma antigen concentration increases.

[0006] Thus, a pH-dependent antigen-binding antibody binds to multiple antigens with a single antibody and can promote the elimination of antigens from plasma compared to a normal antibody, and therefore has effects that normal antibodies cannot achieve. However, no antibody engineering techniques have been reported to date that can further improve the effect of this pH-dependent antigen-binding antibody in repeatedly binding to antigens and in promoting the elimination of antigens from plasma.

[0007] IgG antibodies have a long plasma retention time by binding to FcRn. Binding between IgG and FcRn is observed only under acidic conditions (pH 6.0), and almost no binding is observed under neutral conditions (pH 7.4). IgG antibodies are nonspecifically taken up by cells, but return to the cell surface by binding to FcRn in endosomes under acidic conditions in endosomes, and dissociate from FcRn under neutral conditions in plasma. If a mutation is introduced into the Fc region of IgG to lose the binding to FcRn under acidic pH conditions, the antibody will no longer be recycled from endosomes to plasma, and the plasma retention time of the antibody will be significantly impaired. As a method for improving the plasma retention of IgG antibodies, a method for improving the binding to FcRn under acidic pH conditions has been reported. By introducing amino acid substitutions into the Fc region of IgG antibodies to improve the binding to FcRn under acidic pH conditions, the recycling efficiency from endosomes to plasma is increased, and as a result, the plasma retention time is improved.

[0008] Many studies have been conducted on the effector functions of IgG class antibodies, antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) and complement-dependent cytotoxicity (hereinafter referred to as CDC), and it has been reported that, among the human IgG class, antibodies of the IgG1 subclass have the highest ADCC and CDC activities (Non-Patent Document 13). In addition, antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG class antibodies, has also been suggested as one of the effector functions of antibodies (Non-Patent Documents 14 and 15). Since IgG1 subclass antibodies are capable of exerting these effector functions against tumors, IgG1 subclass antibodies are used as most antibody drugs against cancer antigens.

[0009] In order for an IgG antibody to mediate ADCC and ADCP activity, the Fc region of the IgG antibody must bind to an antibody receptor (hereinafter referred to as Fcγ receptor or FcγR) present on the surface of effector cells such as killer cells, natural killer cells, activated macrophages, etc. In humans, the protein family of Fcγ receptors has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and each allotype has also been reported (Non-Patent Document 16).

[0010] Enhancement of cytotoxic effector functions such as ADCC and ADCP has attracted attention as a promising means for enhancing the antitumor effect of anticancer antibodies. The importance of effector functions mediated by Fcγ receptors for the antitumor effect of antibodies has been reported using mouse models (Non-Patent Document 17, Non-Patent Document 18). In addition, a correlation was observed between clinical effects in humans and the high affinity polymorphism (V158) and low affinity polymorphism (F158) of FcγRIIIa (Non-Patent Document 19). These reports suggest that antibodies with Fc regions optimized for binding to specific Fcγ receptors mediate stronger effector functions, thereby exerting effective antitumor effects. The balance of the affinity of an antibody for each of activating receptors including FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and inhibitory receptors including FcγRIIb, is an important factor in optimizing the effector function of an antibody. Since it is possible to endow antibodies with the ability to mediate stronger effector functions by increasing their affinity for activated receptors (Non-Patent Document 20), various antibody engineering techniques have been reported to enhance or improve the antitumor activity of antibody drugs against cancer antigens.

[0011] It has been shown that the binding of the Fc region to the Fcγ receptor is important for several amino acid residues in the hinge region and CH2 domain of the antibody and for the sugar chain attached to Asn at EU numbering position 297, which is attached to the CH2 domain (Non-Patent Document 13, Non-Patent Document 21, Non-Patent Document 22). Focusing on this binding site, various Fc region mutants with Fcγ receptor binding properties have been studied, and Fc region mutants with higher affinity for activated Fcγ receptors have been obtained (Patent Document 2, Patent Document 3). For example, Lazar et al. have succeeded in increasing the binding of human IgG1 to human FcγRIIIa (V158) by about 370-fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332, as represented by EU numbering, of human IgG1 with Asp, Leu, and Glu, respectively (Non-Patent Document 23, Patent Document 3). This variant has a binding ratio (A / I ratio) to FcγRIIIa and FcγRIIb of about 9 times that of the native form. Shinkawa et al. have succeeded in increasing the binding to FcγRIIIa by about 100 times by deleting the fucose of the sugar chain attached to Asn at position 297 (EU numbering) (Non-Patent Document 24). These methods make it possible to significantly improve the ADCC activity of human IgG1 compared to native human IgG1.

[0012] Thus, in antibodies targeting membrane antigens, the binding activity to Fcγ receptors plays an important role in the cytotoxic activity, and therefore when cytotoxic activity is required, human IgG1 isotypes with high binding activity to FcγR are used, and it is a widely used technique to further enhance the cytotoxic activity by enhancing the binding activity to Fcγ receptors. On the other hand, in antibodies targeting soluble antigens, the role of the binding activity to Fcγ receptors is not known, and it has been thought that there is no difference in effect between human IgG1, which has high binding activity to Fcγ receptors, and human IgG2 and human IgG4, which have low binding activity to FcγR. Therefore, there have been no attempts to enhance the binding activity to Fcγ receptors in antibodies targeting soluble antigens, and no reports have been published on the effect. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] WO2009 / 125825 [Patent Document 2] WO2000 / 042072 [Patent Document 3] WO2006 / 019447 [Non-patent literature]

[0014] [Non-Patent Document 1] Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic., Nat. Biotechnol. (2005) 23, 1073-1078 [Non-Patent Document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. (2005) 59 (3), 389-396 Non-Patent Document 3 Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. (2005) 20 (1), 17-29 Non-Patent Document 4 Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J. Immunol. (2006) 176 (1), 346-356 Non-Patent Document 5 Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat. Biotechnol. (1997) 15 (7), 637-640 Non-Patent Document 6 Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc. Natl. Acad. Sci. U. S. A. (2005) 102 (24), 8466-8471

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Summary of the Invention

Problems to be Solved by the Invention

[0015] The present invention has been made in consideration of these circumstances, and an object of the present invention is to provide antigen-binding molecules that promote intracellular uptake of bound antigens, antigen-binding molecules that increase the number of antigens that can bind per molecule, antigen-binding molecules with improved pharmacokinetics, antigen-binding molecules that promote intracellular dissociation of extracellularly bound antigens, antigen-binding molecules that promote extracellular release in an unbound state, antigen-binding molecules that have the function of reducing the total or free antigen concentration in plasma, pharmaceutical compositions containing the antigen-binding molecules, and methods for producing them. [Means for solving the problem]

[0016] The present inventors have conducted intensive research to achieve the above-mentioned object, and have created an antigen-binding molecule comprising an antigen-binding domain that has binding activity to human FcRn under acidic pH conditions and whose antigen-binding activity changes depending on ion concentration conditions, and an Fcγ receptor-binding domain that has higher Fcγ receptor-binding activity under neutral pH conditions than the Fcγ receptor-binding domain of the Fc region of native human IgG, in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan. The present inventors have also found a method for promoting intracellular uptake of the antigen to which the antigen-binding molecule is bound, a method for increasing the number of antigens that can be bound by a single antigen-binding molecule, a method for improving the pharmacokinetics of an antigen-binding molecule, a method for promoting dissociation of an antigen bound to an antigen-binding molecule outside a cell from the antigen-binding molecule inside a cell, a method for promoting extracellular release in a state where the antigen is not bound, and a method for reducing the total antigen concentration or free antigen concentration in plasma, which comprises a step of contacting the antigen-binding molecule with a cell expressing an Fcγ receptor in vivo or ex vivo. The present inventors have also found a method for producing an antigen-binding molecule having the above-mentioned properties, and have found the usefulness of a pharmaceutical composition containing such an antigen-binding molecule or an antigen-binding molecule produced by the production method according to the present invention as an active ingredient, thereby completing the present invention.

[0017] That is, more specifically, the present invention provides the following [1] to

[46] . [1] A pharmaceutical composition comprising an antigen-binding molecule comprising an antigen-binding domain that has binding activity to human FcRn under an acidic pH range condition and whose antigen-binding activity changes depending on ion concentration conditions, and an Fcγ receptor-binding domain whose binding activity to Fcγ receptor under a neutral pH range condition is higher than the binding activity to Fcγ receptor of an Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan. [2] The pharmaceutical composition according to [1], wherein the antigen is a soluble antigen. [3] The pharmaceutical composition according to [1] or [2], wherein the ion concentration is a calcium ion concentration. [4] The pharmaceutical composition according to [3], wherein the antigen-binding domain has higher antigen-binding activity under a high calcium ion concentration than under a low calcium ion concentration. [5] The pharmaceutical composition according to [1] or [2], wherein the ion concentration conditions are pH conditions. [6] The pharmaceutical composition according to [5], wherein the antigen-binding domain has higher antigen-binding activity in a neutral pH range than in an acidic pH range. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the antigen-binding molecule has neutralizing activity against the antigen. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the Fcγ receptor binding domain comprises an Fc region of an antibody. [9] The Fc region comprises a amino acid sequence selected from the group consisting of amino acids 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, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 51st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd , 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 29 2nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th The pharmaceutical composition of [8], wherein the Fc region contains at least one or more amino acids selected from the group consisting of positions 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440, which differ from the amino acids at the corresponding positions in the native Fc region.

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

[11] The pharmaceutical composition according to any one of [1] to

[10] , wherein the Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan is an Fc region of native human IgG1, native human IgG2, native human IgG3, or native human IgG4 in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan.

[12] The pharmaceutical composition according to any one of [1] to

[11] , wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

[13] The pharmaceutical composition according to any one of [1] to

[11] , wherein the human Fcγ receptor is FcγRIIb.

[14] The Fc region is a region represented by EU numbering of an Fc region; The amino acid at position 238 is Asp, or The amino acid at position 328 is Glu; The pharmaceutical composition according to any one of [8] to

[13] , wherein the Fc region comprises at least one amino acid selected from the group consisting of:

[15] any of the following methods (i) to (vi), comprising the step of contacting an antigen-binding molecule, which comprises an antigen-binding domain that has human FcRn-binding activity under an acidic pH range condition and whose antigen-binding activity changes depending on ion concentration conditions, and an Fcγ receptor-binding domain whose Fcγ receptor-binding activity under a neutral pH range condition is higher than the Fcγ receptor-binding activity of the Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan, with a cell that expresses an Fcγ receptor, in vivo or in vitro; (i) A method for increasing the number of antigens that can be bound by a single antigen-binding molecule, (ii) a method for eliminating antigens in plasma; (iii) a method for improving the pharmacokinetics of an antigen-binding molecule; (iv) A method for promoting dissociation of an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly. (v) A method for promoting the extracellular release of an antigen-binding molecule in a state where the antigen is not bound to the antigen, or (vi) A method for decreasing the total or free antigen concentration in plasma.

[16] The method according to

[15] , wherein the antigen is a soluble antigen.

[17] The method according to

[15] or

[16] , wherein the ion concentration is calcium ion concentration.

[18] The method according to

[17] , wherein the antigen-binding domain has higher antigen-binding activity under a high calcium ion concentration than under a low calcium ion concentration.

[19] The method according to

[15] or

[16] , wherein the ion concentration conditions are pH conditions.

[20] The method according to

[19] , wherein the antigen-binding domain has higher antigen-binding activity under a neutral pH range condition than under an acidic pH range condition.

[21] The method according to any one of

[15] to

[20] , wherein the antigen-binding molecule has neutralizing activity against the antigen.

[22] The method according to any one of

[15] to

[21] , wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[23] The Fc region comprises a sequence selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, and 251 of the EU numbering site of the Fc region. 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 2 92nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th , 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 33 The method according to

[22] , wherein the Fc region contains at least one or more amino acids selected from the group consisting of 5, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440, which differ from the amino acids at the corresponding positions in the native Fc region.

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

[23] , wherein the Fc region comprises at least one amino acid selected from the group consisting of:

[25] The method according to any one of

[15] to

[24] , wherein the Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan is an Fc region of any one of native human IgG1, native human IgG2, native human IgG3, and native human IgG4 in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan.

[26] The method described in any of

[15] to

[25] , wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

[27] The method according to any one of

[15] to

[25] , wherein the human Fcγ receptor is FcγRIIb.

[28] The Fc region is a region represented by EU numbering of an Fc region: The amino acid at position 238 is Asp, or The amino acid at position 328 is Glu; The method according to any one of

[22] to

[27] , wherein the Fc region comprises at least one amino acid of the following:

[29] A method according to any one of the following (i) to (vii), comprising a step of enhancing the Fcγ receptor-binding activity of an Fcγ receptor-binding domain under a neutral pH condition in an antigen-binding molecule comprising an antigen-binding domain that has human FcRn-binding activity under an acidic pH condition and whose antigen-binding activity changes depending on ion concentration conditions, and an Fcγ receptor-binding domain, more than the Fcγ receptor-binding activity under a neutral pH condition of an Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan; (i) A method for modifying an antigen-binding molecule that enhances intracellular uptake of the antigen to which it is bound; (ii) A method for increasing the number of antigens that can be bound by a single antigen-binding molecule; (iii) A method for increasing the plasma antigen elimination ability of an antigen-binding molecule; (iv) A method for improving the pharmacokinetics of an antigen-binding molecule; (v) A method for promoting dissociation of an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly. (vi) A method for promoting the extracellular release of an antigen-binding molecule that has been taken up into a cell in a state bound to an antigen, in a state where the antigen is not bound to the antigen; or (vii) A method for modifying an antigen-binding molecule capable of reducing the total antigen concentration or free antigen concentration in plasma.

[30] The method according to

[29] , wherein the antigen is a soluble antigen.

[31] The method according to

[29] or

[30] , wherein the ion concentration is calcium ion concentration.

[32] The method according to

[31] , wherein the antigen-binding domain has higher antigen-binding activity under a high calcium ion concentration than under a low calcium ion concentration.

[33] The method according to

[29] or

[30] , wherein the ion concentration conditions are pH conditions.

[34] The method according to

[33] , wherein the antigen-binding domain has higher antigen-binding activity under a neutral pH range condition than under an acidic pH range condition.

[35] The method according to any one of

[29] to

[34] , wherein the antigen-binding molecule has neutralizing activity against the antigen.

[36] The method according to any one of

[29] to

[35] , wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[37] The Fc region comprises a sequence selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 32 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 29th 1st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th , 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 3 The method according to

[36] , wherein the Fc region contains one or more amino acids at positions 34, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 that differ from the amino acids at the corresponding positions in a native Fc region.

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

[33] , wherein the Fc region comprises at least one amino acid selected from the group consisting of:

[39] The method according to any one of

[29] to

[38] , wherein the Fc region of native human IgG in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan is an Fc region of any one of native human IgG1, native human IgG2, native human IgG3, and native human IgG4 in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan.

[40] The method described in any of

[29] to

[39] , wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

[41] The method according to any one of

[29] to

[39] , wherein the human Fcγ receptor is FcγRIIb.

[42] The Fc region comprises a region represented by EU numbering of the Fc region: The amino acid at position 238 is Asp, or The amino acid at position 328 is Glu; The method according to any one of

[36] to

[41] , wherein the Fc region comprises at least one amino acid of the following:

[43] The steps (a) to (f) below, (a) determining the antigen-binding activity of the antigen-binding domain under a high calcium ion concentration condition; (b) determining the antigen-binding activity of the antigen-binding domain under a low calcium ion concentration condition; (c) selecting an antigen-binding domain having a higher antigen-binding activity than that obtained in (a) and (b); (d) linking the polynucleotide encoding the antigen-binding domain selected in (c) to a polynucleotide encoding an Fcγ receptor-binding domain that has human FcRn-binding activity under an acidic pH condition and has higher Fcγ receptor-binding activity under a neutral pH condition than the Fcγ receptor-binding activity of an Fc region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain; (e) culturing a cell into which the vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and (f) recovering the antigen-binding molecule from the culture medium of the cells cultured in (e); A method for producing an antigen-binding molecule comprising the steps of:

[44] The steps (a) to (f) below, (a) obtaining the antigen-binding activity of an antibody under a high calcium ion concentration condition; (b) determining the antigen-binding activity of the antibody under a low calcium ion concentration condition; (c) selecting an antibody having an antigen-binding activity higher than that obtained in (a) and (b); (d) linking the polynucleotide encoding the antigen-binding domain of the antibody selected in (c) to a polynucleotide encoding an Fcγ receptor-binding domain that has human FcRn-binding activity in an acidic pH range and has higher Fcγ receptor-binding activity under a neutral pH range condition than the Fcγ receptor-binding activity of an Fc region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain; (e) culturing a cell into which the vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and (f) recovering the antigen-binding molecule from the culture medium of the cells cultured in (e); A method for producing an antigen-binding molecule comprising the steps of:

[45] The steps (a) to (f) below, (a) determining the antigen-binding activity of the antigen-binding domain under a neutral pH range condition; (b) obtaining the antigen-binding activity of the antigen-binding domain under an acidic pH range condition; (c) selecting an antigen-binding domain whose antigen-binding activity obtained in (a) is higher than that obtained in (b); (d) linking the polynucleotide encoding the antigen-binding domain selected in (c) to a polynucleotide encoding an Fcγ receptor-binding domain that has human FcRn-binding activity under an acidic pH condition and has higher Fcγ receptor-binding activity under a neutral pH condition than the Fcγ receptor-binding activity of an Fc region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain; (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and (f) recovering the antigen-binding molecule from the culture medium of the cells cultured in (e); A method for producing an antigen-binding molecule comprising the steps of:

[46] The steps (a) to (f) below, (a) obtaining the antigen-binding activity of an antibody under a neutral pH range condition; (b) obtaining the antigen-binding activity of the antibody against the antigen under an acidic pH range condition; (c) selecting an antibody having an antigen-binding activity higher than that obtained in (a) and (b); (d) linking the polynucleotide encoding the antigen-binding domain of the antibody selected in (c) to a polynucleotide encoding an Fcγ receptor-binding domain that has human FcRn-binding activity under an acidic pH condition and has higher Fcγ receptor-binding activity under a neutral pH condition than the Fcγ receptor-binding activity of an Fc region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain; (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and (f) recovering the antigen-binding molecule from the culture medium of the cells cultured in (e); A method for producing an antigen-binding molecule comprising the steps of:

[47] The method according to any one of

[43] to

[46] , wherein the antigen is a soluble antigen.

[48] ​​The method for production described in any one of

[43] to

[47] , wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[49] The Fc region comprises a sequence selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, and 251 of the EU numbering site of the Fc region. 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 29th 2nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th The method of producing the Fc region according to

[48] , wherein the Fc region contains at least one amino acid selected from the group consisting of positions 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440, which differ from the amino acids at the corresponding positions in the native Fc region.

[50] The Fc region is a region represented by EU numbering of an Fc region; The amino acid at position 221 is either Lys or Tyr; The amino acid at position 222 is Phe, Trp, Glu, or Tyr; The amino acid at position 223 is Phe, Trp, Glu, or Lys; The amino acid at position 224 is Phe, Trp, Glu, or Tyr; The amino acid at position 225 is either Glu, Lys or Trp; The amino acid at position 227 is Glu, Gly, Lys, or Tyr; The amino acid at position 228 is Glu, Gly, Lys, or Tyr; The amino acid at position 230 is Ala, Glu, Gly, or Tyr; The amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr; The amino acid at position 232 is Glu, Gly, Lys, or Tyr; the amino acid at position 233 is any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 238 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 239 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 240 is Ala, Ile, Met, or Thr; The amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or Tyr; The amino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The amino acid at position 244 is His; The amino acid at position 245 is Ala; The amino acid at position 246 is Asp, Glu, His, or Tyr; the amino acid at position 247 is any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The amino acid at position 249 is Glu, His, Gln, or Tyr; The amino acid at position 250 is either Glu or Gln; The amino acid at position 251 is Phe; The amino acid at position 254 is either Phe, Met, or Tyr; The amino acid at position 255 is either Glu, Leu or Tyr; The amino acid at position 256 is either Ala, Met, or Pro; The amino acid at position 258 is Asp, Glu, His, Ser, or Tyr; The amino acid at position 260 is Asp, Glu, His, or Tyr; The amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; the amino acid at position 263 is Ala, Ile, Met, or Thr; the amino acid at position 264 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 265 is any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 is Ala, Ile, Met, or Thr; the amino acid at position 267 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 268 is Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp; the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 270 is any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 272 is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 273 is either Phe or Ile; the amino acid at position 274 is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 275 is either Leu or Trp; The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 278 is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 279 is Ala; The amino acid at position 280 is any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 is Asp, Lys, Pro, or Tyr; The amino acid at position 282 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 283 is any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; The amino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or Tyr; The amino acid at position 285 is Asp, Glu, Lys, Gln, Trp, or Tyr; The amino acid at position 286 is either Glu, Gly, Pro, or Tyr; The amino acid at position 288 is Asn, Asp, Glu, or Tyr; the amino acid at position 290 is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr, and the amino acid at position 291 is Asp, Glu, Gly, His, Ile, Gln, or Thr; The amino acid at position 292 is Ala, Asp, Glu, Pro, Thr, or Tyr; the amino acid at position 293 is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 294 is any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 296 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the amino acid at position 297 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 298 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 300 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 301 is Asp, Glu, His, or Tyr; The amino acid at position 302 is Ile; The amino acid at position 303 is Asp, Gly, or Tyr; the amino acid at position 304 is Asp, His, Leu, Asn, or Thr; The amino acid at position 305 is either Glu, Ile, Thr or Tyr; the amino acid at position 311 is Ala, Asp, Asn, Thr, Val, or Tyr; The amino acid at position 313 is Phe; The amino acid at position 315 is Leu; The amino acid at position 317 is Glu or Gln; The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; The amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 322 is any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 323 is Ile; the amino acid at position 324 is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 325 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 is any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 328 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 330 is any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 332 is any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 is Ala, Glu, Phe, Ile, Leu, Pro, or Thr; the amino acid at position 335 is Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 336 is either Glu, Lys or Tyr; The amino acid at position 337 is either Glu, His, or Asn; the amino acid at position 339 is Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr; The amino acid at position 376 is either Ala or Val; The amino acid at position 377 is either Gly or Lys; The amino acid at position 378 is Asp; The amino acid at position 379 is Asn; The amino acid at position 380 is Ala, Asn, or Ser; The amino acid at position 382 is either Ala or Ile; The amino acid at position 385 is Glu; The amino acid at position 392 is Thr; The amino acid at position 396 is Leu; The amino acid at position 421 is Lys; The amino acid at position 427 is Asn; The amino acid at position 428 is either Phe or Leu; The amino acid at position 429 is Met; The amino acid at position 434 is Trp; The amino acid at position 436 is Ile, and The amino acid at position 440 is Gly, His, Ile, Leu, or Tyr; The method for producing the Fc region according to claim 49, wherein the Fc region contains at least one amino acid selected from the group consisting of:

[51] The production method according to any one of

[43] to

[50] , wherein the Fcγ receptor binding domain is an Fc region of any of native human IgG1, native human IgG2, native human IgG3, or native human IgG4, in which the glycan attached to position 297 (EU numbering) is a fucose-containing glycan.

[52] A method for producing according to any one of

[43] to

[51] , wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

[53] A method for producing a human Fcγ receptor described in any one of

[43] to

[51] , wherein the human Fcγ receptor is FcγRIIb.

[54] The Fc region comprises a region represented by EU numbering of an Fc region: The amino acid at position 238 is Asp, or The amino acid at position 328 is Glu; A method for producing an Fc region comprising at least one amino acid of any one of

[48] to

[53] . [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a non-limiting mechanism of action by which soluble antigens are eliminated from plasma by administration of an antibody that binds to an antigen in an ion concentration-dependent manner and has enhanced Fcγ receptor binding at neutral pH compared to existing neutralizing antibodies. [Diagram 2] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when H54 / L28-IgG1 or Fv4-IgG1, which binds to human IL-6 receptor in a pH-dependent manner, was administered to the mice. [Diagram 3] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when the mice were administered Fv4-IgG1, which binds to human IL-6 receptor in a pH-dependent manner, Fv4-IgG1-F760, a modified Fv4-IgG1 that lacks mouse FcγR binding, Fv4-IgG1-F1022, a modified Fv4-IgG1 with enhanced mouse FcγR binding, or Fv4-IgG1-Fuc, a low-fucose version of Fv4-IgG1. [Figure 4] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when the mice were administered with antigen-binding molecules comprising, as a heavy chain, Fv4-IgG1, Fv4-IgG1-F1022, or Fv4-IgG1-F1093, a modified form of Fv4-IgG1-F1022 that has improved FcRn binding in the acidic pH range. [Diagram 5]FIG. 1 shows the concentration time course of the antigen-binding molecule in the plasma of human FcRn transgenic mice when the antigen-binding molecule contains, as a heavy chain, Fv4-IgG1, Fv4-IgG1-F1022, or Fv4-IgG1-F1093, a modified form of Fv4-IgG1-F1022 that has improved FcRn binding in the acidic pH range. [Figure 6] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1087, a variant of Fv4-IgG1 with enhanced mouse FcγR binding (particularly enhanced binding to mouse FcγRIIb and mouse FcγRIII), or Fv4-IgG1-F1182, a variant of Fv4-IgG1 with enhanced mouse FcγR binding (particularly enhanced binding to mouse FcγRI and mouse FcγRIV), were administered to the mice. [Figure 7] FIG. 1 shows the time course of concentration of the antigen-binding molecules in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified forms of Fv4-IgG1-F1087 with improved FcRn binding in the acidic pH range, were administered to the mice. [Figure 8] FIG. 1 shows the time course of concentration of the antigen-binding molecules in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1182, or Fv4-IgG1-F1181, a modified form of Fv4-IgG1-F1182 with improved FcRn binding in the acidic pH range, was administered to the mice. [Figure 9] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are modified forms of Fv4-IgG1-F1087 with improved FcRn binding in the acidic pH range, were administered to the mice. [Figure 10] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1182, or Fv4-IgG1-F1181, a modified form of Fv4-IgG1-F1182 with improved FcRn binding in the acidic pH range, was administered to the mice. [Figure 11] FIG. 1 shows the results over time of plasma Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 concentrations in human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 was administered to the mice. [Figure 12] FIG. 1 shows the time course of soluble human IL-6 receptor concentration in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 was administered to the mice. [Figure 13] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of normal mice when Fv4-mIgG1, Fv4-mIgG1-mF44, a modified Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, and Fv4-mIgG1-mF46, a modified Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII, were administered to the mice. [Figure 14] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIII-deficient mice when Fv4-mIgG1, Fv4-mIgG1-mF44, a modified Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, and Fv4-mIgG1-mF46, a modified Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII, were administered to the mice. [Figure 15]FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of Fc receptor γ-chain-deficient mice when Fv4-mIgG1, Fv4-mIgG1-mF44, a modified Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, and Fv4-mIgG1-mF46, a modified Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII, were administered to the mice. [Figure 16] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIIb-deficient mice when the mice were administered Fv4-mIgG1, Fv4-mIgG1-mF44, a modified Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a modified Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 17] FIG. 13 shows the results of evaluating the platelet aggregation activity of omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets derived from donors with the FcγRIIa polymorphism (R / H). [Figure 18] FIG. 13 shows the results of evaluating the platelet aggregation activity of omalizumab-G1d-v3 / IgE immune complexes in a platelet aggregation assay using platelets derived from a donor with the FcγRIIa polymorphism (H / H). [Figure 19] This is a diagram showing the results of evaluating CD62p expression on the membrane surface of washed platelets. The graphs filled with black show the results when platelets were reacted with PBS and then stimulated with ADP, and the graphs with no black dots show the results when platelets were reacted with immune complexes and then stimulated with ADP. [Figure 20] 1 shows the results of evaluating the expression of activated integrin on the membrane surface of washed platelets, with the black bars indicating the results when platelets were reacted with PBS and then stimulated with ADP, and the black bars indicating the results when platelets were reacted with immune complexes and then stimulated with ADP. [Figure 21] FIG. 13 shows the results of evaluating the platelet aggregation ability of omalizumab-G1d-v3 / IgE immune complex and omalizumab-BP230 / IgE immune complex in a platelet aggregation assay using platelets derived from a donor having the FcγRIIa polymorphism (R / H). [Figure 22] This is a diagram showing the results of evaluating CD62p expression on the membrane surface of washed platelets. The gray bars indicate the results when the platelets were reacted with PBS and then stimulated with ADP, while the solid line indicates the results when the platelets were reacted with omalizumab-G1d-v3 / IgE immune complex and the dotted line indicates the results when the platelets were reacted with omalizumab-BP230 / IgE immune complex and then stimulated with ADP. [Diagram 23] This figure shows the results of evaluating the expression of activated integrin on the membrane surface of washed platelets. The gray bars indicate the results when the platelets were reacted with PBS and then stimulated with ADP, while the solid line indicates the results when the platelets were reacted with omalizumab-G1d-v3 / IgE immune complex and the dotted line indicates the results when the platelets were reacted with omalizumab-BP230 / IgE immune complex and then stimulated with ADP. [Figure 24] The horizontal axis shows the relative binding activity of each PD variant to FcγRIIb, and the vertical axis shows the relative binding activity of each PD variant to FcγRIIa R. The binding amount of each PD variant to each FcγR was divided by the binding amount of IL6R-F652 / IL6R-L (IL6R-F652 is an antibody heavy chain containing an altered Fc in which Pro at position 238 (EU numbering) is replaced with Asp, as defined by SEQ ID NO: 142), which is a control antibody before the introduction of alterations, to each FcγR, and the value was further multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The plot of F652 in the figure shows the value for IL6R-F652 / IL6R-L. [Diagram 25]The vertical axis shows the relative binding activity to FcγRIIb of variants obtained by introducing each alteration into GpH7-B3 (SEQ ID NO: 159) / GpL16-k0 (SEQ ID NO: 160) that does not have the P238D alteration, and the horizontal axis shows the relative binding activity to FcγRIIb of variants obtained by introducing each alteration into IL6R-F652 (SEQ ID NO: 142) / IL6R-L that has the P238D alteration. The value of the binding amount of each variant to FcγRIIb was divided by the value of the binding amount of the antibody to FcγRIIb before the alteration was introduced, and the value was further multiplied by 100 to obtain the relative binding activity value. Here, modifications that exert an FcγRIIb binding-enhancing effect when introduced into GpH7-B3 / GpL16-k0 that does not have P238D and when introduced into IL6R-F652 / IL6R-L that has P238D are included in region A, and modifications that exert an FcγRIIb binding-enhancing effect when introduced into GpH7-B3 / GpL16-k0 that does not have P238D but do not exert an FcγRIIb binding-enhancing effect when introduced into IL6R-F652 / IL6R-L that has P238D are included in region B. [Figure 26] 1 shows the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex. [Figure 27] FIG. 1 shows a diagram of a superposition of the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex and the model structure of the Fc (WT) / FcγRIIb extracellular region complex, performed by a least-squares method based on the Cα atomic distances for the FcγRIIb extracellular region and Fc CH2 domain A. [Figure 28] The crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex and the model structure of the Fc (WT) / FcγRIIb extracellular region complex were superimposed by the least-squares method based on the Cα atomic distances for Fc CH2 domain A and Fc CH2 domain B alone, and the detailed structures around P238D were compared. [Figure 29]FIG. 13 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, a hydrogen bond is observed between the main chain of Gly at position 237 (EU numbering) in Fc CH2 domain A and Tyr at position 160 in FcγRIIb. [Diagram 30] FIG. 13 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, electrostatic interaction is observed between Asp at position 270 (EU numbering) in Fc CH2 domain B and Arg at position 131 in FcγRIIb. [Diagram 31] The horizontal axis shows the relative binding activity of each 2B variant to FcγRIIb, and the vertical axis shows the relative binding activity of each 2B variant to FcγRIIa R. The binding amount of each 2B variant to each FcγR was divided by the binding amount of the control antibody before the introduction of the alteration (altered Fc in which Pro at position 238 (EU numbering) was replaced with Asp) to each FcγR, and the resulting value was multiplied by 100 to obtain the relative binding activity of each 2B variant to each FcγR. [Diagram 32] FIG. 13 shows Glu at position 233 (EU numbering) of Fc Chain A and its surrounding residues in the extracellular region of FcγRIIb in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Diagram 33] FIG. 13 shows Ala at position 330 (EU numbering) of Fc Chain A and its surrounding residues in the extracellular region of FcγRIIb in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Diagram 34] FIG. 1 shows the structure of Pro at position 271 (EU numbering) of Fc Chain B, obtained by superimposing the crystal structures of the Fc (P238D) / FcγRIIb extracellular domain complex and the Fc (WT) / FcγRIIIa extracellular domain complex using the least-squares method based on the Cα atomic distances with respect to Fc Chain B. [Diagram 35]This is a diagram of the Fc (P208) / FcγRIIb extracellular region complex determined by X-ray crystal structure analysis. For the CH2 domain and CH3 domain of the Fc portion, the left side is domain A and the right side is domain B. [Diagram 36] The structure of the Fc (P208) / FcγRIIb extracellular region complex determined by X-ray crystal structure analysis and the structure of the Fc (WT) / FcγRIIa extracellular region complex (PDB code: 3RY6) were superimposed by the least squares method based on the Cα atomic distance in the Fc CH2 domain A and compared. The structure drawn with thick lines is the Fc (P208) / FcγRIIb extracellular region complex, and the structure drawn with thin lines is the structure of the Fc (WT) / FcγRIIa extracellular region complex. Note that in the structure of the Fc (WT) / FcγRIIa extracellular region complex, only the Fc CH2 domain A is drawn. [Figure 37] This shows detailed structure of the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, in the vicinity of Asp at position 237 (EU numbering) of Fc portion CH2 domain A, which forms a hydrogen bond in the main chain with Tyr at position 160 of FcγRIIb. [Figure 38] FIG. 1 shows the structure of amino acid residues around the side chain of Asp at position 237 (EU numbering) of Fc portion CH2 domain A, which forms a hydrogen bond in the main chain with Tyr at position 160 of FcγRIIb in the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex. [Figure 39]This figure shows a comparison of the X-ray crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex shown in Example 10 and the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex around the loop from positions 266 to 271 (EU numbering) in the CH2 domain B of the Fc portion, which were superimposed by the least squares method based on the Cα atomic distances. In this loop, Fc (P208) has an H268D alteration at position 268 (EU numbering) and a P271G alteration at position 271 (EU numbering) compared to Fc (P238D). [Diagram 40] FIG. 1 shows the structure around Ser239 in CH2 domain B of the Fc portion in the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density with 2Fo-Fc coefficients obtained by X-ray crystal structure analysis. [Diagram 41] The three-dimensional structure of the Fc (P208) / FcγRIIaR extracellular domain complex determined by X-ray crystal structure analysis and the three-dimensional structure of the Fc (P208) / FcγRIIb extracellular domain complex were superimposed by the least-squares method based on the distances between Cα atoms, and compared. [Diagram 42] FIG. 13 shows a comparison of the X-ray crystal structure of the Fc (P208) / FcγRIIaR extracellular region complex with the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density (2Fo-Fc coefficient) obtained by X-ray crystal structure analysis near Asp at position 237 (EU numbering) in CH2 domain A of the Fc portion. [Diagram 43] FIG. 13 shows a comparison of the X-ray crystal structure of the Fc (P208) / FcγRIIaR extracellular region complex with the X-ray crystal structure of the Fc (P208) / FcγRIIb extracellular region complex, together with the electron density (2Fo-Fc coefficient) obtained by X-ray crystal structure analysis near Asp at position 237 (EU numbering) in CH2 domain B of the Fc portion. [Diagram 44] 1 shows a comparison of the constant region sequences of G1d and G4d, in which the amino acids enclosed in bold frames indicate the positions where the amino acid residues differ between G1d and G4d. [Diagram 45] FIG. 1 shows time courses of plasma antibody concentrations of GA2-IgG1 and GA2-F1087 in normal mice. [Figure 46] FIG. 1 shows time courses of plasma hIgA concentration in normal mice administered with GA2-IgG1 and GA2-F1087. [Figure 47] FIG. 1 shows time courses of plasma antibody concentrations of 278-IgG1 and 278-F1087 in C57BL / 6J mice. [Figure 48] FIG. 1 shows the time course of plasma hIgE(Asp6) concentration in C57BL / 6J mice administered 278-IgG1 and 278-F1087. [Figure 49] 1 shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody determined by X-ray crystal structure analysis, (i) shows the heavy chain CDR3 in the crystal structure obtained under crystallization conditions in the presence of calcium ions, and (ii) shows the heavy chain CDR3 in the crystal structure obtained under crystallization conditions in the absence of calcium ions. [Figure 50] FIG. 1 shows the time course of plasma antibody concentrations in normal mice administered with H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody. [Figure 51] FIG. 1 shows the time course of soluble human IL-6 receptor (hsIL-6R) concentration in plasma of normal mice administered with H54 / L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL#9-IgG1 antibody. [Figure 52] 1 shows ion exchange chromatograms of an antibody comprising a human Vk5-2 sequence and an antibody comprising a h Vk5-2_L65 sequence in which the glycosylation sequence in the human Vk5-2 sequence has been modified. The solid line represents a chromatogram of the antibody comprising the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 67 and light chain: hVk5-2, SEQ ID NO: 4), and the dashed line represents a chromatogram of the antibody having the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 67), light chain: hVk5-2_L65 (SEQ ID NO: 70)). [Figure 53A]FIG. 1 shows ion exchange chromatograms of an antibody comprising the LfVk1_Ca sequence (heavy chain: GC_H, SEQ ID NO: 51; and light chain: LfVk1_Ca, SEQ ID NO: 83) and an antibody comprising a sequence in which the Asp (D) residue in the LfVk1_Ca sequence has been modified to an Ala (A) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is set as the main peak, and the y-axis is normalized to the main peak. FIG. 1 shows a chromatogram of an antibody comprising LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 53B] FIG. 13 shows a chromatogram of an antibody comprising LfVk1_Ca1 (sequence number: 85) as a light chain. [Figure 53C] FIG. 13 shows a chromatogram of an antibody containing LfVk1_Ca2 (sequence number: 86) as a light chain. [Fig. 53D] FIG. 13 shows a chromatogram of an antibody comprising LfVk1_Ca3 (sequence number: 87) as a light chain. [Figure 54A] FIG. 1 shows ion exchange chromatograms of an antibody comprising the LfVk1_Ca sequence (heavy chain: GC_H, SEQ ID NO: 51 and light chain: LfVk1_Ca, SEQ ID NO: 83) and an antibody comprising the LfVk1_Ca6 sequence (heavy chain: GC_H, SEQ ID NO: 51 and light chain: LfVk1_Ca6, SEQ ID NO: 88) in which the Asp (D) residue at position 30 (Kabat numbering) in the LfVk1_Ca sequence has been modified to a Ser (S) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is set as the main peak, and the y-axis is normalized to the main peak. FIG. 1 shows a chromatogram of an antibody comprising LfVk1_Ca (SEQ ID NO: 83) as the light chain. [Figure 54B] FIG. 13 shows a chromatogram of an antibody comprising LfVk1_Ca6 (sequence number: 88) as a light chain. [Figure 55]This is a diagram showing the relationship between the amino acid distribution (indicated as Library) and the designed amino acid distribution (indicated as Design) of the sequence information of 290 clones isolated from E. coli into which a Ca-dependent antigen-binding antibody gene library has been introduced. The horizontal axis shows the amino acid site represented by Kabat numbering. The vertical axis shows the amino acid distribution ratio. [Figure 56] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 6RC1IgG_019 antibody under high calcium ion concentration condition (1.2 mM). The horizontal axis shows time, and the vertical axis shows RU value. [Figure 57] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 6RC1IgG_019 antibody under low calcium ion concentration condition (3 μM). The horizontal axis shows time, and the vertical axis shows RU value. [Figure 58] This is a diagram showing the relationship between the amino acid distribution (indicated as Library) of the sequence information of 132 clones isolated from E. coli into which a pH-dependent antigen-binding antibody gene library has been introduced, and the designed amino acid distribution (indicated as Design). The horizontal axis shows the amino acid site represented by Kabat numbering, and the vertical axis shows the amino acid distribution ratio. [Figure 59] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 7.4. The horizontal axis shows time, and the vertical axis shows RU value. [Figure 60] 1 shows sensorgrams of anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 6.0, where the horizontal axis indicates time and the vertical axis indicates RU value. [Figure 61A]FIG. 1 depicts a graphical representation of the ECL response of sera isolated from 15 or 30 independent rheumatoid arthritis patients to native and modified Fc. The graphs show native Fc (Figure 61A), Fv4-YTE (Figure 61B), Fv4-F1166 (=YTE + Q438R / S440E) (Figure 61C), Fv4-F1167 (=YTE + S424N) (Figure 61D), Fv4-LS (Figure 61E), Fv4-F1170 (=LS + Q438R / S440E) (Figure 61F), Fv4-F1171 (=LS + S424N) (Figure 61G), Fv4-N434H (Figure 61H), Fv4-F1172 (=N434H + Q438R / S440E) (Figure 61I), Fv4-F1173 (=N434H + 61C depicts a graphical representation of the ECL response to IgG4-associated IgG4 (S424N) (Figure 61J). [Figure 61B] This is a continuation of Figure 61A. [Figure 61C] This is a continuation of Figure 61B. [Fig.61D] This is a continuation of Figure 61C. [Figure 61E] This is a continuation of Figure 61D. [Fig.61F] This is a continuation of Figure 61E. [Figure 61G] This is a continuation of Figure 61F. [Fig.61H] This is a continuation of Figure 61G. [Fig.61I] This is a continuation of Figure 61H. [Fig.61J] This is a continuation of Figure 61I. [Figure 62A] 62A-62G show graphs of ECL responses of sera isolated from 30 independent rheumatoid arthritis patients to modified Fc. The graphs show graphs of ECL responses to Fv4-LS (FIG. 62A), Fv4-F1380 (FIG. 62B), Fv4-F1384 (FIG. 62C), Fv4-F1385 (FIG. 62D), Fv4-F1386 (FIG. 62E), Fv4-F1388 (FIG. 62F), and Fv4-F1389 (FIG. 62G), respectively. [Figure 62B] This is a continuation of Figure 62A. [Figure 62C]This is a figure showing a continuation of Figure 62B. [Fig.62D] This is a continuation of Figure 62C. [Figure 62E] This is a continuation of Figure 62D. [Fig.62F] This is a continuation of Figure 62E. [Fig.62G] This is a continuation of Figure 62F. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following definitions and detailed descriptions are provided to facilitate the understanding of the invention described herein.

[0020] amino acid As used herein, amino acids are represented by one-letter or three-letter codes, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0021] Amino acid modification To modify amino acids in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. Amino acids can be added, deleted, and / or substituted as appropriate by these known methods. Substitution of an amino acid residue refers to substitution with a different amino acid residue, and is intended to modify, for example, the following points (a) to (c): (a) the backbone structure of the polypeptide in the form of sheet or helical regions; (b) the charge or hydrophobicity at the target site, or (c) Side chain size.

[0022] Amino acid residues are classified into the following groups based on the properties of the side chains contained in their structure: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromaticity: Trp, Tyr, Phe.

[0023] Substitution of amino acid residues within each of these groups is called conservative substitution, while substitution of amino acid residues between other groups is called non-conservative substitution. The substitution in the present invention may be conservative substitution, non-conservative substitution, or a combination of conservative and non-conservative substitution. In addition, as a method for modifying an amino acid by substituting an amino acid other than a natural amino acid, several known methods may also be adopted (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also preferably used.

[0024] Furthermore, as an expression for an amino acid modification, an expression using the one-letter code of the amino acid before and after the modification before and after a number indicating a specific position can be appropriately used. For example, the modification P238D used when making an amino acid substitution in the Fc region contained in the antibody constant region indicates the substitution of Pro at position 238 according to EU numbering with Asp. That is, the number indicates the position of the amino acid according to EU numbering, the one-letter code of the amino acid written before the number indicates the amino acid before the substitution, and the one-letter code of the amino acid written after the number indicates the amino acid after the substitution.

[0025] and / or As used herein, the meaning of the term "and / or" refers to the combination of the terms before and after the phrase "and / or," including any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids at positions 326, 328, and / or 428 are substituted" includes the following amino acid modification variations; (a) 326th place, (b) 328th place, (c) 428th place, (d) 326th place and 328th place, (e) 326th place and 428th place, (f) 328th place and 428th place, (g) 326th place, 328th place and 428th place.

[0026] antigen As used herein, the structure of an "antigen" is not limited to a specific structure as long as it contains an epitope to which an antigen-binding domain binds. In another sense, an antigen may be either inorganic or organic, but a soluble antigen present in the body fluid of a living organism in a form to which the antigen-binding molecule of the present invention can bind is preferred. Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV,UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement control factor (Decay acceleratingfactor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalka In, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NCADHERIN, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Virus rus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thyroid gland TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, Acetylcholine receptor, AdipoR1, AdipoR2, ADP ribosyl cyclase-1, alpha-4 / beta-7 integrin、alpha-5 / beta-1 integrin、alpha-v / beta-6 integrin、alphavbeta1 integrin、Angiopoietin ligand-2、Angptl2、Anthrax、Cadh erin、Carbonic anhydrase-IX、CD105、CD155、CD158a、CD37、CD49b、CD51、CD70、CD72、Claudin 18、Clostridium difficile toxin、CS1、Delta-like protein ligand 4、DHICA oxidase、Dickkopf-1 ligand、Dipeptidyl peptidase IV、EPOR、F protein of RSV、Factor Ia、FasL、Folate receptor alpha、Glucagon receptor、Glucagon-like peptide 1 receptor、Glutamate carboxypeptidase II、GMCSFR、Hepatitis C virus E2 glycoprotein、Hepcidin、IL-17 receptor、IL-22 receptor、IL-23 receptor、IL-3 receptor、Kit tyrosine kinase、Leucine Rich Alpha-2-Glycoprotein 1 (LRG1)、Lysosphingolipid receptor、Membrane glycoprotein OX2、Mesothelin、MET、MICA、MUC-16、Myelin associated glycoprotein、Neuropilin-1、Neuropilin-2、Nogo receptor、PLXNA1、PLXNA2、PLXNA3、PLXNA4A、PLXNA4B 、PLXNB1、PLXNB2、PLXNB3 、PLXNC1 、PLXND1 、Programmed cell death ligand 1、Proprotein convertase PC9、P-selectin glycoprotein ligand-1、RAGE、Reticulon 4、RF、RON-8、SEMA3A、SEMA3B、SEMA3C、SEMA3D、SEMA3E、SEMA3F、SEMA3G、SEMA4A、SEMA4B、SEMA4C、SEMA4D、SEMA4F、SEMA4G、SEMA5A、SEMA5B、SEMA6A、SEMA6B、SEMA6C、SEMA6D、SEMA7A、ShigaExamples of the soluble antigen include, for example, soluble antigens that exist in the body fluids of a living body without being anchored to cells, such as toxin II, sphingosine-1-phosphate receptor-1, ST2, staphylococcal lipoteichoic acid, tenascin, TG2, thymic stromal lymphoprotein receptor, TNF superfamily receptor 12A, transmembrane glycoprotein NMB, TREM-1, TREM-2, trophoblast glycoprotein, TSH receptor, TTR, tubulin, ULBP2, and receptors for hormones and growth factors. Among the receptors, soluble antigens that exist in the body fluids of a living body through some mechanism, including digestion by proteases, of receptors expressed on the cell surface, are also suitable as soluble antigens in the present invention. Examples of such molecules include the soluble IL-6R molecule described herein (J. Immunol. (1994) 152, 4958-4968), CD20, CD52 (Br. J. Haematol. (2003) 123 (5), 850-857), and the like. In addition to molecules that are inherently expressed in vivo, soluble antigens of the present invention also include antigens presented by or on infectious organisms such as viruses, and infectious molecules such as prions that are present in body fluids of the organism. Suitable body fluids include blood, plasma, serum, urine, lymph, saliva, tears, and the like.

[0027] Epitope An epitope, which means an antigenic determinant present in an antigen, means a site on an antigen to which an antigen-binding domain in an antigen-binding molecule disclosed herein binds. Thus, for example, an epitope can be defined by its structure. In addition, an epitope can also be defined by the binding activity to an antigen in an antigen-binding molecule that recognizes the epitope. When an antigen is a peptide or polypeptide, it is also possible to specify the epitope by the amino acid residues that constitute the epitope. In addition, when an epitope is a sugar chain, it is also possible to specify the epitope by a specific sugar chain structure.

[0028] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope. A linear epitope typically comprises at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0029] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids that comprise the epitope is not the single defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by an antibody that defines the epitope). Conformational epitopes may include an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (Ed.).

[0030] Binding activity Methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for IL-6R are exemplified below, but methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for an antigen other than IL-6R can also be appropriately carried out in accordance with the examples below.

[0031] For example, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For the above purpose, a linear peptide consisting of an amino acid sequence constituting the extracellular domain of IL-6R is synthesized. The peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region in the cDNA of IL-6R that encodes an amino acid sequence corresponding to the extracellular domain. Next, the binding activity between the linear peptide consisting of the amino acid sequence constituting the extracellular domain and the test antigen-binding molecule containing an antigen-binding domain for IL-6R is evaluated. For example, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding molecule to IL-6R-expressing cells. The binding activity of the antigen-binding molecule to the linear peptide can be determined by these tests.

[0032] Furthermore, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a conformational epitope can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. Examples of such cases include when a test antigen-binding molecule containing an antigen-binding domain for IL-6R strongly binds to IL-6R-expressing cells upon contact with the cells, while the antigen-binding molecule does not substantially bind to a linear peptide consisting of an amino acid sequence constituting the extracellular domain of immobilized IL-6R. Here, "not substantially binding" refers to a binding activity that is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.

[0033] Methods for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to IL-6R-expressing cells include, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, the binding activity can be evaluated by the principle of ELISA or FACS (fluorescence activated cell sorting) using IL-6R-expressing cells as antigens.

[0034] In the ELISA format, the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to IL-6R-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzyme reaction. That is, the test antigen-binding molecule is added to an ELISA plate on which IL-6R-expressing cells are immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody binding titer to IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule to IL-6R-expressing cells to be compared.

[0035] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected by a flow cytometer. Known examples of flow cytometers include the following: FACSCanto II FACSAriaTM FACSArrayTM FACSVantageTM SE FACSCaliburTM (both are product names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are product names of Beckman Coulter)

[0036] For example, the following method is an example of a suitable method for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to an antigen. First, the test antigen-binding molecule is stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule that has been reacted with cells expressing IL-6R. The test antigen-binding molecule is diluted with a suitable buffer solution to prepare the antigen-binding molecule at a desired concentration for use. For example, the antigen-binding molecule may be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). That is, the binding activity of the test antigen-binding molecule represented by the amount of binding of the test antigen-binding molecule can be measured by obtaining the Geometric Mean value.

[0037] Whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules is detected by cross-blocking assays, etc. For example, competitive ELISA assays are preferred cross-blocking assays.

[0038] Specifically, in the cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to the IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to wells coated with IL-6R protein.

[0039] The amount of the test antigen-binding molecule bound to the well via the IL-6R protein can be easily measured by labeling the antigen-binding molecule in advance. For example, a biotin-labeled antigen-binding molecule can be measured by using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly called competitive ELISA assays. The antigen-binding molecule can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.

[0040] If a competing antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain for IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of the candidate competing antigen-binding molecule, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competing antigen-binding molecule or competes for binding to the same epitope.

[0041] When the structure of the epitope to which a test antigen-binding molecule containing an antigen-binding domain for IL-6R binds has been identified, whether the test antigen-binding molecule and a control antigen-binding molecule share an epitope can be assessed by comparing the binding activity of both antigen-binding molecules against a peptide in which an amino acid mutation has been introduced into the peptide constituting the epitope.

[0042] Such binding activity can be measured, for example, by comparing the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced in the above-mentioned ELISA format. As a method other than ELISA, the binding activity to the mutant peptide bound to a column can also be measured by flowing the test antigen-binding molecule and the control antigen-binding molecule down the column and then quantifying the antigen-binding molecule eluted in the eluate. Methods of adsorbing a mutant peptide to a column, for example as a fusion peptide with GST, are known.

[0043] In addition, when the identified epitope is a conformational epitope, the fact that the test antigen-binding molecule and the control antigen-binding molecule share the epitope can be evaluated by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to a cell suspension in which these cells are suspended in an appropriate buffer such as PBS. Then, an FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added to the cell suspension that has been appropriately washed with a buffer. The fluorescence intensity and cell number of the cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test antigen-binding molecule and the control antigen-binding molecule are used at the desired concentration by appropriately diluting them with a suitable buffer. For example, they are used at any concentration between 10 μg / ml and 10 ng / ml. The amount of the labeled antibody bound to the cell is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the geometric mean value, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, represented by the amount of bound labeled antibody, can be measured.

[0044] In this method, for example, "not substantially binding to mutant IL-6R-expressing cells" can be determined by the following method. First, the test antigen-binding molecule and the control antigen-binding molecule bound to the cells expressing mutant IL-6R are stained with a labeled antibody. Then, the fluorescence intensity of the cells is detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The increase rate of fluorescence intensity due to the binding of the antigen-binding molecule can be obtained by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the antigen-binding molecule based on the following formula. ΔGeo-Mean=Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules)

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

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

[0047] Antigen-binding domain In the present specification, the term "antigen-binding domain" may refer to any domain of any structure as long as it binds to the target antigen. Examples of such domains include the variable regions of the heavy and light chains of an antibody, a module called an A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in the body (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to a protein in fibronectin, a glycoprotein expressed in the cell membrane, Affibody (WO1995 / 001937) using an IgG-binding domain consisting of a bundle of three helices consisting of 58 amino acids of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeat (AR)) which are regions exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a turn containing 33 amino acid residues, two antiparallel helices, and loop subunits are repeatedly stacked. Suitable examples of the antigen-binding domain of the present invention include an antigen-binding domain comprising the heavy and light chain variable regions of an antibody (WO2002 / 020565), anticalin (WO2003 / 029462), which is a four loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich-repeat (LRR) modules are repeatedly stacked in the variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and serves as the adaptive immune system of jawless animals such as lampreys and hagfish (WO2008 / 016854). Suitable examples of the antigen-binding domain of the present invention include an antigen-binding domain comprising the heavy and light chain variable regions of an antibody.Suitable examples of such antigen-binding domains include "single chain Fv (scFv)," "single chain antibody," "Fv," "single chain Fv 2 (scFv2)," "Fab," and "F(ab')2."

[0048] The antigen-binding domains in the antigen-binding molecule of the present invention can bind to the same epitope. Here, the same epitope can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. It can also be present in a protein consisting of the 20th to 365th amino acids of the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, the antigen-binding domains in the antigen-binding molecule of the present invention can bind to different epitopes. Here, the different epitopes can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. It can also be present in a protein consisting of the 20th to 365th amino acids of the amino acid sequence set forth in SEQ ID NO: 1.

[0049] specific "Specific" refers to a state in which one of the specifically binding molecules does not show any significant binding to molecules other than the one or more molecules to which it binds. It is also used when the antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. In addition, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0050] antibody In the present specification, an antibody refers to an immunoglobulin that is naturally occurring or that is partially or completely synthetically produced. Antibodies can be isolated from natural sources such as plasma or serum in which they are naturally present, or from the culture supernatant of hybridoma cells that produce the antibodies, or can be partially or completely synthesized by using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine classes (isotypes) of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. The antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4 of these isotypes. The constant region of IgG also includes naturally occurring mutants thereof. As the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present invention. In particular, for the sequence of human IgG1, the amino acid sequence at positions 356-358 (EU numbering) may be DEL or EEM.

[0051] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be appropriately produced according to the following examples.

[0052] The anti-IL-6R antibody can be obtained as a polyclonal or monoclonal antibody by known means. As the anti-IL-6R antibody, a monoclonal antibody derived from a mammal can be preferably produced. The monoclonal antibody derived from a mammal includes those produced by hybridomas and those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. The monoclonal antibody of the present invention includes "humanized antibodies" and "chimeric antibodies".

[0053] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Hybridomas producing anti-IL-6R antibodies can then be selected by screening for monoclonal antibody-producing cells using a conventional screening method.

[0054] Specifically, monoclonal antibodies are produced, for example, as follows. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for obtaining antibodies, can be obtained by expressing the IL-6R gene, the nucleotide sequence of which is disclosed in SEQ ID NO: 2. That is, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968), is expressed instead of the IL-6R protein represented by SEQ ID NO: 1. Purified natural IL-6R protein can also be used as a sensitizing antigen.

[0055] The purified IL-6R protein can be used as a sensitizing antigen for immunization of a mammal. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. It can also be obtained by decomposing the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be selected from the amino acid sequence corresponding to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids constituting the peptide to be used as a sensitizing antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, a peptide of 8 to 50, preferably 10 to 30 residues can be used as a sensitizing antigen.

[0056] In addition, a fusion protein in which a desired partial polypeptide or peptide of the IL-6R protein is fused with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). A method for obtaining IL-6R used as a sensitizing antigen and a method for immunization using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0057] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. In general, rodents such as mice, rats, hamsters, rabbits, monkeys, etc. are preferably used.

[0058] The above-mentioned animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, immunization is performed by administering the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, etc., is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. In addition, a suitable carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0059] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the body of the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages: -It can maintain the structure of membrane proteins such as IL-6R and provide immune stimulation. -No need to purify the immunogen

[0060] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be suitably used. As a method for administering a vector to a living body, a commonly used method can be used. For example, DNA immunization is performed by introducing gold particles to which an expression vector is adsorbed into the cells of an individual animal to be immunized using a gene gun. Furthermore, an antibody that recognizes IL-6R can also be produced using the method described in International Publication WO2003 / 104453.

[0061] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As preferred immune cells, splenocytes in particular can be used.

[0062] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells are preferably provided with an appropriate selection marker for screening. The selection marker refers to a trait that allows (or does not allow) a cell to survive under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, they can continue to synthesize DNA by utilizing the salvage circuit of normal cells, and therefore grow even in HAT selection medium.

[0063] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0064] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

[0065] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0066] More specifically, the cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

[0067] The ratio of immune cells to myeloma cells may be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, or other usual culture medium used for this type of cell culture may be used, and serum supplements such as fetal calf serum (FCS) may be suitably added.

[0068] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., average molecular weight of about 1000 to 6000) pre-warmed to about 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, the appropriate culture medium listed above is successively added, and the procedure of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like that are undesirable for hybridoma growth.

[0069] The hybridomas thus obtained can be selected by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture can be continued using the above HAT medium for a sufficient time (usually several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die. Then, screening and single cloning of hybridomas producing the desired antibody are performed by a conventional limiting dilution method.

[0070] The hybridomas thus obtained can be selected by using a selection medium according to the selection marker possessed by the myeloma used in the cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing in HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used in the cell fusion, cells that have successfully fused with normal cells can selectively grow in the HAT culture medium. The culture is continued using the above-mentioned HAT culture medium for a sufficient time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Next, screening and single cloning of hybridomas that produce the desired antibody can be performed by the usual limiting dilution method.

[0071] Screening and monocloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such a monoclonal antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows the binding of an antibody to a cell surface to be measured by analyzing cells contacted with a fluorescent antibody with laser light and measuring the fluorescence emitted by individual cells.

[0072] To screen for hybridomas producing the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. The preferred cells for screening are mammalian cells in which IL-6R is forcibly expressed. By using non-transformed mammalian cells used as host cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas producing antibodies that do not bind to host cells but bind to IL-6R-forcibly expressing cells, hybridomas producing IL-6R monoclonal antibodies can be obtained.

[0073] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of a hybridoma is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. When the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody that has the ability to bind to the antigen and are selected by these screenings can be cloned by limiting dilution or the like.

[0074] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium, and can be stored for a long period of time in liquid nitrogen.

[0075] The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining highly pure antibodies.

[0076] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody gene is incorporated into an appropriate vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0077] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from a hybridoma cell that produces the anti-IL-6R antibody. To do this, generally, total RNA is first extracted from the hybridoma. For example, the following method can be used to extract mRNA from cells. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0078] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from a hybridoma. cDNA encoding an antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo Co., Ltd.) or the like. In addition, for the synthesis and amplification of cDNA, a SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be appropriately used. Furthermore, during the synthesis of such cDNA, appropriate restriction enzyme sites (described below) can be introduced at both ends of the cDNA.

[0079] The desired cDNA fragment is purified from the obtained PCR product and then ligated to a vector DNA. The recombinant vector is thus prepared, introduced into E. coli or the like, and colonies are selected, after which the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector has the base sequence of the desired cDNA is confirmed by a known method, for example, the dideoxynucleotide chain termination method.

[0080] To obtain genes encoding variable regions, it is easy to use the 5'-RACE method using primers for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template to obtain a 5'-RACE cDNA library. A commercially available kit such as the SMART RACE cDNA Amplification Kit can be used to synthesize the 5'-RACE cDNA library.

[0081] The resulting 5'-RACE cDNA library is used as a template to amplify antibody genes by PCR. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0082] Specifically, for example, when the objective is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains and κ and λ chains as light chains can be used. To amplify the variable region genes of IgG, a primer that anneals to a portion corresponding to the constant region close to the variable region is generally used as the 3' primer. On the other hand, a primer included in the 5' RACE cDNA library construction kit is used as the 5' primer.

[0083] Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The desired antibody can be screened using the binding activity of the reconstituted immunoglobulin to IL-6R as an index. For example, when the aim is to obtain an antibody against IL-6R, it is more preferable that the binding of the antibody to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, as follows; (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) Selecting an antibody that binds to IL-6R-expressing cells.

[0084] Methods for detecting the binding between an antibody and IL-6R-expressing cells are known. Specifically, the binding between an antibody and IL-6R-expressing cells can be detected by techniques such as FACS described above. Fixed specimens of IL-6R-expressing cells can be appropriately used to evaluate the binding activity of an antibody.

[0085] As a method for screening antibodies using binding activity as an index, a panning method using a phage vector is also preferably used. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell group, a screening method using a phage vector is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form a single chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contacting this phage with a desired antigen, the phage bound to the antigen can be recovered to recover DNA encoding an scFv having the desired binding activity. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.

[0086] After obtaining a cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene. Furthermore, in order to insert one copy of the digested fragment into a vector in the correct direction, it is preferable to insert a restriction enzyme that gives a sticky end. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. At this time, if a gene encoding an antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody whose constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibody of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing the two genes digested with the same combination of restriction enzymes in frame.

[0087] To produce an anti-IL-6R monoclonal antibody, an antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for expressing an antibody include, for example, enhancers and promoters. In addition, a suitable signal sequence can be added to the amino terminus so that the expressed antibody is secreted outside the cell. In the examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted outside the cell as a mature polypeptide. Next, a suitable host cell is transformed with this expression vector to obtain a recombinant cell expressing DNA encoding an anti-IL-6R antibody.

[0088] For the expression of antibody genes, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. An antibody molecule comprising an H chain and an L chain can be expressed by simultaneously transforming (co-transfecting) the same host cell with vectors incorporating the H chain and the L chain. Alternatively, a host cell can be transformed by incorporating DNAs encoding the H chain and the L chain into a single expression vector (see International Publication WO 1994 / 011523).

[0089] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. Any of these expression systems can be applied to isolate the antigen-binding domain of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells include the following cells: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK (human embryonic kidney) 293, Freestyle TM293 etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0090] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. For transformation of plant cells, callus cultured cells can be appropriately used.

[0091] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris - Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0092] In addition, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be appropriately used. An expression vector containing the target antibody gene is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0093] In addition to the above-mentioned host cells, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by in-frame insertion into a gene encoding a protein that is produced specifically in milk. For example, goat β-casein can be used as a protein secreted into milk. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The desired antibody can be obtained as a fusion protein with a milk protein from the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. Also, a hormone can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0094] When the antigen-binding molecule described herein is administered to humans, an antigen-binding domain derived from a recombinant antibody that has been artificially modified for the purpose of reducing heterologous antigenicity against humans, etc., may be appropriately used as the antigen-binding domain in the molecule. Examples of recombinant antibodies include humanized antibodies. These modified antibodies are appropriately produced using known methods.

[0095] The variable region of an antibody used to prepare an antigen-binding domain in an antigen-binding molecule described herein is usually composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are regions that essentially determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences constituting FRs often show high identity even between antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of a certain antibody can be transplanted to another antibody by transplanting CDRs.

[0096] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies in which the CDR of an animal other than human, for example, a mouse antibody, is transplanted to a human antibody are known. General gene recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for transplanting the CDR of a mouse antibody to a human FR. In overlap extension PCR, a base sequence encoding the CDR of the mouse antibody to be transplanted is added to a primer for synthesizing the FR of a human antibody. Primers are prepared for each of the four FRs. In general, in transplanting mouse CDRs to human FRs, it is considered to be advantageous to select a human FR that is highly identical to the mouse FR in terms of maintaining the function of the CDR. That is, it is generally preferable to use a human FR that is composed of an amino acid sequence that is highly identical to the amino acid sequence of the FR adjacent to the mouse CDR to be transplanted.

[0097] The base sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The base sequences encoding mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform a complementary strand synthesis reaction. Through this reaction, the human FRs are linked via the mouse CDR sequences.

[0098] Finally, the V region gene in which three CDRs and four FRs are linked is amplified in its entirety by primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences added thereto. A human antibody expression vector can be prepared by inserting the DNA obtained as described above and a DNA encoding a human antibody C region into an expression vector so as to fuse in frame. After introducing the integration vector into a host to establish a recombinant cell, the recombinant cell is cultured and the DNA encoding the humanized antibody is expressed, whereby the humanized antibody is produced in the culture of the cultured cells (see European Patent Publication EP239400 and International Publication WO1996 / 002576).

[0099] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select the FR of a human antibody in which the CDR forms a good antigen-binding site when linked via the CDR. If necessary, amino acid residues in the FR can be substituted so that the CDR of the reshaped human antibody forms a suitable antigen-binding site. For example, a mutation in the amino acid sequence can be introduced into the FR by applying the PCR method used for transplanting mouse CDR to human FR. Specifically, a partial base sequence mutation can be introduced into a primer annealing to the FR. A base sequence mutation is introduced into the FR synthesized by such a primer. A mutant FR sequence having desired properties can be selected by measuring and evaluating the antigen-binding activity of a mutant antibody with amino acid substitution by the above method (Cancer Res., (1993) 53, 851-856).

[0100] Furthermore, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0101] Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv) by phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined. After determining the DNA sequence of the scFv that binds to an antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell as listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0102] As a method for obtaining antibody genes, in addition to the above, techniques for B cell cloning (identification and cloning of the coding sequence of each antibody, isolation thereof, and use for constructing expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4), etc.) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or WO2008 / 081008 can be used as appropriate.

[0103] EU numbering and Kabat numbering According to the method used in the present invention, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). In the present specification, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids of the variable region are represented according to the Kabat numbering, and the amino acids of the constant region are represented according to the EU numbering based on the Kabat amino acid positions.

[0104] Ion concentration conditions Metal ion concentration conditions In one embodiment of the present invention, the ion concentration refers to the metal ion concentration. The term "metal ion" refers to ions of elements belonging to Group I, such as alkali metals and copper group excluding hydrogen, Group II, such as alkaline earth metals and zinc group, Group III, such as boron, Group IV, such as carbon and silicon, Group VIII, such as iron group and platinum group, and each of the A subgroups of Groups V, VI, and VII, as well as metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become positive ions, which is called ionization tendency. Metals with a high ionization tendency are said to be chemically active.

[0105] An example of a suitable metal ion in the present invention is calcium ion. Calcium ions are involved in the regulation of many vital phenomena, including the contraction of muscles such as skeletal muscles, smooth muscles, and cardiac muscles, activation of leukocytes such as movement and phagocytosis, activation of platelets such as deformation and secretion, activation of lymphocytes, activation of mast cells such as histamine secretion, cell responses via catecholamine α receptors and acetylcholine receptors, exocytosis, release of transmitters from neuronal terminals, and axonal flow of neurons. Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known as intracellular calcium ion receptors that have multiple calcium ion binding sites and are thought to have originated from a common origin in molecular evolution, and many binding motifs are also known. Well-known examples include the cadherin domain, the EF hand found in calmodulin, the C2 domain found in protein kinase C, the Gla domain found in the blood coagulation protein Factor IX, the C-type lectins found in the asialoglycoprotein receptor and mannose-binding receptor, the A domain found in the LDL receptor, annexin, thrombospondin type 3 domain, and EGF-like domain.

[0106] In the present invention, when the metal ion is a calcium ion, the calcium ion concentration conditions include low calcium ion concentration conditions and high calcium ion concentration conditions. The term "the binding activity changes depending on the calcium ion concentration conditions" refers to the change in the binding activity of the antigen-binding molecule to the antigen due to the difference between low calcium ion concentration conditions and high calcium ion concentration conditions. For example, the binding activity of the antigen-binding molecule to the antigen under high calcium ion concentration conditions is higher than that under low calcium ion concentration conditions. In addition, the binding activity of the antigen-binding molecule to the antigen under low calcium ion concentration conditions is also higher than that under high calcium ion concentration conditions.

[0107] In the present specification, the high calcium ion concentration is not limited to a specific numerical value, but may be preferably a concentration selected from between 100 μM and 10 mM. In another embodiment, it may be a concentration selected from between 200 μM and 5 mM. In another embodiment, it may be a concentration selected from between 500 μM and 2.5 mM, and in another embodiment, it may be a concentration selected from between 200 μM and 2 mM. It may also be a concentration selected from between 400 μM and 1.5 mM. In particular, a concentration selected from between 500 μM and 2.5 mM, which is close to the calcium ion concentration in plasma (blood) in the living body, is preferably mentioned.

[0108] In the present specification, the low calcium ion concentration is not limited to a specific numerical value, but may be preferably a concentration selected from between 0.1 μM and 30 μM. In another embodiment, it may be a concentration selected from between 0.2 μM and 20 μM. In another embodiment, it may be a concentration selected from between 0.5 μM and 10 μM, and in another embodiment, it may be a concentration selected from between 1 μM and 5 μM. It may also be a concentration selected from between 2 μM and 4 μM. In particular, a concentration selected from between 1 μM and 5 μM, which is close to the ionized calcium concentration in early endosomes in vivo, is preferably mentioned.

[0109] In the present invention, "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" means that the antigen-binding activity of the antigen-binding molecule at a calcium ion concentration selected from between 0.1 μM and 30 μM is weaker than that at a calcium ion concentration selected from between 100 μM and 10 mM. Preferably, the antigen-binding activity of the antigen-binding molecule at a calcium ion concentration selected from between 0.5 μM and 10 μM is weaker than that at a calcium ion concentration selected from between 200 μM and 5 mM, and more preferably, the antigen-binding activity at a calcium ion concentration in early endosomes in a living body is weaker than that at a calcium ion concentration in plasma in a living body. Specifically, the antigen-binding activity of the antigen-binding molecule at a calcium ion concentration selected from between 1 μM and 5 μM is weaker than that at a calcium ion concentration selected from between 500 μM and 2.5 mM.

[0110] Whether or not the antigen-binding activity of an antigen-binding molecule changes depending on the metal ion concentration can be determined by using a known measurement method such as that described above in the section on binding activity. For example, to confirm that the antigen-binding activity of an antigen-binding molecule changes more significantly under high calcium ion concentrations than under low calcium ion concentrations, the antigen-binding activities of the antigen-binding molecule under low and high calcium ion concentrations are compared.

[0111] Furthermore, in the present invention, the expression "the antigen-binding activity under a low calcium ion concentration condition is lower than that under a high calcium ion concentration condition" can also be expressed as "the antigen-binding activity of an antigen-binding molecule under a high calcium ion concentration condition is higher than that under a low calcium ion concentration condition." Note that in the present invention, "the antigen-binding activity under a low calcium ion concentration condition is lower than that under a high calcium ion concentration condition" may also be described as "the antigen-binding ability under a low calcium ion concentration condition is weaker than that under a high calcium ion concentration condition," and "the antigen-binding activity under a low calcium ion concentration condition is made lower than that under a high calcium ion concentration condition" may also be described as "the antigen-binding ability under a low calcium ion concentration condition is made weaker than that under a high calcium ion concentration condition."

[0112] Conditions other than calcium ion concentration when measuring binding activity to an antigen can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of HEPES buffer and 37°C. For example, it can be measured using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by flowing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, the binding activity to the membrane antigen can be evaluated by flowing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0113] In the antigen-binding molecules of the present invention, the ratio of antigen-binding activity under low calcium ion concentration conditions to that under high calcium ion concentration conditions is not particularly limited, so long as the antigen-binding activity under low calcium ion concentration conditions is weaker than that under high calcium ion concentration conditions, but the ratio of the KD (Dissociation constant) for the antigen under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, KD (3 μM Ca) / KD (2 mM Ca), is preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the KD (3 μM Ca) / KD (2 mM Ca) value is not particularly limited, and may be any value, such as 400, 1000, or 10,000, as long as it can be produced by those skilled in the art. It can also be specified by the value of KD (3 μM Ca) / KD (1.2 mM Ca). That is, the value of KD (3 μM Ca) / KD (1.2 mM Ca) is 2 or more, more preferably the value of KD (3 μM Ca) / KD (1.2 mM Ca) is 10 or more, and even more preferably the value of KD (3 μM Ca) / KD (1.2 mM Ca) is 40 or more. There is no particular upper limit to the value of KD (3 μM Ca) / KD (1.2 mM Ca), and any value such as 400, 1000, 10000, etc. may be used as long as it can be produced by the techniques of those skilled in the art.

[0114] As the value of binding activity to an antigen, when the antigen is a soluble antigen, the KD (dissociation constant) can be used, whereas when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and the apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0115] In addition, as another index showing the ratio of the antigen-binding activity of the antigen-binding molecule of the present invention under low calcium concentration conditions to that under high calcium concentration conditions, for example, the dissociation rate constant kd (Dissociation rate constant) can also be suitably used. When kd (Dissociation rate constant) is used instead of KD (Dissociation constant) as an index showing the ratio of binding activity, the value of kd (low calcium concentration condition) / kd (high calcium concentration condition), which is the ratio of kd (Dissociation rate constant) under low calcium concentration conditions to kd (Dissociation rate constant) under high calcium concentration conditions, is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (low calcium concentration condition) / kd (high calcium concentration condition) is not particularly limited, and may be any value, such as 50, 100, 200, etc., as long as it can be produced within the technical common knowledge of a person skilled in the art.

[0116] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble antigen, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), flow cytometer, etc. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different calcium ion concentrations, it is preferable to keep the conditions other than the calcium concentration the same.

[0117] For example, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than its antigen-binding activity at a high calcium ion concentration, which is one embodiment provided by the present invention, can be obtained by screening for antigen-binding domains or antibodies comprising the following steps (a) to (c). (a) obtaining the antigen-binding activity of an antigen-binding domain or antibody under a low calcium concentration condition; (b) obtaining the antigen-binding activity of the antigen-binding domain or antibody under a high calcium concentration condition; and (c) selecting an antigen-binding domain or antibody whose antigen-binding activity is lower under a low calcium concentration condition than under a high calcium concentration condition.

[0118] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than its antigen-binding activity at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (c). (a) contacting an antigen-binding domain or an antibody, or a library thereof, with an antigen under a high calcium concentration condition; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under low calcium concentration conditions; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0119] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d). (a) contacting a library of antigen-binding domains or antibodies with an antigen under low calcium concentration conditions; (b) selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to an antigen under high calcium concentration conditions; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0120] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antibodies with a column onto which an antigen is immobilized under high calcium concentration conditions; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under low calcium concentration conditions; and (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0121] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains or antibodies through a column having an antigen immobilized thereon under low calcium concentration conditions; (b) recovering the antigen-binding domain or antibody that is eluted without binding to the column in step (a); (c) allowing the antigen-binding domain or antibody recovered in step (b) to bind to an antigen under high calcium concentration conditions; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0122] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under high calcium concentration conditions; (b) obtaining the antigen-binding domain or antibody bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under low calcium concentration conditions; and (d) isolating in step (c) antigen-binding domains or antibodies whose antigen-binding activity is weaker than the criterion selected in step (b).

[0123] The above steps may be repeated two or more times. Thus, the present invention provides an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, obtained by the above-mentioned screening method further comprising the step of repeating steps (a) to (c) or (a) to (d) two or more times. The number of times that steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is usually within 10 times.

[0124] In the screening method of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under low calcium concentration conditions is not particularly limited as long as the ionized calcium concentration is between 0.1 μM and 30 μM, but a preferred ionized calcium concentration is between 0.5 μM and 10 μM. A more preferred ionized calcium concentration is the ionized calcium concentration in early endosomes in vivo, specifically, the antigen-binding activity at 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding domain or antibody under high calcium concentration conditions is not particularly limited as long as the ionized calcium concentration is between 100 μM and 10 mM, but a preferred ionized calcium concentration is between 200 μM and 5 mM. A more preferred ionized calcium concentration is the ionized calcium concentration in plasma in vivo, specifically, the antigen-binding activity at 0.5 mM to 2.5 mM.

[0125] The antigen-binding activity of an antigen-binding domain or antibody can be measured by a method known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate constant), apparent kd (Apparent dissociation rate constant), etc. These can be measured by a method known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, FACS, etc. can be used.

[0126] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition means the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0127] As long as the antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition, the difference between the antigen-binding activity under a high calcium concentration condition and that under a low calcium concentration condition is not particularly limited; however, the antigen-binding activity under a high calcium concentration condition is preferably at least twice, more preferably at least 10 times, and even more preferably at least 40 times that of the antigen-binding activity under a low calcium concentration condition.

[0128] The antigen-binding domain or antibody of the present invention to be screened by the above screening method may be any antigen-binding domain or antibody, and it is possible to screen, for example, the above-mentioned antigen-binding domains or antibodies. For example, an antigen-binding domain or antibody having a native sequence may be screened, or an antigen-binding domain or antibody with a substituted amino acid sequence may be screened.

[0129] Library According to one embodiment, the antigen-binding domain or antibody of the present invention can be obtained from a library mainly composed of a plurality of antigen-binding molecules with different sequences, each of which contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on an ion concentration condition. Preferred examples of ion concentrations include metal ion concentration and hydrogen ion concentration.

[0130] As used herein, the term "library" refers to multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, or a nucleic acid or polynucleotide encoding these sequences. The sequences of the multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules contained in a library are not a single sequence, but are antigen-binding molecules or fusion polypeptides containing antigen-binding molecules with different sequences.

[0131] As used herein, the term "different sequences" in the description of multiple antigen-binding molecules with different sequences means that the sequences of the individual antigen-binding molecules in a library are different from each other. In other words, the number of different sequences in a library reflects the number of independent clones with different sequences in the library, and is sometimes referred to as the "library size." In a typical phage display library, the number of sequences is 10 6 From 10 12 By applying known techniques such as ribosome display, the library size can be increased to 10 14 The number of phage particles used in panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess, also called the "library equivalent number", indicates that there may be 10 to 10,000 individual clones having the same amino acid sequence. Thus, in the present invention, the term "different in sequence" means that the sequences of the individual antigen-binding molecules in the library excluding the library equivalent number are different from each other, more specifically, that there are 10 antigen-binding molecules having sequences different from each other. 6 From 10 14 Molecular, preferably 10 7 From 10 12 molecule, more preferably 10 8 From 10 11 Molecular weight, particularly preferably 10 8 From 10 12 It means to exist.

[0132] Furthermore, the term "plurality" in the description of a library mainly composed of a plurality of antigen-binding molecules of the present invention generally refers to a collection of two or more types of the substance, for example, the antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors, or viruses of the present invention. For example, if two or more substances differ from each other in terms of a specific trait, it indicates that there are two or more types of the substance. An example may be a mutant amino acid observed at a specific amino acid position in an amino acid sequence. For example, if there are two or more antigen-binding molecules of the present invention that are substantially the same, preferably identical, except for flexible residues or specific mutant amino acids at highly diverse amino acid positions exposed on the surface, then there are a plurality of antigen-binding molecules of the present invention. In another example, if there are two or more polynucleotide molecules of the present invention that are substantially the same, preferably identical, except for bases encoding flexible residues or bases encoding specific mutant amino acids at highly diverse amino acid positions exposed on the surface, then there are a plurality of polynucleotide molecules of the present invention.

[0133] Furthermore, the term "mainly consisting of" in the description of a library of the present invention consisting mainly of a plurality of antigen-binding molecules reflects the number of antigen-binding molecules whose antigen-binding activity differs depending on ion concentration conditions among the number of independent clones with different sequences in the library. Specifically, the number of antigen-binding molecules exhibiting such binding activity in the library is at least 10 4 It is preferable that the antigen-binding domain of the present invention has at least 10 antigen-binding molecules that exhibit such binding activity. 5 More preferably, the antigen-binding domain of the present invention can be obtained from a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 6 The antigen-binding domain of the present invention can be obtained from a library in which at least 10 antigen-binding molecules exhibiting such binding activity are present. 7 Preferably, the antigen-binding domain of the present invention can be obtained from a library in which at least 10 antigen-binding molecules exhibit such binding activity are present.8 The antigen-binding domain of the present invention can be obtained from a library in which the antigen-binding molecule is present. In another expression, it can be suitably expressed as the ratio of antigen-binding molecules in which the binding activity of the antigen-binding molecule to the antigen differs depending on the ion concentration condition, among the number of independent clones in the library with different sequences. Specifically, the antigen-binding domain of the present invention can be obtained from a library in which the antigen-binding molecule exhibiting such binding activity is contained in 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, even more preferably 2% to 20%, and particularly preferably 4% to 10% of the number of independent clones in the library with different sequences. In the case of a fusion polypeptide, a polynucleotide molecule, or a vector, it can be expressed as the number of molecules or the ratio in the whole molecule, as described above. In the case of a virus, it can be expressed as the number of virus individuals or the ratio in the whole individuals, as described above.

[0134] Amino acids whose binding activity to antigens of antigen-binding domains changes depending on calcium ion concentration

[0135] The antigen-binding domain or antibody of the present invention to be screened by the above-mentioned screening method may be prepared in any manner. For example, when the metal ion is a calcium ion concentration, it is possible to use pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries in which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced into these antibodies or libraries (libraries with an increased content of amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acids, libraries in which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced at specific sites, etc.).

[0136] As described above, examples of amino acids that change the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions include, for example, when the metal ion is a calcium ion, any type of amino acid can be used as long as it forms a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490), Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J. (1990) 265, 261-265), Bairoch and Cox (FEBS Lett. (1990) 269, 454-456), Davis (New Biol. (1990) 2, 410-419), Schaefer et al. (Genomics (1995) 25, 638-643), Economou et al. (EMBO J. (1990) 9, 349-354), Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). That is, any known calcium-binding motif, such as ASGPR, CD23, MBR, and C-type lectins such as DC-SIGN, may be contained in the antigen-binding molecules of the present invention. In addition to the above, preferred examples of such calcium-binding motifs include the calcium-binding motif contained in the antigen-binding domain of SEQ ID NO: 62.

[0137] Furthermore, preferred examples of amino acids whose antigen-binding activity changes depending on calcium ion concentration include amino acids having metal chelating activity, such as serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

[0138] The position of the antigen-binding domain containing the amino acid is not limited to a specific position, and may be any position in the heavy chain variable region or light chain variable region forming the antigen-binding domain, as long as the binding activity of the antigen-binding molecule to the antigen is changed depending on the calcium ion concentration. That is, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which an amino acid that changes the binding activity of the antigen-binding molecule to the antigen depending on the calcium ion concentration is contained in the antigen-binding domain of the heavy chain. In another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which the amino acid is contained in the CDR3 of the heavy chain. In another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which the amino acid is contained in the 95th, 96th, 100a, and / or 101st positions as represented by the Kabat numbering of the CDR3 of the heavy chain.

[0139] In a non-limiting embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration condition is contained in the antigen-binding domain of the light chain. In another embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which the amino acid is contained in the light chain CDR1. In another embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, in which the amino acid is contained in the light chain CDR1 at position 30, 31, and / or 32 as represented by the Kabat numbering.

[0140] In another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences in which the amino acid residue is contained in the light chain CDR2. In another embodiment, a library is provided which mainly composed of antigen-binding molecules having different sequences in which the amino acid residue is contained at position 50 according to the Kabat numbering in the light chain CDR2.

[0141] In yet another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in the light chain CDR3. In another embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained at position 92 according to the Kabat numbering in the light chain CDR3.

[0142] In another embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in two or three CDRs selected from the above-described light chain CDR1, CDR2, and CDR3. Furthermore, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in any one or more of positions 30, 31, 32, 50, and / or 92 of the light chain as defined by the Kabat numbering.

[0143] In a particularly preferred embodiment, it is desirable that the framework sequence of the light chain and / or heavy chain variable region of the antigen-binding molecule has a human germline framework sequence. Therefore, in one aspect of the present invention, if the framework sequence is a completely human sequence, it is considered that the antigen-binding molecule of the present invention will cause little or no immunogenic reaction when administered to humans (e.g., for the treatment of a disease). In the above sense, "comprising a germline sequence" of the present invention means that a part of the framework sequence of the present invention is identical to a part of any human germline framework sequence. For example, an antigen-binding molecule of the present invention "comprising a germline sequence" is also an antigen-binding molecule of the present invention in which the heavy chain FR2 sequence of multiple different human germline framework sequences is combined.

[0144] Examples of frameworks include currently known fully human framework region sequences, which are included in websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ). These framework region sequences can be appropriately used as germline sequences contained in the antigen-binding molecules of the present invention. Germline sequences can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798), Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461), and Cox et al. (Nat. Genetics (1994) 7, 162-168)). Suitable germline sequences can be appropriately selected from Vκ, which is classified into seven subgroups, Vλ, which is classified into ten subgroups, and VH, which is classified into seven subgroups.

[0145] Fully human VH sequences include, but are not limited to, VH1 subgroups (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), VH2 subgroups (e.g., VH2-5, VH2-26, VH2-70), VH3 subgroups (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-3), VH4 subgroups (VH4-5, VH4-6, VH4-70, VH4-8, VH4-9, VH4-10, VH4-11, VH4-13, VH4-15, VH4-16, VH4-20, VH4-21, VH4-23, VH4-3), VH4-3 subgroups (VH4-3, V ... Preferred examples of VH sequences include those of the 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). These are also described in known documents (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)) and others, and those skilled in the art can appropriately design the antigen-binding molecules of the present invention based on this sequence information. Other completely human frameworks or framework subregions can also be suitably used.

[0146] Examples of fully human VK sequences include, but are not limited to, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18, which are classified into the Vk1 subgroup, and A1, A2, A3, A5, A7, A17, and A19, which are classified into the Vk2 subgroup. Preferred examples thereof include 18, A19, A23, O1, O11, A11, A27, L2, L6, L10, L16, L20, L25 classified into the Vk3 subgroup, B3 classified into the Vk4 subgroup, B2 classified into the Vk5 subgroup (also referred to as Vk5-2 in this specification)), and A10, A14, A26, etc. classified into the VK6 subgroup (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028), Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022), and Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

[0147] Examples of fully human VL sequences include, but are not limited to, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22, which are classified into the VL1 subgroup; V2-1, V2-6, V2-7, and V2- Preferred examples of such polypeptides include V1-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, V3-2, V3-3, and V3-4 classified into the VL3 subgroup, V4-1, V4-2, V4-3, V4-4, and V4-6 classified into the VL4 subgroup, and V5-1, V5-2, V5-4, and V5-6 classified into the VL5 subgroup (Kawasaki et al., Genome Res. (1997) 7, 250-261).

[0148] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used together with the "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention. Other examples of fully human frameworks that can be used together with the "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY, POM, etc. (e.g., Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250) mentioned above).

[0149] Although the present invention is not bound by any particular theory, it is believed that one reason why the use of germline sequences is expected to eliminate adverse immune responses in most individuals is that somatic mutations frequently occur in the variable regions of immunoglobulins as a result of the affinity maturation step that occurs during a normal immune response. These mutations occur primarily around the CDRs, whose sequences are hypervariable, but also affect residues in the framework regions. Although these framework mutations are not present in the germline genes, they are unlikely to be immunogenic in patients because the normal human population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are expected to be less immunogenic or non-immunogenic in patients. To maximize the chances of immune tolerance, genes encoding the variable regions can be selected from a commonly present set of functional germline genes.

[0150] To prepare an antigen-binding molecule of the present invention in which the framework sequence contains amino acids that change the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration conditions, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used.

[0151] For example, a library containing a plurality of antigen-binding molecules of the present invention having different sequences can be prepared by combining a light chain variable region selected as a framework sequence that contains in advance at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on calcium ion concentration conditions with a heavy chain variable region prepared as a randomized variable region sequence library. As a non-limiting example, when the ion concentration is calcium ion concentration, a suitable example is a library that combines a light chain variable region sequence belonging to the Vk5-2 family, represented by the light chain variable region sequence set forth in SEQ ID NO: 62 (Vk5-2), with a heavy chain variable region prepared as a randomized variable region sequence library.

[0152] In addition, the light chain variable region sequence selected as a framework sequence that contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration can be designed to contain various amino acids as residues other than the amino acid residue. In the present invention, such residues are referred to as flexible residues. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence described in SEQ ID NO: 62 (Vk5-2) include the amino acid residues listed in Table 1 or Table 2.

[0153] [Table 1]

[0154] [Table 2]

[0155] As used herein, flexible residues refer to variations in amino acid residues present at positions where amino acids are highly diverse on the light and heavy chain variable regions with several different amino acids present at that position when comparing the amino acid sequences of known and / or natural antibodies or antigen-binding domains. Highly diverse positions are generally present in CDR regions. In one embodiment, data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful for determining highly diverse positions of known and / or natural antibodies. In addition, multiple databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html) provide collected sequences and their arrangements of many human light and heavy chains, and information on these sequences and their arrangements is useful for determining highly diverse positions in the present invention. According to the present invention, an amino acid position is said to be highly diverse if it has a diversity of preferably about 2 to about 20, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 possible different amino acid residues at that position. In some embodiments, an amino acid position may have a diversity of preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, preferably about 12 possible different amino acid residues.

[0156] In addition, a library containing a plurality of antigen-binding molecules of the present invention having different sequences can be prepared by combining a light chain variable region into which at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on the ion concentration conditions is introduced with a heavy chain variable region prepared as a randomized variable region sequence library. As a non-limiting example, when the ion concentration is calcium ion concentration, a library combining a light chain variable region sequence in which a specific residue in a germline such as SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), or SEQ ID NO: 8 (Vk4) is replaced with at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on the calcium ion concentration, and a heavy chain variable region prepared as a randomized variable region sequence library is preferably exemplified. Non-limiting examples of the amino acid residue include amino acid residues contained in CDR1 of the light chain. Other non-limiting examples of the amino acid residue include amino acid residues contained in CDR2 of the light chain. Further, other non-limiting examples of such amino acid residues include amino acid residues contained in the light chain CDR3.

[0157] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include the amino acid residues at positions 30, 31, and / or 32 as indicated by the Kabat numbering in the CDR1 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR2 include the amino acid residue at position 50 as indicated by the Kabat numbering in the CDR2 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR3 include the amino acid residue at position 92 as indicated by the Kabat numbering in the CDR3 of the light chain variable region. In addition, as long as these amino acid residues form a calcium-binding motif and / or the binding activity of the antigen-binding molecule to an antigen changes depending on the calcium ion concentration condition, these amino acid residues may be contained alone or in combination of two or more of these amino acids. Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known to have multiple calcium ion-binding sites and are thought to have been derived from a common origin in molecular evolution, and it is also possible to design the light chain CDR1, CDR2, and / or CDR3 so as to include the binding motif. For example, for the above purpose, a cadherin domain, an EF hand contained in calmodulin, a C2 domain contained in protein kinase C, a Gla domain contained in the blood coagulation protein Factor IX, a C-type lectin contained in the asialoglycoprotein receptor or the mannose-binding receptor, an A domain contained in the LDL receptor, annexin, a thrombospondin type 3 domain, and an EGF-like domain can be appropriately used.

[0158] Even when a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration conditions is introduced is combined with a heavy chain variable region prepared as a randomized variable region sequence library, it is possible to design the sequence of the light chain variable region to contain flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence include the amino acid residues listed in Table 1 or Table 2.

[0159] A suitable example of the heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed based on antibody genes derived from lymphocytes of animals immunized with a specific antigen, infectious disease patients, humans vaccinated and with increased blood antibody titers, cancer patients, and autoimmune disease patients can be preferably used as the randomized variable region library.

[0160] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of an appropriate length can also be suitably used as a randomized variable region library. In this case, since the diversity of the gene sequence of CDR3 of the heavy chain is observed, it is also possible to replace only the sequence of CDR3. The criterion for generating amino acid diversity in the variable region of an antigen-binding molecule is to provide diversity to amino acid residues at positions exposed on the surface of the antigen-binding molecule. The surface-exposed position refers to a position that is judged to be capable of being exposed on the surface and / or capable of contacting with an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally its CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions may be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558). Determination of surface-exposed positions may be performed using suitable software for protein modeling and three-dimensional structural information obtained from the antibody. A suitable software available for such purposes is the SYBYL Biopolymer Module software (Tripos Associates). Typically and preferably, where an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Additionally, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0161] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy subjects and have an unbiased repertoire, can also be particularly preferably used as a randomized variable region library (Gejima et al. (Human Antibodies (2002) 11, 121-129) and Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)). The amino acid sequence containing a naive sequence described in the present invention refers to an amino acid sequence obtained from such a naive library.

[0162] In one embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library containing a plurality of antigen-binding molecules of the present invention having different sequences by combining a heavy chain variable region selected as a framework sequence that contains in advance "at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on the ion concentration condition" with a light chain variable region prepared as a randomized variable region sequence library. As a non-limiting example, when the ion concentration is calcium ion concentration, a library that combines a heavy chain variable region sequence described in SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) with a light chain variable region prepared as a randomized variable region sequence library is preferably mentioned. Alternatively, instead of the light chain variable region prepared as a randomized variable region sequence library, it can be prepared by appropriately selecting from light chain variable regions having germline sequences. A suitable example is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) with a light chain variable region having a germline sequence.

[0163] In addition, it is also possible to design the heavy chain variable region sequence selected as the framework sequence that already contains the above-mentioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration condition" so that it contains flexible residues. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration condition, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the heavy chain variable region sequence described in SEQ ID NO: 9 (6RL#9-IgG1) include all amino acid residues of heavy chain CDR1 and CDR2, as well as amino acid residues of CDR3 other than positions 95, 96, and / or 100a of heavy chain CDR3. Alternatively, non-limiting examples of flexible residues to be introduced into the heavy chain variable region sequence set forth in SEQ ID NO: 10 (6KC4-1#85-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues in CDR3 other than positions 95 and / or 101 of heavy chain CDR3.

[0164] In addition, a library containing a plurality of antigen-binding molecules with different sequences can also be prepared by combining a heavy chain variable region into which the "at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on the ion concentration" has been introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence. As a non-limiting example, when the ion concentration is calcium ion concentration, for example, a library combining a heavy chain variable region sequence in which a specific residue in the heavy chain variable region is substituted with at least one amino acid residue that changes the binding activity of the antigen-binding molecule to an antigen depending on the calcium ion concentration and a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence is preferably exemplified. Non-limiting examples of the amino acid residue include amino acid residues contained in CDR1 of the heavy chain. Another non-limiting example of the amino acid residue includes amino acid residues contained in CDR2 of the heavy chain. Another non-limiting example of the amino acid residue includes amino acid residues contained in CDR3 of the heavy chain. Non-limiting examples of such amino acid residues contained in the heavy chain CDR3 include amino acids at positions 95, 96, 100a, and / or 101 as represented by the Kabat numbering in the CDR3 of the heavy chain variable region. Furthermore, as long as these amino acid residues form a calcium-binding motif and / or the antigen-binding activity of the antigen-binding molecule changes depending on the calcium ion concentration condition, these amino acid residues may be contained alone or in combination of two or more of these amino acids.

[0165] Even when combining a heavy chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions is introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence, it is possible to design the heavy chain variable region sequence to include flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be included in the CDR sequence and / or FR sequence of the heavy chain. In addition, a randomized variable region library may be suitably used as the amino acid sequence of CDR1, CDR2 and / or CDR3 of the heavy chain variable region other than the amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions. When a germline sequence is used as the light chain variable region, non-limiting examples include germline sequences such as SEQ ID NO:5 (Vk1), SEQ ID NO:6 (Vk2), SEQ ID NO:7 (Vk3), and SEQ ID NO:8 (Vk4).

[0166] As the amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration, any amino acid can be suitably used as long as it forms a calcium-binding motif, and specific examples of such amino acids include amino acids having electron-donating properties, such as serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0167] Hydrogen ion concentration conditions In one embodiment of the present invention, the ion concentration condition refers to the hydrogen ion concentration condition or the pH condition. In the present invention, the concentration condition of protons, i.e., the atomic nuclei of hydrogen atoms, is treated as synonymous with the hydrogen exponent (pH) condition. When the activity of hydrogen ions in an aqueous solution is expressed as aH+, pH is defined as -log10aH+. When the ionic strength in an aqueous solution is (for example, 10 -3If the pH is lower than 7, aH+ is approximately equal to the hydrogen ion strength. For example, the ionic product of water at 25°C and 1 atm is Kw=aH+aOH=10-14, so for pure water, aH+=aOH=10-7. In this case, pH=7 is neutral, and an aqueous solution with a pH less than 7 is acidic, and an aqueous solution with a pH greater than 7 is alkaline.

[0168] In the present invention, when pH conditions are used as ion concentration conditions, examples of pH conditions include high hydrogen ion concentration or low pH, i.e., acidic pH conditions, and low hydrogen ion concentration or high pH, ​​i.e., neutral pH conditions. The term "the binding activity changes depending on the pH condition" refers to the change in the binding activity of an antigen-binding molecule to an antigen due to the difference between high hydrogen ion concentration or low pH (acidic pH range) and low hydrogen ion concentration or high pH (neutral pH range). For example, the binding activity of an antigen-binding molecule to an antigen in a neutral pH range is higher than that in an acidic pH range. In addition, the binding activity of an antigen-binding molecule to an antigen in an acidic pH range is higher than that in a neutral pH range.

[0169] In the present specification, the neutral pH range is not limited to a specific numerical value, but may be preferably selected from the range of pH 6.7 to pH 10.0. In another embodiment, it may be selected from the range of pH 6.7 to pH 9.5. In another embodiment, it may be selected from the range of pH 7.0 to pH 9.0, and in another embodiment, it may be selected from the range of pH 7.0 to pH 8.0. In particular, pH 7.4, which is close to the pH in plasma (blood) in the living body, is preferable.

[0170] In the present specification, the acidic pH range is not limited to a specific numerical value, but may be preferably selected from the range of pH 4.0 to pH 6.5. In another embodiment, it may be selected from the range of pH 4.5 to pH 6.5. In a different embodiment, it may be selected from the range of pH 5.0 to pH 6.5, and in another embodiment, it may be selected from the range of pH 5.5 to pH 6.5. In particular, a pH of 5.8, which is close to the ionized calcium concentration in early endosomes in vivo, is preferably mentioned.

[0171] In the present invention, the antigen-binding activity of an antigen-binding molecule at a high proton concentration or low pH (acidic pH range) is lower than that at a low proton concentration or high pH (neutral pH range) means that the antigen-binding activity of the antigen-binding molecule at a pH selected from between pH 4.0 and pH 6.5 is weaker than that at a pH selected from between pH 6.7 and pH 10.0. Preferably, the antigen-binding activity of the antigen-binding molecule at a pH selected from between pH 4.5 and pH 6.5 is weaker than that at a pH selected from between pH 6.7 and pH 9.5, more preferably, the antigen-binding activity of the antigen-binding molecule at a pH selected from between pH 5.0 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 9.0. Also, preferably, the antigen-binding activity of the antigen-binding molecule at a pH selected from between pH 5.5 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 8.0. Particularly preferably, it means that the antigen-binding activity at a pH within an early endosome in a living body is weaker than the antigen-binding activity at a pH in plasma in a living body, specifically, it means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.

[0172] Whether or not the binding activity of an antigen-binding molecule to an antigen changes depending on pH conditions can be determined by using a known measurement method, for example, as described in the section on binding activity above. That is, the binding activity is measured under different pH conditions in the measurement method. For example, to confirm that the binding activity of an antigen-binding molecule to an antigen under neutral pH conditions changes more than the binding activity of an antigen-binding molecule to an antigen under acidic pH conditions, the binding activities of the antigen-binding molecule to an antigen under acidic pH conditions and neutral pH conditions are compared.

[0173] Furthermore, in the present invention, the expression "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range" can also be expressed as "the antigen-binding activity of the antigen-binding molecule at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range." In the present invention, "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be described as "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weaker than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" and "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is made lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be described as "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is made weaker than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range".

[0174] Conditions other than hydrogen ion concentration or pH when measuring the binding activity to an antigen can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of HEPES buffer and 37°C. For example, it can be measured using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by flowing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, the binding activity to the membrane antigen can be evaluated by flowing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0175] In the antigen-binding molecules of the present invention, as long as the antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, is weaker than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, the ratio of the antigen-binding activity under a high proton concentration or low pH, i.e., an acidic pH range, to the antigen-binding activity under a low proton concentration or high pH, ​​i.e., a neutral pH range, is not particularly limited; however, the ratio of the KD (Dissociation constant) for the antigen at a high proton concentration or low pH, i.e., an acidic pH range, to the KD at a low proton concentration or high pH, ​​i.e., a neutral pH range, KD (pH5.8) / KD (pH7.4), is preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the KD (pH 5.8) / KD (pH 7.4) value is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by those skilled in the art.

[0176] As the value of binding activity to an antigen, when the antigen is a soluble antigen, the KD (dissociation constant) can be used, whereas when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and the apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0177] As another indicator showing the ratio of the antigen-binding activity of the antigen-binding molecule of the present invention at a high proton concentration or low pH, i.e., an acidic pH range condition, to that at a low proton concentration or high pH, ​​i.e., a neutral pH range condition, for example, the dissociation rate constant kd (Dissociation rate constant) can also be suitably used. When kd (Dissociation rate constant) is used instead of KD (Dissociation constant) as an indicator showing the binding activity ratio, the value of kd (in an acidic pH range condition) / kd (in a neutral pH range condition), which is the ratio of kd (Dissociation rate constant) at a high proton concentration or low pH, i.e., an acidic pH range condition, to that at a low proton concentration or high pH, ​​i.e., a neutral pH range condition, is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (in an acidic pH range) / kd (in a neutral pH range) is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be prepared within the technical common sense of a person skilled in the art.

[0178] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble antigen, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), flow cytometer, etc. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different hydrogen ion concentrations, i.e., pH, it is preferable that the conditions other than hydrogen ion concentration, i.e., pH, are the same.

[0179] For example, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by screening for antigen-binding domains or antibodies comprising the following steps (a) to (c). (a) obtaining the antigen-binding activity of an antigen-binding domain or antibody under an acidic pH range condition; (b) obtaining the antigen-binding activity of the antigen-binding domain or antibody under a neutral pH condition; and (c) selecting an antigen-binding domain or antibody whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range.

[0180] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (c). (a) contacting an antigen-binding domain or an antibody or a library thereof with an antigen under a neutral pH range condition; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under an acidic pH condition; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0181] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d). (a) contacting a library of antigen-binding domains or antibodies with an antigen under an acidic pH range condition; (b) selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a); (c) binding the antigen-binding domain or antibody selected in step (b) to an antigen under a neutral pH range condition; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0182] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antibodies with a column on which an antigen is immobilized under a neutral pH condition; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under an acidic pH condition; and (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0183] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (d). (a) passing a library of antigen-binding domains or antibodies through an antigen-immobilized column under an acidic pH condition; (b) recovering the antigen-binding domain or antibody that is eluted without binding to the column in step (a); (c) binding the antigen-binding domain or antibody recovered in step (b) to an antigen under a neutral pH range condition; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0184] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (d). (a) contacting a library of antigen-binding domains or antibodies with an antigen under a neutral pH range condition; (b) obtaining the antigen-binding domain or antibody bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under an acidic pH condition; and (d) isolating in step (c) antigen-binding domains or antibodies whose antigen-binding activity is weaker than the criterion selected in step (b).

[0185] The above steps may be repeated two or more times. Thus, the present invention provides an antigen-binding domain or antibody whose antigen-binding activity in an acidic pH range is lower than its antigen-binding activity in a neutral pH range, which is obtained by the above-mentioned screening method further comprising the step of repeating steps (a) to (c) or (a) to (d) two or more times. The number of times that steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is usually within 10 times.

[0186] In the screening method of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under high hydrogen ion concentration conditions or low pH, i.e., in the acidic pH range, is not particularly limited as long as it is an antigen-binding activity between pH 4.0 and 6.5, but a preferred pH can be an antigen-binding activity between pH 4.5 and 6.6. Another preferred pH can be an antigen-binding activity between pH 5.0 and 6.5, and furthermore, an antigen-binding activity between pH 5.5 and 6.5. A more preferred pH can be the pH in early endosomes in vivo, and specifically, an antigen-binding activity at pH 5.8 can be mentioned. In addition, the antigen-binding activity of an antigen-binding domain or antibody under low hydrogen ion concentration conditions or high pH, ​​i.e., in the neutral pH range, is not particularly limited as long as it is an antigen-binding activity between pH 6.7 and 10, but a preferred pH can be an antigen-binding activity between pH 6.7 and 9.5. Another preferred pH can be an antigen-binding activity between pH 7.0 and 9.5, and furthermore, an antigen-binding activity between pH 7.0 and 8.0. An example of a more preferable pH is the pH in plasma in a living body, specifically, the antigen-binding activity at a pH of 7.4.

[0187] The antigen-binding activity of an antigen-binding domain or antibody can be measured by a method known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate constant), apparent kd (Apparent dissociation rate constant), etc. These can be measured by a method known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, FACS, etc. can be used.

[0188] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., an acidic pH range, means the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., a neutral pH range.

[0189] As long as the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, the difference between the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range and the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is not particularly limited, but the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range is preferably at least 2-fold, more preferably at least 10-fold, and even more preferably at least 40-fold, of the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range.

[0190] The antigen-binding domain or antibody of the present invention to be screened by the above screening method may be any antigen-binding domain or antibody, and it is possible to screen, for example, the above-mentioned antigen-binding domains or antibodies. For example, an antigen-binding domain or antibody having a native sequence may be screened, or an antigen-binding domain or antibody with a substituted amino acid sequence may be screened.

[0191] The antigen-binding domain or antibody of the present invention to be screened by the above-mentioned screening method may be prepared in any manner, and for example, pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced into these antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites, etc.).

[0192] Preferred methods for obtaining antigen-binding domains or antibodies whose antigen-binding activity at low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at high proton concentration or low pH, i.e., in an acidic pH range, from antigen-binding domains or antibodies prepared from hybridomas obtained by immunizing animals or B cells from immunized animals include antigen-binding molecules or antibodies in which at least one amino acid in the antigen-binding domain or antibody is substituted with an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid mutation, as described in WO2009 / 125825, or in which an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is inserted into the antigen-binding domain or antibody.

[0193] The position at which an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a mutation of a non-natural amino acid is introduced is not particularly limited, and may be any site as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range compared to before the substitution or insertion (the value of KD(acidic pH range) / KD(neutral pH range) is increased, or the value of kd(acidic pH range) / kd(neutral pH range) is increased). For example, when the antigen-binding molecule is an antibody, suitable sites include the variable region and CDR of the antibody. The number of amino acids to be substituted with amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, or the number of amino acids to be inserted, can be appropriately determined by those skilled in the art. An amino acid may be substituted with a single amino acid (e.g., histidine or glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0, an amino acid (e.g., histidine or glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 may be inserted, an amino acid may be substituted with two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0, or two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 may be inserted. In addition to substitution with or insertion of amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, deletion, addition, insertion and / or substitution of other amino acids may be performed at the same time.Substitution with amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, or insertion of amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids can be randomly performed by methods such as histidine scanning, in which alanine in alanine scanning is replaced with histidine or the like, known to those skilled in the art. From antigen-binding domains or antibodies into which mutations such as substitution or insertion of amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been randomly introduced, antigen-binding molecules with increased KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / kd (neutral pH range) values ​​compared to before mutation can be selected.

[0194] Preferred examples of antigen-binding molecules that have been mutated to amino acids whose side chains have a pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids as described above and have lower antigen-binding activity in an acidic pH range than in a neutral pH range include antigen-binding molecules whose antigen-binding activity in a neutral pH range after mutation to amino acids whose side chains have a pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids is equivalent to the antigen-binding activity in a neutral pH range before mutation to amino acids whose side chains have a pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids. In the present invention, an antigen-binding molecule after mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid has antigen-binding activity equivalent to that of an antigen-binding molecule before mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid means that, when the antigen-binding activity of the antigen-binding molecule before mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is taken as 100%, the antigen-binding activity of the antigen-binding molecule after mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is at least 10%, preferably 50% or more, more preferably 80% or more, and more preferably 90% or more. The antigen-binding activity at pH 7.4 after mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid may be higher than the antigen-binding activity at pH 7.4 before mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid. When the antigen-binding activity of an antigen-binding molecule is reduced by substitution or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, the antigen-binding activity can be made equivalent to the antigen-binding activity before substitution or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid by substitution, deletion, addition, and / or insertion of one or more amino acids in the antigen-binding molecule.The present invention also includes antigen-binding molecules whose binding activity is equivalent to that of the antigen-binding molecules obtained by substituting or inserting an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, followed by substituting, deleting, adding, and / or inserting one or more amino acids.

[0195] Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, another preferred embodiment of the antigen-binding molecule having lower antigen-binding activity in an acidic pH range than in a neutral pH range includes a method in which the antibody constant region contained in the antigen-binding molecule is modified. Specific examples of the antibody constant region after modification preferably include the constant regions set forth in SEQ ID NOs: 11, 12, 13, or 14.

[0196] Amino acids that change the binding activity of antigen-binding domains to antigens depending on hydrogen ion concentration conditions The antigen-binding domain or antibody of the present invention to be screened by the above-mentioned screening method may be prepared in any manner. For example, when the ion concentration conditions are hydrogen ion concentration conditions or pH conditions, it is possible to use pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries in which mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been introduced into these antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, libraries in which mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been introduced at specific sites, etc.).

[0197] In one non-limiting embodiment of the present invention, a library comprising a plurality of antigen-binding molecules of the present invention having different sequences can also be prepared by combining a light chain variable region into which "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library.

[0198] Non-limiting examples of such amino acid residues include amino acid residues contained in CDR1 of the light chain. Other non-limiting examples of such amino acid residues include amino acid residues contained in CDR2 of the light chain. Another non-limiting example of such amino acid residues includes amino acid residues contained in CDR3 of the light chain.

[0199] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include amino acid residues at positions 24, 27, 28, 31, 32, and / or 34 as defined by Kabat numbering in the CDR1 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR2 include amino acid residues at positions 50, 51, 52, 53, 54, 55, and / or 56 as defined by Kabat numbering in the CDR2 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR3 include amino acid residues at positions 89, 90, 91, 92, 93, 94, and / or 95A as defined by Kabat numbering in the CDR3 of the light chain variable region. Furthermore, as long as the binding activity of the antigen-binding molecule to an antigen changes depending on the hydrogen ion concentration condition, these amino acid residues may be contained alone or in combination of two or more of these amino acids.

[0200] Even when a light chain variable region into which the above-mentioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced is combined with a heavy chain variable region prepared as a randomized variable region sequence library, it is possible to design the sequence of the light chain variable region to contain flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on hydrogen ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence include the amino acid residues listed in Table 3 or Table 4. In addition, germline sequences such as Vk1 (SEQ ID NO: 5), Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7), and Vk4 (SEQ ID NO: 8) may be suitably used as amino acid sequences of the light chain variable region other than the amino acid residues that change the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions and the flexible residues.

[0201] [Table 3] (The positions represent the Kabat numbering.)

[0202] [Table 4] (The positions represent the Kabat numbering.)

[0203] Any amino acid residue can be suitably used as the amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions. Specific examples of such amino acid residues include amino acids whose side chain pKa is 4.0 to 8.0. Suitable examples of such electron-donating amino acids include natural amino acids such as histidine and glutamic acid, as well as histidine analogs (US20090035836) and unnatural amino acids such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21) and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768). Particularly suitable examples of such amino acid residues include amino acids whose side chains have a pKa of 6.0 to 7.0. Suitable examples of such electron-donating amino acids include histidine.

[0204] For modifying amino acids in the antigen-binding domain, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. In addition, as a method for modifying amino acids by substituting amino acids other than natural amino acids, several known methods can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, can be suitably used.

[0205] A suitable example of the heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed based on antibody genes derived from lymphocytes of animals immunized with a specific antigen, infectious disease patients, humans vaccinated and with increased blood antibody titers, cancer patients, and autoimmune disease patients can be preferably used as the randomized variable region library.

[0206] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of an appropriate length can also be suitably used as a randomized variable region library. In this case, since the diversity of the gene sequence of CDR3 of the heavy chain is observed, it is also possible to replace only the sequence of CDR3. The criterion for generating amino acid diversity in the variable region of an antigen-binding molecule is to provide diversity to amino acid residues at positions exposed on the surface of the antigen-binding molecule. The term "surface-exposed position" refers to a position that is judged to be capable of being exposed on the surface and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions may be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558). Determination of surface-exposed positions may be performed using suitable software for protein modeling and three-dimensional structural information obtained from the antibody. A suitable software available for such purposes is the SYBYL Biopolymer Module software (Tripos Associates). Typically and preferably, where an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Additionally, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0207] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy subjects and have a bias-free repertoire, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344).

[0208] Neutralizing activity In a non-limiting embodiment of the present invention, an antigen-binding molecule having neutralizing activity against an antigen, comprising an antigen-binding domain having binding activity against human FcRn in an acidic pH range and whose binding activity against an antigen changes depending on ion concentration conditions, and an Fcγ receptor-binding domain having higher binding activity against an Fcγ receptor under a neutral pH range condition than the binding activity against an Fcγ receptor of the Fc region of a native human IgG in which the sugar chain bound to position 297 (EU numbering) is a fucose-containing sugar chain, and a pharmaceutical composition comprising the antigen-binding molecule are provided. In general, neutralizing activity refers to the activity of inhibiting the biological activity of a ligand having biological activity against cells, such as a virus or a toxin. That is, a substance having neutralizing activity refers to a substance that binds to the ligand or a receptor to which the ligand binds, and inhibits the binding of the ligand to the receptor. A receptor whose binding to a ligand is prevented by neutralizing activity cannot exert biological activity through the receptor. When the antigen-binding molecule is an antibody, an antibody having such neutralizing activity is generally called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand between the presence and absence of the test substance.

[0209] For example, IL-6, which is represented by SEQ ID NO: 15, is considered to be the main ligand of IL-6R. IL-6R, a type I membrane protein whose amino terminus forms the extracellular domain, forms a heterotetramer with gp130 receptor, whose dimerization is induced by IL-6 (HEINRICH et al. (Biochem. J. (1998) 334, 297-314)). The formation of the heterotetramer activates Jak associated with the gp130 receptor. Jak performs autophosphorylation and receptor phosphorylation. The phosphorylation sites of the receptor and Jak act as binding sites for molecules belonging to the Stat family that have SH2, such as Stat3, MAP kinase, PI3 / Akt, and other proteins and adaptors that have SH2. Next, Stat bound to the gp130 receptor is phosphorylated by Jak. The phosphorylated Stat forms a dimer and migrates into the nucleus, where it regulates the transcription of target genes. Jak or Stat can also participate in signaling cascades through other classes of receptors. Deregulated IL-6 signaling cascades are observed in the pathology of autoimmune diseases, inflammation, and cancers such as multiple myeloma and prostate cancer. Stat3, which can act as an oncogene, is constitutively activated in many cancers. In prostate cancer and multiple myeloma, there is crosstalk between the signaling cascade from IL-6R and the signaling cascade from members of the epidermal growth factor receptor (EGFR) family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

[0210] Since such intracellular signal cascades differ for each cell type, a target molecule can be set appropriately for each target cell of interest, and is not limited to the above factors. Neutralizing activity can be evaluated by measuring the activation of an in vivo signal. In addition, the activation of an in vivo signal can be detected using the transcription induction effect on a target gene present downstream of the in vivo signal cascade as an index. The change in the transcription activity of a target gene can be detected by the principle of a reporter assay. Specifically, a reporter gene such as GFP (Green Fluorescence Protein) or luciferase is placed downstream of the transcription factor or promoter region of the target gene, and the reporter activity is measured, thereby measuring the change in the transcription activity as the reporter activity. A commercially available kit for measuring the activation of an in vivo signal can be used appropriately (e.g., Mercury Pathway Profiling Luciferase System (Clontech), etc.).

[0211] Furthermore, as a method for measuring the neutralizing activity of receptor ligands such as the EGF receptor family, which usually act on a signal cascade that promotes cell proliferation, the neutralizing activity of a neutralizing antibody can be evaluated by measuring the proliferation activity of the target cells. For example, the following method is preferably used as a method for evaluating or measuring the inhibitory effect based on the neutralizing activity of an anti-HB-EGF antibody on the proliferation of cells whose proliferation is promoted by a growth factor of the EGF family, such as HB-EGF. As a method for evaluating or measuring the cell proliferation inhibitory activity in a test tube, a method is used in which the incorporation of [3H]-labeled thymidine added to a culture medium by living cells is measured as an index of DNA replication ability. As a more convenient method, a dye exclusion method in which the ability to exclude dyes such as trypan blue from cells is measured under a microscope, or the MTT method is used. The latter utilizes the ability of living cells to convert the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue formazan product. More specifically, after a certain period of time has elapsed since the test antibody was added together with the ligand to the culture medium of the test cells, an MTT solution is added to the culture medium and allowed to stand for a certain period of time to allow the MTT to be taken up by the cells. As a result, the yellow compound MTT is converted to a blue compound by succinate dehydrogenase in the mitochondria of the cells. This blue product is dissolved and colored, and its absorbance is measured to provide an index of the number of live cells. In addition to MTT, reagents such as MTS, XTT, WST-1, and WST-8 are also commercially available (e.g., Nacalai Tesque) and can be used suitably. When measuring the activity, a conjugated antibody having the same isotype as the anti-HB-EGF antibody and not having the cell proliferation inhibitory activity is used as a control antibody in the same manner as the anti-HB-EGF antibody, and the activity can be determined by the anti-HB-EGF antibody showing stronger cell proliferation inhibitory activity than the control antibody.

[0212] As the cells for evaluating the activity, for example, the RMG-1 cell line, which is an ovarian cancer cell line whose proliferation is promoted by HB-EGF, and mouse Ba / F3 cells transformed with a vector ligated so as to express a gene encoding hEGFR / mG-CSFR, which is a fusion protein in which the extracellular domain of human EGFR and the intracellular domain of mouse GCSF receptor are fused in frame, can be suitably used. Thus, those skilled in the art can use the cells for evaluating the activity in the measurement of the cell proliferation activity by appropriately selecting the cells.

[0213] Since the antigen-binding molecule provided by the present invention can eliminate an antigen from plasma, it is not necessarily required that the antigen-binding molecule itself has neutralizing activity. However, it is more preferable that the antigen-binding molecule exerts neutralizing activity against the antigen until the antigen is taken up into a cell expressing an Fcγ receptor by endocytosis mediated by the Fcγ receptor, thereby blocking the function of the antigen present in plasma.

[0214] In addition, the antigen-binding molecule provided by the present invention can promote the dissociation of an antigen bound to the antigen-binding molecule outside a cell from the antigen-binding molecule inside the cell, and therefore the antigen dissociated from the antigen-binding molecule inside the cell is degraded in the lysosome. Therefore, it is not necessarily required that the antigen-binding molecule itself has neutralizing activity. However, it is more preferable that the function of the antigen present in the plasma is blocked by exerting neutralizing activity against the antigen until the antigen is taken up into a cell expressing the Fcγ receptor together with the antigen-binding molecule by endocytosis mediated by the Fcγ receptor.

[0215] Furthermore, since the antigen-binding molecule provided by the present invention can reduce the total antigen concentration or free antigen concentration in plasma, it is not necessarily required that the antigen-binding molecule itself has neutralizing activity. However, it is more preferable that the antigen-binding molecule exerts neutralizing activity against the antigen during the period until the antigen is taken up into a cell expressing an Fcγ receptor by endocytosis mediated by the Fcγ receptor, thereby blocking the function of the antigen present in plasma.

[0216] Fcγ receptor The term Fcγ receptor (FcγR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, and refers to any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI (CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. FcγRs may be from any organism, including, but not limited to, humans, mice, rats, rabbits and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (FcγRIV, CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. Suitable examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16) and / or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI are shown in SEQ ID NOs: 16 (NM_000566.3) and 17 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of human FcγRIIa (allotype H131) are shown in SEQ ID NOs: 18 (BC020823.1) and 19 (AAH20823.1), respectively (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 19 is substituted with Arg). The polynucleotide sequence and amino acid sequence of FcγRIIb are shown in SEQ ID NOs: 19 (NM_000566.3) and 17 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of FcγRIIIa are set forth in SEQ ID NOs: 20 (BC146678.1) and 21 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIa are set forth in SEQ ID NOs: 22 (BC033678.1) and 23 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIb are set forth in SEQ ID NOs: 24 (BC128562.1) and 25 (AAI28563.1), respectively (RefSeq accession numbers are indicated in parentheses). Whether or not an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the above-described FACS or ELISA format, as well as by the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) or the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0217] Also, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of the Fc ligand to Fc 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), a family of Fc receptors that are homologous to FcγR (Davis et al., (2002) Immunological Reviews 190, 123-136) and FCRL (Annu Rev Immunol, 2007; 25: 525-60). Fc ligands may also include as yet undiscovered molecules that bind to Fc.

[0218] FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), are associated with an α chain that binds to the Fc portion of IgG and a common γ chain that has ITAMs that transmit activation signals intracellularly. On the other hand, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its own cytoplasmic domain. These receptors are expressed on many immune cells, such as macrophages, mast cells, and antigen-presenting cells. The activation signals transmitted by the binding of these receptors to the Fc portion of IgG promote the phagocytic ability of macrophages, the production of inflammatory cytokines, the degranulation of mast cells, and the enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are also referred to as activating Fcγ receptors in the present invention.

[0219] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM that transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses activation signals from the BCR, resulting in the inhibition of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic ability and the ability to produce inflammatory cytokines. In the present invention, Fcγ receptors that have the ability to transmit inhibitory signals as described above are also called inhibitory Fcγ receptors.

[0220] Binding activity to Fcγ receptors The binding activity of the FcγR-binding domain contained in the antigen-binding molecule of the present invention to any one of human Fcγ receptors, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb, can be confirmed by the FACS and ELISA formats described above, as well as ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). In these assays, the extracellular domain of the human Fcγ receptor can be used as a soluble antigen.

[0221] The ALPHA screen is carried out by ALPHA technology using two beads, donor and acceptor, based on the following principle: A luminescence signal is detected only when a molecule bound to a donor bead biologically interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

[0222] For example, an antigen-binding molecule containing a biotin-labeled Fc region is bound to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a competing antigen-binding molecule containing an Fc region variant, an antigen-binding molecule having a native Fc region and an Fcγ receptor interact to generate a signal at 520-620 nm. An antigen-binding molecule containing an untagged Fc region variant competes with the interaction between an antigen-binding molecule having a native Fc region and an Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of the competition. It is known to biotinylate an antigen-binding molecule such as an antibody using Sulfo-NHS-biotin or the like. The method of tagging Fcγ receptor with GST may be appropriately selected by expressing a fusion gene in which a polynucleotide encoding Fcγ receptor and a polynucleotide encoding GST are fused in frame in a vector operably linked to the fusion gene in cells or the like, and purifying the fusion gene using a glutathione column, etc. The obtained signal is suitably analyzed by fitting the signal to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0223] One of the substances (ligand) for which interaction is to be observed is fixed on the gold film of a sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass. When the other substance (analyte) for which interaction is to be observed is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond is dissociated, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be calculated from the curve of the sensorgram, and the affinity (KD) can be calculated from the ratio of these constants. Inhibition measurement methods are also preferably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0224] Fcγ receptor binding domain An Fcγ receptor-binding domain having a higher binding activity to an Fcγ receptor than that of the Fc region of a native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain can be prepared by modifying the amino acids in the Fc region of a native human IgG. Furthermore, the Fcγ receptor-binding domain may be any domain having any structure among the antigen-binding domains described above that are characterized by binding to an Fcγ receptor. In such a case, the domain may be prepared without the need for introducing amino acid modifications, and further modifications may be introduced to increase affinity to the Fcγ receptor. Examples of such Fcγ receptor-binding domains include Fab fragment antibodies that bind to FcγRIIIa, 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, camel-derived single domain antibodies and single chain Fv antibodies, and FcγRI-binding cyclic peptides as described in FASEB J. 2009 Feb;23(2):575-85. Whether or not the binding activity of an Fcγ receptor-binding domain to FcγR is higher than the binding activity of an Fc region of a native human IgG in which the sugar chain bound to position 297 (EU numbering) is a fucose-containing sugar chain, can be determined appropriately using the method described in the above section on binding activity.

[0225] In the present invention, the Fc region of human IgG is a suitable example of the starting Fcγ receptor binding domain. In the present invention, the "amino acid modification" or "amino acid modification" of the Fc region includes modification to an amino acid sequence different from the amino acid sequence of the starting Fc region. Any Fc region can be used as the starting Fc region as long as the modified variant of the starting Fc region can bind to a human Fcγ receptor in a neutral pH range. In addition, an Fc region that has already been modified and further modified can also be used suitably as the Fc region of the present invention. The starting Fc region may refer to the polypeptide itself, a composition containing the starting Fc region, or an amino acid sequence encoding the starting Fc region. The starting Fc region may include known Fc regions produced by recombination as outlined in the section on antibodies. The origin of the starting Fc region may be obtained from any organism of a non-human animal or from a human, but is not limited thereto. Preferably, the any organism is preferably an organism selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In another embodiment, the starting Fcγ receptor binding domain can also be obtained from cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the starting Fc region can be obtained from human IgG1, but is not limited to a specific class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be appropriately used as the starting Fcγ receptor binding domain. Similarly, in the present specification, it means that the Fc region of any class or subclass of IgG from any organism can be preferably used as the starting Fc region.Examples of naturally occurring IgG variants or engineered forms are described in the known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0226] Examples of modifications include one or more mutations, such as substitution of amino acid residues different from those of the starting Fc region, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the amino acids of the starting Fc region. Preferably, the amino acid sequence of the modified Fc region includes an amino acid sequence that includes at least a portion of a non-naturally occurring Fc region. Such variants necessarily have less than 100% sequence identity or similarity with the starting Fc region. In a preferred embodiment, the variant has an amino acid sequence with about 75% to less than 100% amino acid sequence identity or similarity, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100%, and most preferably about 95% to less than 100% identity or similarity with the amino acid sequence of the starting Fc region. In a non-limiting aspect of the present invention, there is at least one amino acid difference between the starting Fc region and the modified Fc region of the present invention. The amino acid differences between the starting Fc region and the modified Fc region can also be suitably identified by specific amino acid differences at amino acid residue positions specified by the EU numbering system described above.

[0227] For the modification of amino acids in the Fc region, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. In addition, as a method for modifying amino acids by substituting amino acids other than natural amino acids, several known methods can be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also preferably used.

[0228] The Fc region having binding activity to Fcγ receptors in a neutral pH range contained in the antigen-binding molecule of the present invention can be obtained by any method, but specifically, an Fc region having binding activity to Fcγ receptors in a neutral pH range can be obtained by modifying amino acids of a human IgG immunoglobulin used as a starting Fc region. Preferred examples of the Fc region of an IgG immunoglobulin for modification include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and modified forms thereof). The Fc region of IgG also includes naturally occurring mutants thereof. As for the Fc region of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present invention. In particular, as for the sequence of human IgG1, the amino acid sequence at positions 356-358 (EU numbering) may be DEL or EEM.

[0229] As for modification to other amino acids, amino acids at any position may be modified as long as they have Fcγ receptor-binding activity in a neutral pH range or can enhance Fcγ receptor-binding activity in a neutral pH range. When the antigen-binding molecule contains a human IgG1 Fc region as the human Fc region, it is preferable that the antigen-binding molecule contains a modification that has the effect of enhancing Fcγ receptor binding in a neutral pH range compared to the binding activity of the starting Fc region of human IgG1. Amino acid modifications for enhancing binding activity to Fcγ receptors under neutral pH conditions have been reported in, for example, 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.

[0230] Examples of amino acids that can be modified in this way include those at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 49th, 250th, 251st, 254th, 255th, 256th, 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th , 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286 288th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 3 04th, 305th, 311th, 313rd, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, Examples of such amino acids include at least one or more amino acids selected from the group consisting of positions 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440. Modification of these amino acids enhances the binding of the Fc region of IgG immunoglobulin to an Fcγ receptor under a neutral pH range condition.

[0231] Particularly preferred modifications for use in the present invention include, for example, those represented by the EU numbering of the Fc region: The amino acid at position 221 is either Lys or Tyr; The amino acid at position 222 is Phe, Trp, Glu, or Tyr; The amino acid at position 223 is Phe, Trp, Glu, or Lys; The amino acid at position 224 is Phe, Trp, Glu, or Tyr; The amino acid at position 225 is either Glu, Lys or Trp; The amino acid at position 227 is Glu, Gly, Lys, or Tyr; The amino acid at position 228 is Glu, Gly, Lys, or Tyr; The amino acid at position 230 is Ala, Glu, Gly, or Tyr; The amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr; The amino acid at position 232 is Glu, Gly, Lys, or Tyr; the amino acid at position 233 is any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 238 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 239 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 240 is Ala, Ile, Met, or Thr; The amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or Tyr; The amino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; The amino acid at position 244 is His; The amino acid at position 245 is Ala; The amino acid at position 246 is Asp, Glu, His, or Tyr; the amino acid at position 247 is any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr; The amino acid at position 249 is Glu, His, Gln, or Tyr; The amino acid at position 250 is either Glu or Gln; The amino acid at position 251 is Phe; The amino acid at position 254 is either Phe, Met, or Tyr; The amino acid at position 255 is either Glu, Leu or Tyr; The amino acid at position 256 is either Ala, Met, or Pro; The amino acid at position 258 is Asp, Glu, His, Ser, or Tyr; The amino acid at position 260 is Asp, Glu, His, or Tyr; The amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; the amino acid at position 263 is Ala, Ile, Met, or Thr; the amino acid at position 264 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position 265 is any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 is Ala, Ile, Met, or Thr; the amino acid at position 267 is Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 268 is Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp; the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 270 is any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 272 is Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 273 is either Phe or Ile; the amino acid at position 274 is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 275 is either Leu or Trp; The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 278 is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 279 is Ala; The amino acid at position 280 is any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 is Asp, Lys, Pro, or Tyr; The amino acid at position 282 is Glu, Gly, Lys, Pro, or Tyr; the amino acid at position 283 is any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; The amino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or Tyr; The amino acid at position 285 is Asp, Glu, Lys, Gln, Trp, or Tyr; The amino acid at position 286 is either Glu, Gly, Pro, or Tyr; The amino acid at position 288 is Asn, Asp, Glu, or Tyr; the amino acid at position 290 is Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr, and the amino acid at position 291 is Asp, Glu, Gly, His, Ile, Gln, or Thr; The amino acid at position 292 is Ala, Asp, Glu, Pro, Thr, or Tyr; the amino acid at position 293 is Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 294 is any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 296 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the amino acid at position 297 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 298 is any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 300 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; The amino acid at position 301 is Asp, Glu, His, or Tyr; The amino acid at position 302 is Ile; The amino acid at position 303 is Asp, Gly, or Tyr; the amino acid at position 304 is Asp, His, Leu, Asn, or Thr; The amino acid at position 305 is either Glu, Ile, Thr or Tyr; the amino acid at position 311 is Ala, Asp, Asn, Thr, Val, or Tyr; The amino acid at position 313 is Phe; The amino acid at position 315 is Leu; The amino acid at position 317 is Glu or Gln; The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; The amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 322 is any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 323 is Ile; the amino acid at position 324 is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 325 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 is any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 328 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 330 is any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 332 is any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 is any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 is Ala, Glu, Phe, Ile, L...

Claims

[Claim 1] The invention described herein.

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