RU2772771C1 - Antigen-binding molecule for accelerating antigen loss - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including an antigen-binding molecule for antigen elimination from plasma, a method for reducing the total concentration of an antigen or concentration of a free antigen in plasma, a method for increasing the capability of an antigen-binding molecule of eliminating plasma antigens, a method for producing an antigen-binding molecule for reducing the total concentration of an antigen or concentration of a free antigen in plasma (options). In one of embodiment, the antigen-binding molecule contains an antigen-binding domain, where the domain has antigen-binding activity that changes depending on pH, where a value of KD (pH 5.8) / KD (pH 7.4) equal to 2 or more is a KD ratio for an antigen in conditions of a range of acidic pH values to KD in conditions of a range of neutral pH values.

EFFECT: invention expands the arsenal of means for antigen elimination from plasma.

28 cl, 81 dwg, 54 tbl, 52 ex

Description

Technical field

The present invention relates to antigen-binding molecules with enhanced intracellular uptake of bound antigen, antigen-binding molecules in which the number of antigens that can bind per molecule is increased, antigen-binding molecules with improved pharmacokinetics, antigen-binding molecules with enhanced intracellular dissociation of intracellularly bound antigen, antigen-binding molecules with enhanced extracellular release in a non-antigen-bound state, antigen-binding molecules having the function of reducing total antigen concentration or free plasma antigen concentration, pharmaceutical compositions containing such antigen-binding molecules, and methods for their preparation.

State of the art

Antibodies are attracting attention as drugs because they are highly stable in plasma and have few side effects. At present, a number of such IgG-type pharmaceuticals are commercially available, and many antibody-based drugs are being developed today (Non-Patent Documents 1 and 2). Meanwhile, various methods applicable to the second generation of antibody-based drugs have been described, including methods that enhance effector function, antigen-binding ability, pharmacokinetics, and stability, and methods that reduce the risk of developing immunogenicity (Non-Patent Document 3). As a rule, the required dose of drug based antibodies is very high. This, in turn, has led to problems such as high production costs as well as difficulties in obtaining subcutaneous formulations. In theory, the dose of an antibody-based pharmaceutical can be reduced by improving the pharmacokinetics of the antibody or by increasing the affinity between antibodies and antigens.

The literature describes methods for improving the pharmacokinetics of antibodies using artificial amino acid substitutions in the constant regions (non-patent documents 4 and 5). Similarly, affinity maturation is described as a method for improving antigen-binding ability or antigen neutralization activity (non-patent document b). This method makes it possible to increase the antigen-binding capacity by introducing an amino acid mutation in the CDR of the variable region, and the like. Increasing the antigen-binding capacity provides an improvement in in vitro biological activity or allows a dose reduction, and further provides an improved in vivo efficacy (Non-Patent Document 7).

The ability of a single antibody molecule to neutralize an antigen depends on its affinity. By increasing the affinity, the antigen can be neutralized with less antibody. Various methods can be used to increase the affinity of an antibody (non-patent document b). Further, if affinity could be made infinite by covalently binding an antibody to an antigen, one antibody molecule could neutralize one antigen molecule (a divalent antibody could neutralize two antigen molecules). However, the neutralization stoichiometry of one antibody against one antigen (one bivalent antibody against two antigens) is a limiting factor for the prior methods, and thus it is not possible to completely neutralize an antigen with less antibody than antigen. In other words, the affinity enhancing effect has a limiting value (Non-Patent Document 9). To increase the period of action of the neutralizing effect of a neutralizing antibody over a certain period, the antibody must be administered at a dose higher than the amount of antigen produced in the body during the same period. Thus, in the case of improving only the pharmacokinetics of antibodies or affinity maturation technology, as described above, there is a limitation in terms of reducing the required dose of the antibody. Thus, in order to maintain the effect of antibodies in neutralizing an antigen for a given period of time with antibodies in an amount less than the amount of antigen, a single antibody must neutralize a plurality of antigens. Recently, an antibody that binds to an antigen in a pH-dependent manner has been described as a novel method for achieving the above-described object (Patent Document 1). Antibodies with pH-dependent antigen binding that bind strongly to antigen under neutral plasma conditions and dissociate from antigen under acidic conditions in the endosome can dissociate from antigen in the endosome. When an antibody with pH-dependent antigen binding dissociates from the antigen and is recirculated to plasma via the FcRn, it can re-bind to another antibody. Thus, an antibody with pH dependent antigen binding can repeatedly bind to multiple antigens.

In addition, the retention time of the antigen in plasma is very short compared to the retention time of antibodies recirculating to plasma by binding to FcRn. If an antibody with such a long plasma retention time binds an antigen, the plasma retention time of the antigen-antibody complex is increased to the same retention time as that of the antibody. Thus, due to binding to the antibody, the retention time of the antigen increases, and thus the plasma concentration of the antigen increases.

Thus, antibodies with pH-dependent antigen binding have effects that cannot be achieved with normal antibodies because they can accelerate the elimination of antigens from plasma compared to normal antigens by binding a single antibody to multiple antigens. However, methods for engineering antibodies to improve the effects of antibodies with pH-dependent antigen binding, which can repeatedly bind antigens and which accelerate the elimination of antigens from plasma, have not been described to date.

IgG antibodies have a long plasma retention time due to their 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 taken up into cells non-specifically, but by binding to FcRn in the endosome under acidic conditions within the endosome, they return to the cell surface and dissociate from the FcRn under neutral conditions in plasma. When the Fc region of IgG is mutated such that binding to FcRn in the acidic pH range is lost, recycling of the antibody from the endosome to plasma does not occur and the plasma retention time of the antibody is significantly reduced. A method for increasing FcRn binding under conditions of an acidic pH range is described as a method for increasing the plasma retention time of an IgG antibody. Increasing binding to FcRn under conditions of the acidic pH range by introducing amino acid substitutions in the Fc region of an IgG antibody leads to an increased efficiency in recycling the antibody from the endosome to plasma and, as a result, increases the plasma retention time.

So far, there have been many studies on antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) and complement-dependent cytotoxicity (hereinafter referred to as CDC), which are effector functions of IgG class antibodies. In the human IgG class, IgG1 half-class antibodies have been described to have the highest ADCC activity and CDC activity (Non-Patent Document 13). Moreover, one of the effector functions of antibodies is also believed to be antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG-class antibodies (Non-Patent Documents 14 and 15). Because IgG1 subclass antibodies can induce these effector functions against tumors, IgG1 subclass antibodies are used for most pharmaceuticals against cancer antigens.

In order for IgG antibodies to mediate ADCC and ADCP activity, the Fc region of an IgG antibody must bind to antibody receptors (hereinafter referred to as Fcγ receptor or FcγR) that are present on the surface of effector cells such as killer cells, natural killer cells and activated macrophages. In humans, FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms are described as members of the Fcγ receptor family, and their corresponding allotypes are also described (Non-Patent Document 16).

Enhancement of cytotoxic effector functions such as ADCC and ADCP has attracted attention as promising agents for enhancing the antitumor effects of antibodies against

malignant tumor. The importance of Fcγ receptor-mediated effector functions targeted by the antitumor effects of antibodies has been described using mouse models (Non-Patent Documents 17 and 18). Moreover, clinical effects in humans were found to correlate with the high affinity polymorphic allotype (VI58) and the low affinity polymorphic allotype (F158) of FcγRIIIa (Non-Patent Document 19). These reports indicate that antibodies with an Fc region optimized for binding to specific Fcγ receptors mediate more potent effector functions and thus exhibit more effective antitumor effects. The balance between the affinity of antibodies against activating receptors, including FcγRIa, FcγRIIa, FcγRIIIa and FcγRIIIb, and inhibitory receptors, including FcγRIIb, is an important factor in optimizing antibody effector functions. Increasing affinity for activating receptors can provide antibodies with the property of mediating more potent effector functions (Non-Patent Document 20), and thus has been described in various reports to date as a modification method by engineering antibodies to increase or enhance the antitumor activity of pharmaceuticals by antibodies against cancer antigens.

Regarding the binding between the Fc region and the Fcγ receptor, several residues in the antibody hinge region and the antibody CH2 domain and the sugar chain linked to Asn at position 297 (EU numbering) in the CH2 domain have been shown to be important (non-patent papers 13, 21 and 22). Focusing on these binding sites, research has hitherto been carried out on Fc region mutants having different Fcγ receptor binding properties, and Fc region mutants with higher affinity for the activating Fcγ receptor have been obtained (Patent Documents 2 and 3). For example, Lazar et al. successfully increased the binding of human IgG1 to human FcγRIIIa (V158) approximately 370-fold by replacing Ser at position 239, Ala at position 330, and He at position 332 (EU numbering) of human IgG1 with Asp, Leu, and Glu, respectively (non-patent document 19 and patent document 2). The FcγRIIIa to FcγRIIb binding ratio (A/1 ratio) for this mutant was approximately 9 times higher than for the wild type. Moreover, Shinkawa et al. successfully increased binding to FcγRIIIa up to about 100-fold by removing fucose from the sugar chain linked to Asn at position 297 (EU numbering) (non-patent document 24). These methods can significantly increase the activity of human IgG1 against ADCC compared to naturally occurring human IgG1.

Thus, since Fcγ receptor binding activity plays an important role in cytotoxic activity in antibodies targeting membrane-type antigens, a human IgG1 isotype with high FcγR binding activity is used when cytotoxic activity is required. Increase in cytotoxic

activity by enhancing Fcγ receptor binding activity is also a widely used method. On the other hand, the role played by Fcγ receptor binding activity in antibodies targeting soluble antigens is unknown, and it is believed that there is no difference between human IgG1 with high Fcγ receptor binding activity and human IgG2 and human IgG4 with low FcγR binding activity. . Thus, no attempt has been made to date to enhance the Fcγ receptor binding activity for antibodies targeting soluble antigens, and their effects have not been described.

[Prior Art Documents]

Patent Documents

[Patent document 1] WO 2009/125825

[Patent document 2] WO 2000/042072

[Patent document 3] WO 2006/019447

Non-Patent Documents

[Non-Patent Document 1] Janice M Reichert, Clark J Rosenweig, 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

[Non-Patent Document 7] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA., Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract., J. Mol. Biol. (2007) 368, 652-665

[Non-Patent Document 8] Hanson CV, Nishiyama Y, Paul S., Catalytic antibodies and their applications., Curr Opin Biotechnol. (2005) 16(6), 631-636

[Non-Patent Document 9] Rathanaswami P, Roalstad S, Roskos L, Su QJ, Lackie S, Babcook J., Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8., Biochem. Biophys. Res. commun. (2005) 334(4), 1004-1013

[Non-Patent Document 10] Dall'Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA, Wu H, Kiener PA, Langermann S., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences ., J. Immunol. (2002) 169(9), 5171-5180

[Non-Patent Document 11] Yeung YA, Leabman MK, Marvin JS, Qiu J, Adams CW, Lien S, Starovasnik MA, Lowman HB., Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates., J. Immunol. (2009) 182 (12), 7663-7671

[Non-Patent Document 12] Datta-Mannan A, Witcher DR, Tang Y, Watkins J, Wroblewski VJ., Monoclonal antibody clearance. Impact of modulating the interaction of IgG with the neonatal Fc receptor., J. Biol. Chem. (2007) 282(3), 1709-1717

[Non-Patent Document 13] Clark, M., Antibody Engineering IgG Effector Mechanisms., Chemical Immunology (1997) 65, 88-110

[Non-Patent Document 14] Horton HM, Bernett MJ, Pong E, Peipp M, Karki S, Chu SY, Richards JO, Vostiar I, Joyce PF, Repp R, Desjarlais JR, Zhukovsky EA., Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia., Cancer Res. (2008) 68, 8049-8057

[Non-Patent Document 15] Zalevsky J, Leung IW, Karki S, Chu SY, Zhukovsky EA, Desjarlais JR, Carmichael DF, Lawrence CE., The impact of Fc engineering on an anti-CD19 antibody: increased Fey receptor affinity enhances B-cell clearing in nonhuman primates., Blood (2009) 113, 3735-3743

[Non-Patent Document 16] Jefferis R, Lund J., Interaction sites on human IgG-Fc for FcgammaR: current models., Immunol. Lett. (2002) 82, 57-65

[Non-Patent Document 17] Clynes, R., Yoshizumi, T., Moroi, Y., Houghton, A.N., and Ravetch, J.V., Fc Receptors are required for passive and active immunity to melanoma., Proc. Natl. Acad. sci. U. S. A. (1998) 95, 652-656

[Non-Patent Document 18] Clynes RA, Towers TL, Presta LG, Ravetch JV., Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets., Nat. Med. (2000) 6, 443-446

[Non-Patent Document 19] Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H., Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene., Blood ( 2002) 99, 754-758

[Non-Patent Document 20] Nimmerjahn F, Ravetch JV., Divergent immunoglobulin g subclass activity through selective Fc receptor binding., Science (2005) 310, 1510-1512

[Non-Patent Document 21] Greenwood J, Clark M, Waldmann H., Structural motifs involved in human IgG antibody effector functions., Eur. J. Immunol. (1993) 23, 1098-1104

[Non-Patent Document 22] Morgan A, Jones ND, Nesbitt AM, Chaplin L, Bodmer MW, Emtage JS., The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for Clq, Fc gamma RI and Fc gamma RIII binding., Immunology (1995) 86, 319-324

[Non-Patent Document 23] Lazar GA, Dang W, Karki S, Vafa 0, Peng JS, Hyun L, Chan C, Chung HS, Eivazi A, Yoder SC, Vielmetter J, Carmichael DF, Hayes RJ, Dahiyat BI., Engineered antibody Fc variants with enhanced effector function., Proc. Nat. Acad. sci. U. S. A. (2006) 103, 4005-4010

[Non-Patent Document 24] Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K., The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity., J. Biol. Chem. (2003) 278, 3466-3473

The essence of the invention

[Problems Solved by the Invention]

The present invention has been made in view of the circumstances described above. It is an object of the present invention to provide an antigen-binding molecule with enhanced intracellular uptake of bound antigen, antigen-binding molecules in which the number of antigens that can bind per molecule is increased, antigen-binding molecules with improved pharmacokinetics, antigen-binding molecules with enhanced intracellular dissociation of extracellularly bound antigen, antigen-binding molecules with enhanced extracellular release in a non-antigen bound state, antigen-binding molecules having the function of reducing total antigen concentration or free antigen plasma concentration, pharmaceutical compositions containing such antigen-binding molecules, and methods for their preparation.

[Troubleshooting Tools]

As a result of careful research to achieve the above objects, the present inventors have provided an antigen-binding molecule comprising an antigen-binding domain having human FcRn-binding activity under acidic pH range conditions, where the antigen-binding activity of the antigen-binding molecule varies with ion concentration, and binding Fcγ- a receptor domain having an Fcγ receptor binding activity under neutral pH conditions higher than that of the Fcγ receptor binding domain of the native human IgG Fc region, in which the sugar chain attached at position 297 (EU numbering) is a fucose-containing sugar chain. Moreover, the present inventors have developed a method for enhancing the intracellular uptake of bound antigens, a method for increasing the number of antigens that can bind to a single antigen-binding molecule, a method for improving the pharmacokinetics of an antigen-binding molecule, a method for accelerating the intracellular dissociation of an antigen that has extracellularly bound to an antigen-binding molecule from an antigen-binding molecule, a method for accelerating extracellular release of an antigen-binding molecule not bound to an antigen; and a method for reducing total antigen concentration and plasma free antigen concentration, wherein the methods include contacting the antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or in vitro. Moreover, the inventors have developed methods for producing antigen-binding molecules having the above-mentioned properties, and have also discovered the use of pharmaceutical compositions containing, as an active ingredient, such an antigen-binding molecule or an antigen-binding molecule obtained by the production method of the present invention, and thereby realized the present invention.

In other words, more specifically, the present invention relates to [1]-[46] below:

[1] A pharmaceutical composition that contains an antigen-binding molecule comprising an antigen-binding domain and an Fcγ receptor-binding domain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions, and wherein the antigen-binding domain has antigen-binding activity that varies depending on the conditions ion concentration, and the Fcγ receptor-binding domain has higher activity at the Fcγ receptor under conditions of the neutral pH range than the Fc region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain.

[2] The pharmaceutical composition according to [1], wherein the antigen is a soluble antigen.

[3] The pharmaceutical composition according to [1] or [2], where the ion concentration is the concentration of calcium ions.

[4] The pharmaceutical composition according to [3], wherein the antigen-binding domain is an antigen-binding domain in which the antigen-binding activity under high calcium ion conditions is higher than under low calcium ion conditions.

[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 is an antigen-binding domain in which the antigen-binding activity under neutral pH range conditions is higher than under acidic pH range conditions.

[7] The pharmaceutical composition according to any one of [1] to [6], wherein the antigen-binding molecule has a neutralizing activity against an 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 pharmaceutical composition according to [8], wherein the Fc region is an Fc region in which at least one or more amino acids are selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 324, 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region.

[10] The pharmaceutical composition according to [9], wherein the Fc region is an Fc region that contains at least one or more amino acids selected from the group consisting of:

either Lys or Tyr at amino acid position 221;

any of Phe, Trp, Glu and Tyr at amino acid position 222;

any of Phe, Trp, Glu and Lys at amino acid position 203;

any of Phe, Trp, Glu and Tyr at amino acid position 224;

any of Glu, Lys and Trp at amino acid position 225;

any of Glu, Gly, Lys and Tyr at amino acid position 227;

any of Glu, Gly, Lys and Tyr at amino acid position 228;

any of Ala, Glu, Gly and Tyr at amino acid position 230;

any of Glu, Gly, Lys, Pro and Tyr at the amino acid position

any of Glu, Gly, Lys and Tyr at amino acid position 2-32;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 236;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

any of Ala, Ile, Met and Thr at amino acid position 240;

any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

His at amino acid position 244;

Ala at amino acid position 245;

any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

any of Glu, His, Gln and Tyr at amino acid position 249; either Glu or Gln at amino acid position 250; Phe at amino acid position 251;

any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258;

any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

any of Ala, Ile, Met and Thr at amino acid position 263;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 264;

any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 265;

any of Ala, Ile, Met and Thr at amino acid position 266;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;

any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

either Phe or Not at amino acid position 273;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

either Leu or Trp at amino acid position 275;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

Ala at amino acid position 279;

any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

any of Glu, Gly, Pro and Tyr at amino acid position 286;

any of Asn, Asp, Glu and Tyr at amino acid position 288;

any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Not at amino acid position 302;

any of Asp, Gly and Tyr at amino acid position 303;

any of Asp, His, Leu, Asn and Thr at amino acid position 304;

any of Glu, Ile, Thr and Tyr at amino acid position 305;

any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

Phe at amino acid position 313; Leu at amino acid position 315;

either Glu or Gln at amino acid position 317;

any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

Not at amino acid position 32 3;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

any of Glu, Lys and Tyr at amino acid position 336;

any of Glu, His and Asn at amino acid position 337;

any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

either Ala or Val at amino acid position 376;

either Gly or Lys at amino acid position 377;

Asp at amino acid position 378;

Asn at amino acid position 379;

any of Ala, Asn and Ser at amino acid position 380;

either Ala or Ile at amino acid position 382;

Glu at amino acid position 385;

Thr at amino acid position 392;

]Leu at amino acid position 396;

Lys at amino acid position 421;

Asn at amino acid position 427;

either Phe or Leu at amino acid position 428;

Met at amino acid position 429;

Trp at amino acid position 434;

Not at amino acid position 436; and

any of Gly, His, Ile, Leu and Tyr at amino acid position 440,

in the region of the Fc region according to the EU numbering.

[11] The pharmaceutical composition according to any one of [1] to [10], wherein the Fc region of native human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain is Fc- a region of any of native human IgG1, native human IgG2, native human IgG3 and native human IgG4, wherein the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain.

[12] The pharmaceutical composition according to any one of [1]-[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]-[11], wherein the human Fcγ receptor is FcγRIIb.

[14] The pharmaceutical composition according to any one of [8]-[13], wherein the Fc region is an Fc region that contains at least one or more of

Asp at amino acid position 238 and

Glu at amino acid position 328

in the region of the Fc region according to the EU numbering.

[15] A method comprising the step of contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn-binding activity under conditions of an acidic pH range and contains an antigen-binding domain whose antigen-binding activity varies depending on concentration conditions ions, and an Fcγ receptor binding domain that has higher Fcγ receptor binding activity under neutral pH range conditions compared to the native human IgG Fc region in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain, which is a method according to any of:

(i) a method for increasing the amount of antigens that a single antigen-binding molecule can bind;

(ii) a method for eliminating plasma antigens;

(iii) a method for improving the pharmacokinetics of an antigen-binding molecule;

(iv) a method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen is extracellularly bound to the antigen-binding molecule;

(v) a method for accelerating the release of an antigen-binding molecule not associated with an antigen to the outside of the cell; and

(vi) a method of reducing the total concentration of antigen or the concentration of free antigen in plasma.

[16] The method according to [15], wherein the antigen is a soluble antigen.

[17] The method according to [15] or [16], where the ion concentration is the concentration of calcium ions.

[18] The method according to [17], wherein the antigen-binding domain is an antigen-binding domain in which the antigen-binding activity under high calcium ion conditions is higher than under low calcium ion conditions.

[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 is an antigen-binding domain in which the antigen-binding activity under neutral pH range conditions is higher than under acidic pH range conditions.

[21] The method according to any one of [15]-[20], wherein the antigen-binding molecule has neutralizing activity against the antigen.

[22] The method according to any one of [15]-[21], wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[23] The method according to [22], wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 324, 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region.

[24] The method according to [23], wherein the Fc region is an Fc region that contains at least one or more amino acids selected from the group consisting of:

either Lys or Tyr at amino acid position 221;

any of Phe, Trp, Glu and Tyr at amino acid position 222;

any of Phe, Trp, Glu and Lys at amino acid position 223;

any of Phe, Trp, Glu and Tyr at amino acid position 224;

any of Glu, Lys and Trp at amino acid position 225;

any of Glu, Gly, Lys and Tyr at amino acid position 227;

any of Glu, Gly, Lys and Tyr at amino acid position 228;

any of Ala, Glu, Gly and Tyr at amino acid position 230;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231;

any of Glu, Gly, Lys and Tyr at amino acid position 232; any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 238;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

any of Ala, Ile, Met and Thr at amino acid position 240;

any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

His at amino acid position 244;

Ala at amino acid position 245;

any of Asp, Glu, His and Tyr at amino acid position 246;

any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

any of Glu, His, Gln and Tyr at amino acid position 249;

either Glu or Gln at amino acid position 250;

Phe at amino acid position 251;

any of Phe, Met and Tyr at amino acid position 254;

any of Glu, Leu and Tyr at amino acid position 255;

any of Ala, Met and Pro at amino acid position 256;

any of Asp, Glu, His, Ser and Tyr at amino acid position 258;

any of Asp, Glu, His and Tyr at amino acid position 260;

any of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

any of Ala, Ile, Met and Thr at amino acid position 263;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264;

any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265;

any of Ala, Ile, Met and Thr at amino acid position 266;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271;

any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

either Phe or Not at amino acid position 273;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

either Leu or Trp at amino acid position 275;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

Ala at amino acid position 279;

any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

any of Glu, Gly, Pro and Tyr at amino acid position 286;

any of Asn, Asp, Glu and Tyr at amino acid position 288;

any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Not at amino acid position 302;

any of Asp, Gly and Tyr at amino acid position 303;

any of Asp, His, Leu, Asn and Thr at amino acid position 304;

any of Glu, Ile, Thr and Tyr at amino acid position 305;

any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

Phe at amino acid position 313;

Leu at amino acid position 315;

either Glu or Gln at amino acid position 317;

any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

Not at amino acid position 323;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

any of Glu, Lys and Tyr at amino acid position 336;

any of Glu, His and Asn at amino acid position 337;

any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

either Ala or Val at amino acid position 376;

either Gly or Lys at amino acid position 377;

Asp at amino acid position 378;

Asn at amino acid position 379;

any of Ala, Asn and Ser at amino acid position 380;

either Ala or Not at amino acid position 382;

Glu at amino acid position 385;

Thr at amino acid position 392;

Leu at amino acid position 396;

Lys at amino acid position 421;

Asn at amino acid position 427;

either Phe or Leu at amino acid position 428;

Met at amino acid position 429;

Trp at amino acid position 434;

Ile at amino acid position 436; and

any of Gly, His, Ile, Leu and Tyr at amino acid position 440,

in the region of the Fc region according to the EU numbering.

[25] The method according to any one of [15]-[24], wherein the Fc region of native human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain is the Fc region according to any one of native human IgG1, native human IgG2, native human IgG3 and native human IgG4, wherein the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain.

[26] The method according to any of [15]-[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]-[25], wherein the human Fcγ receptor is FcγRIIb.

[28] The method according to any one of [22]-[27], wherein the Fc region is an Fc region that contains at least one or more of

Asp at amino acid position 238 and

Glu at amino acid position 328

in the region of the Fc region according to the EU numbering.

[29] A method comprising the step of enhancing the Fcγ receptor binding activity under conditions of a neutral pH range of the Fcγ receptor binding domain in the antigen binding molecule compared to a native human IgG Fc region in which the sugar chain linked at position 297 according to EU numbered, is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions, and contains an Fcγ receptor-binding domain and an antigen-binding domain whose antigen-binding activity varies with ion concentration conditions, which is a method according to any of:

(i) a method of modifying the antigen-binding molecule, where the intracellular uptake of the antigen to which it binds is enhanced;

(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 ability of the antigen-binding molecule to eliminate plasma antigens;

(iv) a method for improving the pharmacokinetics of an antigen-binding molecule;

(v) a method of accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen is extracellularly bound to the antigen-binding molecule;

(vi) a method for accelerating the release to the outside of the cell of an antigen-binding molecule not bound to an antigen, wherein the antigen-binding molecule has been taken into the cell in an antigen-bound form; and

(vii) a method of altering the antigen-binding molecule, which may reduce the total antigen concentration or the free antigen plasma concentration.

[30] The method according to [29], wherein the antigen is a soluble antigen.

[31] The method according to [29] or [30], where the ion concentration is the concentration of calcium ions.

[32] The method of [31], wherein the antigen-binding domain is an antigen-binding domain in which the antigen-binding activity under high calcium ion conditions is higher than under low calcium ion conditions.

[33] The method according to [29] or [30], wherein the ion concentration conditions are pH conditions.

[34] The method of [33], wherein the antigen-binding domain is an antigen-binding domain in which the antigen-binding activity under neutral pH range conditions is higher than under acidic pH range conditions.

[35] The method according to any one of [29]-[34], wherein the antigen-binding molecule has neutralizing activity against the antigen.

[36] The method according to any one of [29]-[35], wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[37] The method according to [36], wherein the Fc region is an Fc region in which at least one or more amino acids are selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 , 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324 , 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differs from the amino acids in the corresponding regions in the native Fc region.

[38] The method according to [33], wherein the Fc region is an Fc region containing at least one or more amino acids selected from the group consisting of:

either Lys or Tyr at amino acid position 221;

any of Phe, Trp, Glu and Tyr at amino acid position 222;

any of Phe, Trp, Glu and Lys at amino acid position 223;

any of Phe, Trp, Glu and Tyr at amino acid position 224;

any of Glu, Lys and Trp at amino acid position 225;

any of Glu, Gly, Lys and Tyr at amino acid position 227;

any of Glu, Gly, Lys and Tyr at amino acid position 228;

any of Ala, Glu, Gly and Tyr at amino acid position 230;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231;

any of Glu, Gly, Lys and Tyr at amino acid position 232;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,

Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

any of Ala, Ile, Met and Thr at amino acid position 240;

any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

His at amino acid position 244;

Ala at amino acid position 245;

any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

any of Glu, His, Gln and Tyr at amino acid position 249;

either Glu or Gin at amino acid position 250;

Phe at amino acid position 251;

any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258;

any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

any of Ala, Ile, Met and Thr at amino acid position 263;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264;

any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265;

any of Ala, Ile, Met and Thr at amino acid position 266;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;

any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

either Phe or Not at amino acid position 273;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 274;

either Leu or Trp at amino acid position 275;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

Ala at amino acid position 279;

any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

any of Glu, Gly, Pro and Tyr at amino acid position 286;

any of Asn, Asp, Glu and Tyr at amino acid position 288;

any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Not at amino acid position 302;

any of Asp, Gly and Tyr at amino acid position 303;

any of Asp, His, Leu, Asn and Thr at amino acid position 304;

any of Glu, Ile, Thr and Tyr at amino acid position 305;

any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

Phe at amino acid position 313;

Leu at amino acid position 315;

either Glu or Gln at amino acid position 317;

any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

Ile at amino acid position 32 3;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329; any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,

Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 330;

any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

any of Glu, Lys and Tyr at amino acid position 336;

any of Glu, His and Asn at amino acid position 337;

any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

either Ala or Val at amino acid position 376;

either Gly or Lys at amino acid position 377;

Asp at amino acid position 378;

Asn at amino acid position 379;

any of Ala, Asn and Ser at amino acid position 380;

either Ala or Ile at amino acid position 382;

Glu at amino acid position 385;

Thr at amino acid position 392;

Leu at amino acid position 396;

Lys at amino acid position 421;

Asn at amino acid position 427;

either Phe or Leu at amino acid position 428;

Met at amino acid position 429;

Trp at amino acid position 434;

Ile at amino acid position 436; and

any of Gly, His, Ile, Leu and Tyr at amino acid position 440,

in the region of the Fc region according to the EU numbering.

[39] The method according to any one of [29]-[38], wherein the Fc region of native human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain is the Fc region according to any one of native human IgGl, native human IgG2, native human IgG3 and native human IgG4, wherein the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain.

[40] The method according to any one of [29]-[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]-[39], wherein the human Fcγ receptor is FcγRIIb.

[42] The method according to any one of [36]-[41], wherein the Fc region is an Fc region that contains at least one or more of:

Asp at amino acid position 238 and

Glu at amino acid position 328

in the region of the Fc region according to the EU numbering.

[43] A method for producing an antigen-binding molecule, which comprises the steps of:

(a) determining the antigen-binding activity of the antigen-binding domain under conditions of high concentration of calcium ions;

(b) determining the antigen-binding activity of the antigen-binding domain under low calcium ion conditions;

(c) selecting an antigen-binding domain for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen-binding domain selected according to (c) with a polynucleotide encoding an Fcγ receptor binding domain having human FcRn binding activity under acidic pH range conditions and in which Fcγ receptor binding activity under neutral pH range conditions. the pH is higher than that of the Fc region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from the culture according to (e).

[44] A method for producing an antigen-binding molecule, which includes the steps of:

(a) determining the antigen-binding activity of the antibody under conditions of high concentration of calcium ions;

(b) determining the antigen-binding activity of the antibody under low calcium ion conditions;

(c) selecting an antibody for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen-binding domain of an antibody selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having a human FcRn binding activity in an acidic pH range and in which an Fcγ receptor binding activity under neutral range conditions The pH is higher than the Fc region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and (f) collecting antigen-binding molecules from the cell culture according to (e).

[45] A method for producing an antigen-binding molecule, which includes the steps of:

(a) determining the antigen-binding activity of the antigen-binding domain under neutral pH conditions;

(b) determining the antigen-binding activity of the antigen-binding domain under acidic pH conditions;

(c) selecting an antigen-binding domain for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen-binding domain selected according to (c) with a polynucleotide encoding an Fcγ receptor binding domain having human FcRn binding activity under acidic pH range conditions and in which Fcγ receptor binding activity under neutral pH range conditions. the pH is higher than the Fc region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from the cell culture according to (e).

[46] A method for producing an antigen-binding molecule, which includes the steps of:

(a) determining the antigen-binding activity of the antibody under neutral pH conditions;

(b) determining the antigen-binding activity of the antibody under acidic pH conditions;

(c) selecting an antibody for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antibody antigen-binding domain selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having human FcRn binding activity under conditions of an acidic pH range, wherein the Fcγ receptor binding activity under conditions of the neutral pH is higher than the Fc region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from the cell culture according to (e).

[47] The production method according to any one of [43] to [46], wherein the antigen is a soluble antigen.

[48] The production method according to any one of [43]-[47], wherein the Fcγ receptor binding domain comprises an Fc region of an antibody.

[49] The production method according to [48], wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227 , 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258 , 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286 , 288 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 , 324, 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region.

[50] The production method according to [49], wherein the Fc region contains at least one or more amino acids selected from the group consisting of:

either Lys or Tyr at amino acid position 221;

any of Phe, Trp, Glu and Tyr at amino acid position 222;

any of Phe, Trp, Glu and Lys at amino acid position 223;

any of Phe, Trp, Glu and Tyr at amino acid position 224;

any of Glu, Lys and Trp at amino acid position 225;

any of Glu, Gly, Lys and Tyr at amino acid position 227;

any of Glu, Gly, Lys and Tyr at amino acid position 228;

any of Ala, Glu, Gly and Tyr at amino acid position 230;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231;

any of Glu, Gly, Lys and Tyr at amino acid position 232;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

any of Ala, Ile, Met and Thr at amino acid position 240;

any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

His at amino acid position 244;

Ala at amino acid position 245;

any of Asp, Glu, His and Tyr at amino acid position 246;

any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

any of Glu, His, Gln and Tyr at amino acid position 249;

either Glu or Gln at amino acid position 250;

Phe at amino acid position 251;

any of Phe, Met and Tyr at amino acid position 254;

any of Glu, Leu and Tyr at amino acid position 255;

any of Ala, Met and Pro at amino acid position 256;

any of Asp, Glu, His, Ser and Tyr at amino acid position 258;

any of Asp, Glu, His and Tyr at amino acid position 260;

any of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

any of Ala, Ile, Met and Thr at amino acid position 263;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264;

any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265;

any of Ala, Ile, Met and Thr at amino acid position 266;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271;

any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

either Phe or Ile at amino acid position 273;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

either Leu or Trp at amino acid position 275;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

Ala at amino acid position 279;

any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

any of Asp, Lys, Pro and Tyr at amino acid position 281;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

any of Glu, Gly, Pro and Tyr at amino acid position 286;

any of Asn, Asp, Glu and Tyr at amino acid position 288;

any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300;

any of Asp, Glu, His and Tyr at amino acid position 301;

Ile at amino acid position 302;

any of Asp, Gly and Tyr at amino acid position 303;

any of Asp, His, Leu, Asn and Thr at amino acid position 304;

any of Glu, Ile, Thr and Tyr at amino acid position 305;

any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

Phe at amino acid position 313;

Leu at amino acid position 315;

either Glu or Gln at amino acid position 317;

any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

Ile at amino acid position 323;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

any of Glu, Lys and Tyr at amino acid position 336;

any of Glu, His and Asn at amino acid position 337;

any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

either Ala or Val at amino acid position 376;

either Gly or Lys at amino acid position 377;

Asp at amino acid position 378;

Asn at amino acid position 379;

any of Ala, Asn and Ser at amino acid position 380;

either Ala or Ile at amino acid position 382;

Glu at amino acid position 385;

Thr at amino acid position 392;

Leu at amino acid position 396;

Lys at amino acid position 421;

Asn at amino acid position 427;

either Phe or Leu at amino acid position 428;

Met at amino acid position 429;

Trp at amino acid position 434;

Ile at amino acid position 436; and

any of Gly, His, Ile, Leu and Tyr at amino acid position 440,

in the region of the Fc region according to the EU numbering.

[51] The production method according to any one of [43]-[50], wherein the Fcγ receptor binding domain is an Fc region of any of native human IgG1, native human IgG2, native human IgG3, and native human IgG4, wherein the sugar chain, linked at position 297 according to EU numbering is a fucose-containing sugar chain.

[52] The production method according to any of [43]-[51], wherein the human Fcγ receptor is FcγRIa, FcγRIIa (R), FcγRIIa (H), FcγRIIb, FcγRIIIa (V), or FcγRIIIa (F).

[53] The production method according to any one of [43]-[51], wherein the human Fcγ receptor is FcγRIIb.

[54] The production method according to any one of [48]-[53], wherein the Fc region contains at least one or more amino acids from:

Asp at amino acid position 238 and

Glu at amino acid position 328

in the region of the Fc region according to the EU numbering.

Brief description of the drawings

In FIG. 1 shows an unrestricted mechanism of action for eliminating soluble antigen from plasma by administering an antibody that binds to the antigen in an ion concentration dependent manner and whose binding to the Fcγ receptor is enhanced at neutral pH compared to existing neutralizing antibodies.

In FIG. 2 shows the concentration of human IL-6 receptor over time in plasma of human FcRn transgenic mice injected with Fv4-IgG1, which binds to the human IL-6 receptor in a pH dependent manner, or H54/L28-IgG1.

In FIG. 3 shows the plasma concentration of human IL-6 receptor in human FcRn transgenic mice injected with Fv4-IgG1, which binds to the human IL-6 receptor in a pH dependent manner, Fv4-IgG1-F760, which is a Fv4-IgG1 variant that lacks mouse FcγR binding, Fv4-IgG1-F1022, which is a Fv4-IgG1 variant with enhanced mouse FcγR binding, or Fv4-IgG1-Fuc, which is a low-fucose Fv4-IgG1 antibody.

In FIG. 4 shows the plasma concentration of human IL-6 receptor in transgenic mice with human FcRn injected with Fv4-IgG1 or antigen-binding molecules containing Fv4-IgGl-F1022 or Fv4-IgG1-F1093 as a heavy chain, which is a variant of Fv4-IgG1- F1022 with increased FcRn binding in the acidic pH range.

In FIG. 5 shows the plasma concentration over time of injected antigen-binding molecules in transgenic mice with human FcRn injected with Fv4-IgG1 or antigen-binding molecules containing Fv4-IgG1-F1022 or Fv4-IgG1-F1093 as a heavy chain, which is a variant of Fv4- IgG1-F1022 with increased FcRn binding in the acidic pH range.

In FIG. 6 shows the plasma concentration of human IL-6 receptor in transgenic mice with human FcRn injected with Fv4-IgG1, Fv4-IgG1-F1087, which is an Fv4-IgG1 variant with increased mouse FcγR binding (specifically, increased mouse FcγRIIb binding and FcγRIII binding). mice) and Fv4-IgG1-F1182, which is an Fv4-IgG1 variant with enhanced FcγR binding (specifically, enhanced FcγRI binding and FcγRIV binding).

In FIG. 7 shows the plasma concentration over time of injected antigen-binding molecules in human FcRn transgenic mice injected with Fv4-IgG1, Fv4-IgG1-F1087 and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are Fv4-IgG1-F1087 variants. with increased FcRn binding in the acidic pH range.

In FIG. 8 shows the plasma concentration over time of administered antigen-binding molecules in transgenic mice with human FcRn injected with Fv4-IgG1, Fv4-IgG1-F1182 and Fv4-IgG1-F1181, which is a variant of Fv4-IgG1-F1182 with increased FcRn binding in the acid range. pH values.

In FIG. 9 shows the plasma concentration of human IL-6 receptor in transgenic mice with human FcRn injected with Fv4-IgG1, Fv4-IgG1-F1087 and Fv4-IgG1-F1180 and Fv4-IgG1-F1412, which are variants of Fv4-IgG1-F1087 with increased FcRn binding in the acidic pH range.

In FIG. 10 shows the plasma concentration of human IL-6 receptor in transgenic mice with human FcRn injected with Fv4-IgG1, Fv4-IgG1-F1182 and Fv4-IgG1-F1181, which is a variant of Fv4-IgG1-F1182 with increased FcRn binding, in the range of acidic pH values.

In FIG. 11 shows the results of the change in plasma concentration of Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 in a human FcRn transgenic mouse when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 was administered to mice.

In FIG. 12 shows the results of the change in plasma concentration of soluble human IL-6 receptor in a human FcRn transgenic mouse when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 was administered to mice.

In FIG. 13 shows the plasma concentration of human IL-6 receptor in normal mice injected with Fv4-mIgG1, Fv4-mIgG1-mF44, which is a Fv4-mIgG1 variant with enhanced mouse FcγRIIb binding and mouse FcγRIII binding, and Fv4-mIgG1-mF46, which is Fv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouse FcγRIII binding.

In FIG. 14 shows the plasma concentration of human IL-6 receptor in FcγRIII deficient mice injected with Fv4-mIgG1, Fv4-mIgG1-mF44, which is an Fv4-mIgG1 variant with enhanced mouse FcγRIIb binding and mouse FcγRIII binding, and Fv4-mIgG1-mF46, which is an Fv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouse FcγRIII binding.

In FIG. 15 shows the plasma concentration of human IL-6 receptor in Fc receptor γ-chain deficient mice injected with Fv4-mIgG1, Fv4-mIgG1-mF44, which is a Fv4-mIgG1 variant with enhanced mouse FcγRIIb binding and mouse FcγRIII binding, and Fv4 -mIgG1-mF46, which is a Fv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouse FcγRIII binding.

In FIG. 16 shows the plasma concentration of human IL-6 receptor in FcγRIIb deficient mice injected with Fv4-mIgG1, Fv4-mIgG1-mF44, which is an Fv4-mIgG1 variant with enhanced mouse FcγRIIb binding and mouse FcγRIII binding, and Fv4-mIgG1-mF46, which is an Fv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouse FcγRIII binding.

In FIG. 17 shows the result of evaluation of the platelet aggregation ability of the omalizumab-G1d-v3/IgE immunocomplex by a platelet aggregation assay using platelets derived from donors with the FcγRIIa (R/H) allotype.

In FIG. 18 shows the result of evaluation of the platelet aggregation ability of the omalizumab-G1d-v3/IgE immunocomplex by platelet aggregation assay using platelets derived from donors with the FcγRIIa (H/H) allotype.

In FIG. 19 shows the result of evaluation of CD62p expression on the membrane surface of washed platelets. The black shaded area on the graph indicates the result of stimulation with ADP after reaction with PBS. The area that is not filled in the graph indicates the result of stimulation with ADP after reaction with the immunocomplex.

In FIG. 20 shows the result of evaluating the expression of active integrin on the platelet membrane surface. The black shaded area on the graph indicates the result of stimulation with ADP after reaction with PBS. The area that is not filled in the graph indicates the result of stimulation with ADP after reaction with the immunocomplex.

In FIG. 21 shows the results of evaluation of platelet aggregation activity induced by omalizumab-BP230/IgE immunocomplex and omalizumab-G1d-v3/IgE immunocomplex in a platelet aggregation assay using platelets derived from a donor with FcγRIIa (R/H) polymorphism.

In FIG. 22 shows the results of evaluation of CD62p expression on the membrane surface of washed platelets. The gray shaded graph indicates the result when ADP stimulation was performed after the reaction with PBS, the solid line and the dotted line indicate the results when ADP stimulation was performed after the reaction with omalizumab-G1d-v3/IgE and omalizumab-BP230/ immunocomplex IgE, respectively.

In FIG. 23 shows the results of evaluation of activation of integrin expression on the membrane surface of washed platelets. The gray shaded graph indicates the result when ADP stimulation was performed after the reaction with PBS, the solid line and the dotted line indicate the results when ADP stimulation was performed after the reaction with omalizumab-G1d-v3/IgE and omalizumab-BP230/ immunocomplex IgE, respectively.

In FIG. 24 is a graph showing the relative magnitude of FcγRIIb binding activity for each PD variant on the horizontal axis and the relative magnitude of type R FcγRIIa binding activity for each PD variant on the vertical axis. The value of binding of each PD variant to each FcγR was divided by the value of binding of IL6R-F652/IL6R-L, which is the control antibody before modification (IL6R-F652, corresponding to SEQ ID NO: 142, is the heavy chain of the antibody containing the modified Fc with Pro at position 238 (EU numbering) replaced by Asp), with each FcγR; and then the resulting value was multiplied by 100 and used as the relative value of the binding activity for each PD variant with each FcγR. Graph F652 in the figure shows the value for IL6R-F652/IL6R-L.

In FIG. 25 is a graph showing on the vertical axis the relative amount of FcγRIIb binding activity for variants obtained by making each change in GpH7-B3 (SEQ ID NO: 159)/GpL16-k0 (SEQ ID NO: 160) which has no change P238D and the horizontal axis shows the relative amount of FcγRIIb binding activity for the variants obtained by making each change in IL6R-F652 (SEQ ID NO: 142)/IL6R-L having the P238D change. The FcγRIIb binding value of each variant was divided by the FcγRIIb binding value of the pre-modified antibody; and then the obtained value was multiplied by 100 and used as the value of the relative binding activity. In this case, region A contains changes that exhibit the effect of enhancing FcγRIIb binding in both cases when a change is made to GpH7-B3/GpL16-k0 that does not have P238D, and when a change is made to IL6R-F652/IL6R-L that has P238D. Region B contains changes that show the effect of enhancing FcγRIIb binding when introduced into GpH7-B3/GpL16-k0, which does not have P238D, but does not show the effect of enhancing FcγRIIb binding when introduced into IL6R-F652/IL6R-L, which has P238D.

In FIG. 26 shows the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 27 is an overlay of the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, with respect to the FcγRIIb extracellular region and the Fc CH2 domain A by least squares fitting based on the distances between atom pairs Cα.

In FIG. 28 shows a comparison of the detailed structure in the P238D region after superposition of the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, in relation to Fc CH2 domain A only or Fc CH2 domain B only by approximations by the least squares method based on the distances between pairs of Сα atoms.

In FIG. 29 shows that a hydrogen bond can be found between the Gly backbone at position 237 (listed according to EU numbering) in Fc's A CH2 domain, and Tyr at position 160 in FcγRIIb in the crystal structure of the Fc (P238D)/FcγRIIb extracellular region complex.

In FIG. 30 shows that an electrostatic interaction can be detected between Asp at position 270 (listed according to EU numbering) in the CH2 domain B of Fc, and Arg at position 131 of FcγRIIb in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 31 is a graph showing the relative magnitude of FcγRIIb binding activity for each variant 2B on the horizontal axis and the relative magnitude of type R FcγRIIa binding activity for each variant 2B on the vertical axis. The binding value of each variant 2B to each FcγR was divided by the binding value of the control antibody before change (changed Fc with Pro at position 238 (referenced by EU numbering) replaced by Asp) for each FcγR; and then the resulting value was multiplied by 100, and used as the value of the relative binding activity of each option 2B against each FcγR.

In FIG. 32 shows Glu at position 233 (indicated according to EU numbering) in the Fc chain A and surrounding residues in the FcγRIIb extracellular region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 33 shows Ala at position 330 (indicated according to EU numbering) in the Fc chain A and surrounding residues in the FcγRIIb extracellular region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 34 shows the structures of Pro at position 271 (indicated according to EU numbering) of the B chain of Fc after superposition of the crystal structures of the Fc(P238D) complex/FcγRIIb extracellular region and the Fc(WT) complex/FcγRIIIa extracellular region by least-squares fitting based on distances between atom pairs Cα, in relation to the B-chain Fc.

In FIG. 35 is an image of the Fc(P208)/FcγRIIb extracellular region determined by X-ray diffraction analysis. For each of the CH2 and CH3 domains in the Fc region, the domains on the left are labeled the A domain and the domains on the right are labeled the B domain.

In FIG. 36 shows a comparison after superposition of the structures of the Fc complex (P208)/FcγRIIb extracellular region and the Fc complex (WT)/FcγRIIa extracellular region (PDB code: 3RY6) determined by X-ray diffraction analysis, in relation to the CH2 domain A of the Fc region by method approximation least squares based on distances between pairs of Сα atoms. In this diagram, the structure depicted with a thick line shows the Fc (P208) complex/FcγRIIb extracellular region, while the structure depicted with a thin line indicates the Fc (WT) structure/FcγRIIa extracellular region. Only the CH2 domain A of the Fc region is depicted for the Fc(WT)/extracellular FcγRIIa complex.

In FIG. 37 shows the X-ray crystal structure of the Fc (P208)/FcγRIIb extracellular region, detailed structure at the Asp region at position 237 (EU numbering) in the CH2 domain A of the Fc region, which forms a hydrogen bond with Tyr at position 160 in FcγRIIb in the core structure. chains.

In FIG. 38 shows the X-ray crystal structure of the Fc (P208) complex/FcγRIIb extracellular region, the structure of the amino acid residues in the Asp region at position 237 (EU numbering) in the CH2 domain A of the Fc region, which forms a hydrogen bond with Tyr at position 160 in FcγRIIb in the structure main chain.

In FIG. 39 shows a comparison in the loop region at positions 266-271 (EU numbering) after superposition of X-ray crystal structures of the Fc (P238D)/FcγRIIb extracellular domain presented in Example 10 and the Fc (P208)/FcγRIIb extracellular domain in relation to the CH2 domain. In the Fc region, by least squares approximation based on distances between pairs of Cα atoms. Compared to Fc (P238D), Fc (P208) has an H268D change at position 268 (EU numbering) and a P271G change at position 271 (EU numbering) in the loop.

In FIG. 40 is a diagram showing the structure in the Ser239 region in the CH2 domain B of the Fc region in the X-ray crystal structure of the Fc(P208)/FcγRIIb extracellular region, along with electron density determined by X-ray diffraction analysis using the 2Fo-Fc coefficient.

In FIG. 41 shows a comparison after superposition of the 3D structures of the Fc (P208)/FcγRIIaR extracellular region and Fc (P208)/FcγRIIb extracellular region determined by X-ray diffraction analysis by least squares fit based on Cα atom pair distances.

In FIG. 42 shows a comparison in the Asp region at position 237 (EU numbering) in the CH2 domain A of the Fc region between the X-ray crystal structures of the Fc complex (P208)/FcγRIIaR extracellular region and the Fc complex (P208)/FcγRIIb extracellular region, together with electron density, determined by X-ray diffraction analysis using the coefficient 2Fo-Fc.

In FIG. 43 shows a comparison in the Asp region at position 237 (EU numbering) in the CH2 domain of the Fc region between the X-ray crystal structures of the Fc complex (P208)/FcγRIIaR extracellular region and the Fc complex (P208)/FcγRIIb extracellular region, together with electron density, determined by X-ray diffraction analysis using the coefficient 2Fo-Fc.

In FIG. 44 shows a comparison between G1d and G4d constant region sequences. In the diagram, amino acids in bold boxes indicate positions with different amino acid residues between G1d and G4d.

In FIG. 45 shows the change in plasma concentrations of GA2-IgG1 and GA2-F1087 antibodies in normal mice.

In FIG. 46 shows the change in plasma concentration of hIgA in normal mice injected with GA2-IgG1 and GA2-F1087.

In FIG. 47 shows the change in plasma concentrations of 278-IgG1 and 278-F1087 antibodies in C57BL/6J mice.

In FIG. 48 shows the change in plasma concentration of hIgE (Asp6) in C57BL/6J mice treated with 278-IgG1 and 278-F1087.

In FIG. 49 shows the heavy chain CDR3 structure of the 6RL#9 Fab fragment determined by X-ray diffraction analysis. (i) shows the crystal structure of heavy chain CDR3 obtained under crystallization conditions in the presence of calcium ions. (ii) shows the crystal structure of heavy chain CDR3 obtained under crystallization conditions in the absence of calcium ions.

In FIG. 50 shows the plasma concentration over time of each antibody in normal mice treated with H54/L28-IgG1, FH4-IgG1, or 6RL#9-IgG1 antibody.

In FIG. 51 shows plasma concentration over time of soluble human IL-6 receptor (hsIL-6R) in normal mice treated with H54/L28-IgG1, FH4-IgG1, or 6RL#9-IgG1 antibody.

In FIG. 52 shows ion exchange chromatograms for an antibody having the human Vk5-2 sequence and an antibody having the hVk5-2_L65 sequence that has an altered glycosylation sequence in the human Vk5-2 sequence. The solid line shows the chromatogram for an antibody having the sequence of human Vk5-2 (heavy chain: CIM_H (SEQ ID NO: 67); light chain: hVk5-2 (SEQ ID NO: 4)); the dotted line shows the chromatogram for an antibody having the sequence hVk5-2_L65 (heavy chain: CIM_H (SEQ ID NO: 67); light chain: hVk5-2_L65 (SEQ ID NO: 70)).

In FIG. 53A shows ion exchange chromatograms for an antibody having the sequence LfVk1_Ca (heavy chain: GC_H (SEQ ID NO: 51); light chain: LfVk1_Ca (SEQ ID NO: 83)) and an antibody having a sequence in which Asp(D) in the sequence LfVk1_Ca changed to Ala, (A) after storage at 5°C (solid line) or 50°C (dashed line). After storage at 5°C, the highest peak in the chromatogram for each antibody is defined as the main peak and the axis of each ion exchange chromatography was normalized to the main peak. The graph shows a chromatogram for an antibody having LfVk1_Ca (SEQ ID NO: 83) as a light chain.

In FIG. 53B is a chromatogram for an antibody having LfVk1_Cal (SEQ ID NO: 85) as the light chain.

In FIG. 53C is a chromatogram for an antibody having LfVk1_Ca2 (SEQ ID NO: 86) as a light chain.

In FIG. 53D is a chromatogram for an antibody having LfVk1_Ca3 (SEQ ID NO: 87) as a light chain.

In FIG. 54A shows ion exchange chromatograms for an antibody having the sequence LfVk1_Ca (heavy chain: GC_H (SEQ ID NO: 51); light chain: LfVk1_Ca (SEQ ID NO: 83)) and an antibody having the sequence LfVk1_Ca6 (heavy chain: GC_H (SEQ ID NO: 83)) : 51); light chain: LfVk1_Ca6 (SEQ ID NO: 88)), in which Asp (D) at position 30 (Kabat numbering) in the LfVk1_Ca sequence is replaced by Ser (S), after storage at 5°C (solid line) or 50°C (dotted line). After storage at 5° C., the highest peak in the chromatogram for each antibody is defined as the main peak and the axis of each ion exchange chromatogram is normalized to the main peak. The graph shows a chromatogram for an antibody having LfVk1_Ca (SEQ ID NO: 83) as a light chain.

In FIG. 54B is a chromatogram for an antibody having LfVk1_Ca6 (SEQ ID NO: 88) as the light chain.

In FIG. 55 shows the relationship between the model amino acid distribution (referred to as "Model") and the amino acid distribution for sequence information of 290 clones isolated from E. coli that were introduced with an antibody gene library that binds to antigen in a Ca-dependent manner (referred to as "Library"). The position of amino acids is indicated on the horizontal axis (Kabat numbering). The vertical axis indicates the percentage distribution of amino acids.

In FIG. 56 shows sensorgrams for anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 6RC1IgG_019 antibody under conditions of high calcium ion concentration (1.2 mM). The horizontal axis represents time and the vertical axis represents RU.

In FIG. 57 shows sensorgrams for anti-IL-6R antibody (tocilizumab), 6RC1IgG_010 antibody, 6RC1IgG_012 antibody, and 6RC1IgG_019 antibody under conditions of low calcium ion concentration (3 μM). The horizontal axis shows the time and the vertical axis represents the value of RU.

In FIG. 58 shows the relationship between the model amino acid distribution (referred to as "Model") and the amino acid distribution for sequence information of 132 clones isolated from E. coli that were introduced with a library of antibody genes that bind to antigens in a pH-dependent manner (referred to as " Library"). The horizontal axis represents the position of the amino acids (Kabat numbering). The vertical axis represents the percentage distribution of amino acids.

In FIG. 59 shows sensorgrams for anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 7.4. The horizontal axis represents time and the vertical axis represents RU.

In FIG. 60 shows sensorgrams for anti-IL-6R antibody (tocilizumab), 6RpH#01 antibody, 6RpH#02 antibody, and 6RpH#03 antibody at pH 6.0. The horizontal axis shows the time and the vertical axis represents the value of RU.

In FIG. 61A is a plot of ECL responses to native Fc and altered Fc from sera obtained from 15-30 independent rheumatic patients. Plotted ECL responses to native Fc (FIG. 61A), Fv4-YTE (FIG. 61B), Fv4-F1166 (=YTE+Q438R/S440E) (FIG. 61C), Fv4-F1167 (=YTE+S424N) ( Fig. 61D), Fv4-LS (Fig. 61E), Fv4-F1170 (=LS+Q438R/S440E) (Fig. 61F), Fv4-F1171 (=LS+S424N) (Fig. 61G, Fv4-N434H (Fig. 61H), Fv4-F1172 (=N434H+Q438R/S440E) (Fig. 61I), Fv4-F1173 (=N434H+S424N) (Fig. 61J), respectively.

Fig. 61B is a continuation of FIG. 61A.

Fig. 61C is a continuation of FIG. 61B.

Fig. 61D is a continuation of FIG. 61C.

Fig. 61E is a continuation of FIG. 61D.

Fig. 61E is a continuation of FIG. 61E.

Fig. 61G is a continuation of FIG. 61F.

Fig. 61H is a continuation of FIG. 61G.

Fig. 61I is a continuation of FIG. 61H.

Fig. 61J is a continuation of FIG. 61I.

In FIG. 62A is a graph of ECL responses to altered Fc from sera obtained from 30 independent rheumatic patients. Plotted ECL responses to Fv4-LS (FIG. 62A), Fv4-F1380 (FIG. 62B), Fv4-F1384 (FIG. 62C), Fv4-F1385 (FIG. 62D), Fv4-F1386 (FIG. 62E) are shown. , Fv4-F1388 (FIG. 62F) and Fv4-F1389 (FIG. 62G), respectively.

Fig. 62B is a continuation of FIG. 62A.

Fig. 62C is a continuation of FIG. 62V.

Fig. 62D is a continuation of FIG. 62C.

Fig. 62E is a continuation of FIG. 62d.

Fig. 62F is a continuation of FIG. 62E.

Fig. 62G is a continuation of FIG. 62F.

[Methods for carrying out the invention]

The following are definitions and a detailed description to facilitate understanding of the present invention illustrated in the present description.

Amino acids

Within the scope of the present invention, amino acids are described using one or three letter codes or both, e.g. 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 or Val/V.

Amino acid change

For amino acid changes in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlapping PCR. Insertions, deletions and/or substitutions of amino acids are appropriately made using these known methods. Substitution of amino acid residues means the replacement of an amino acid residue with another amino acid residue for the purpose of changing aspects such as the following:

(a) the structure of the main chain of the polypeptide in the region of the helical structure or the region of the layer structure;

(b) the charge or hydrophobicity of a given site; or

(c) side chain length.

Amino acid residues are divided into the following groups based on the properties of the side chains included in their structures:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu and Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn and Gln;

(3) acidic: Asp and Glu;

(4) main: His, Lys and Arg;

(5) residues that affect chain orientation: Gly and Pro; and

(6) aromatic: Trp, Tyr and Phe.

A substitution between amino acid residues in each of these groups is called a conservative substitution. On the other hand, a substitution between amino acid residues from different groups of amino acids is called a non-conservative substitution. Substitutions within the scope of the present invention may be conservative substitutions or non-conservative substitutions, or a combination of conservative and non-conservative substitutions. Moreover, as methods for changing amino acids to non-native amino acids, many known methods can be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003 ) 100(11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA that has a non-native amino acid linked to a complementary amber suppressor tRNA of a UAG codon (an amber codon), which is one of the stop codons, can be used.

Moreover, as a designation for an amino acid change, a designation using one-letter amino acid codes for the amino acid before ligation and the amino acid after the change, before and after the number indicating the specific position, respectively, can be used. For example, the change P238D, which is used when changing the amino acid of the Fc region included in the constant region of the antibody, denotes the replacement of Pro at position 238 (according to EU numbering) with Asp. In other words, the number indicates the position of the amino acid according to EU numbering, the one-letter amino acid code before the number indicates the amino acid before the change, and the one-letter amino acid code after the number indicates the amino acid after the change.

And/or

As used herein, the term "and/or" means the combination of the terms before and after the expression "and/or" and includes each combination where "and" and "or" are suitably combined. In particular, for example, the expression "amino acids at positions 326, 328 and/or 428 are substituted" includes the following variants of amino acid changes:

amino acid(s) at (a) position 326, (b) position 328, (c) position 428, (d) positions 326 and 328, (e) positions 326 and 428, (f) positions 328 and 428 and (g) regulations 326, 328 and 428.

Antigens

As used herein, the structure of an "antigen" is not particularly limited to a particular structure, provided that it includes an epitope that is bound by an antigen-binding domain. In another sense, an antigen may be an inorganic material or an organic material, and preferably it is a soluble antigen that is present in a body fluid and which, in one embodiment, can be bound by an antigen-binding molecule of the present invention. Examples of antigens are the following molecules:

17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, active, A active, AB active, B active , activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF , ALCAM, ALK, ALK-1, ALK-7, alpha-1 antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, BAX, BCA-1, BACAM, 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-6 Vgr-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 neurotrophic factor , BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, cancer-associated carcinoembryonic antigen (CEA), cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17 , CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2 , CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18 , CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54 , CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, toxin Botulinum, Clostridium perfringens toxin, CKb8-l, CLC, CMV, 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-associated tumor antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (decay accelerating factor), des-(1-3)-IGF-I (IGF-1 brain), 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/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activating factor (FAP), Fas, FcRI, FEN-1, ferritin, FGF , FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZ D6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1 ), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp 130, gp72, GRO, releasing factor growth hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, gB HCMV envelope glycoprotein, HCMV dH envelope glycoprotein, UL HCMV, hematopoietic growth factor (HGF), HepB gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, loop V3 gp 120 IIIB HIV, HLA, HLA-DR, HM1,24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, I GF, 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, Alpha-2 integrin, Alpha-3 integrin, Alpha-4 integrin, Alpha-4 integrin /beta1, integrin alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin beta2, interferon-gamma, IP-10, I-TAC, JE, kallikrein 2 , kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KS, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bpl, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT , lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface protein, luteinizing hormone, lymphotoxin-beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MSC-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, Adherin N-C, NCA90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4 or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR , NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGGI, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin , prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, P tk, PTN, R51, RANK, RANKL, RANTES, RANTES, Relaxin A chain, Relaxin B chain, Renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100 , SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (eg, T cell receptor alpha/beta), TdT, TESC, TEM1, TEM5, TEM7, TEM8, TERT, PLAP-like testis alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, pan-specific TGF-beta, 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, thymus 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 R5, 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 CD 120a, p55-60), TNFRSF1B (TNF RII CD 120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (0X40 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 Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (Apo-2 TRAIL ligand, TL2), TNFSF11 (ODF TRANCE/RANK ligand, OPG ligand), TNFSF12 (Apo-3 TWEAK ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (IVEM LIGHT I ligand, LTg), TNFSF15 (TL1A/VEGI ), TNFSF18 (AITR ligand of GITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (gp34 ligand OX40, TXGP1) , TNFSF5 (CD154 CD40 ligand, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Apo-1 Fas ligand, APT1 ligand), TNFSF7 (CD70 CD27 ligand), TNFSF8 (CD153 CD30 ligand), TNFSF9 (CD137 ligand 4 -1BB), TP-1, t-PA, Thro, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor associated CA125, associated tumor antigen expressing Lewis-Y-associated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt -4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6 , WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, αβ, CD81, CD 97, 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 , high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, Clr, Cls, 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 Xlla, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3 , syndecan-4, LPA, SIP; acetylcholine receptor, AdipoRl, AdipoR2, ADP ribosylcyclase-1, alpha-4/beta-7 integrin, alpha-5/beta-1 integrin, alpha-v/beta-6 integrin, alpha-v/beta-1 integrin, angiopoietin ligand -2, Angptl2, Anthrax, cadherin, 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 ligand -1, dipeptidyl peptidase IV, EPOR, RSV F protein, 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, tyrosine kinase Kit, leucine-bloated alpha-2-glycoprotein 1 (LRG1), lysosphingolipid receptor, OX2 membrane glycoprotein, mesothelin, MET, MICA, MUC-16 associated with myelin glycoprotein, neuropilin-1, neuropilin-2, Nogo receptor, PLXNA1, PLXNA2, PLXNA3, PLXNA4A, PLXNA4B, PLXNB1, PLXNB2, PLXNB3, PLXNC1, PLXND1, programmed cell death ligand 1, PC9 proprotein convertase, 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, Shiga-like toxin II, sphingosine-1-phosphate receptor 1, ST2, staphylococcal lipoteichoic acid, tenascin, TG2, thymic stromal lymphoprotein receptor, receptor 12A of the TNF superfamily, NMB transmembrane glycoprotein, TREM-1, TREM-2, trophoblastic glycoprotein, TSH receptor, TTR, tubulin, and ULBP2; and receptors for growth factors and hormones, molecules that exist in their soluble form and are not anchored to cells in body fluids. For example, among receptors, soluble antigens present in body fluid due to some mechanism including protease-mediated cleavage of receptors and the like expressed on the cell surface are also suitable examples of soluble antigens of the present invention. Examples of such molecules may include the soluble IL-6R molecule (J. Immunol. (1994) 152, 4958-4968) and CD20, as well as CD52 (Br. J. Haematol. (2003) 123 (5), 850-857), described in the present description. Moreover, also examples of the soluble antigens of the present invention are not only molecules naturally expressed in a living body, but also soluble antigens existing in the body fluid, which are infectious molecules such as or antigens presented by infectious organisms such as viruses. , or presented on such organisms. Suitable examples of body fluids include blood, plasma, serum, urine, lymph, saliva, and lacrimal fluid.

epitope

"Epitope" means an antigenic determinant in an antigen and refers to the antigenic site to which the antigen-binding domain of the antigen-binding molecule described herein binds. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be indicated by the amino acid residues that form the epitope. Alternatively, when the epitope is a sugar chain, the epitope may be indicated by its defined sugar chain structure.

A linear epitope is an epitope that contains an epitope in which its primary amino acid sequence is recognized. Such a linear epitope typically contains at least three and most often at least five, for example, from about 8 to about 10 or 6-20 amino acids in its particular sequence.

In contrast to a linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of the conformational epitope is not necessarily recognized by the epitope-defining antibody). Conformational epitopes may contain more amino acids than linear epitopes. An antibody that recognizes a conformational epitope recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and/or polypeptide backbones that form the conformational epitope are located next to each other, and the epitope becomes recognizable to the antibody. Methods for determining conformations of epitopes include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance, they are not limited to them. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

Binding Activity

Examples of a method for assessing epitope binding by a test antigen-binding molecule containing an antigen-binding domain for IL-6R are described below. According to the examples below, methods for evaluating epitope binding of an antigen-binding molecule of interest containing an antigen-binding domain to an antigen other than IL-6R can also be appropriately carried out.

For example, that an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R recognizes a linear epitope in an IL-6R molecule can be confirmed, for example, as described below. For the above purpose, a linear peptide is synthesized containing the amino acid sequence forming the extracellular domain of IL-6R. The peptide can be chemically synthesized or can be genetically engineered using the region encoding the amino acid sequence corresponding to the extracellular domain in the IL-6R cDNA. The test antigen-binding molecule containing the antigen-binding domain for IL-6R is then evaluated for its binding activity to a linear peptide containing an amino acid sequence forming an extracellular domain. For example, an immobilized linear peptide can be used as an antigen in an ELISA to evaluate the binding activity of an antigen-binding molecule to a peptide. Alternatively, the binding activity of a linear peptide can be assessed in terms of the level at which the linear peptide inhibits the binding of an antigen-binding molecule to cells expressing IL-6R. These assays can demonstrate the binding activity of an antigen-binding molecule to a linear peptide.

Whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a conformational epitope can be assessed as follows. For the above purpose, cells expressing IL-6R are obtained. The recognition of a conformational epitope by an antigen-binding molecule of interest containing an IL-6R antigen-binding domain can be determined when it binds strongly to IL-6R expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence forming an IL extracellular domain -6R. In the context of the present invention, "substantially does not bind" means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the cell binding activity, expressing human IL-6R.

Methods for evaluating the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to IL-6R expressing cells include, for example, the methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). In particular, the evaluation can be carried out based on the principle of ELISA or fluorescence-activated cell sorting (FACS) using IL-6R expressing cells as antigen.

In an ELISA format, the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to IL-6R expressing cells can be quantified by comparing the signal levels generated by the enzymatic reaction. Specifically, the antigen-binding molecule complex of interest is added to an ELISA plate on which IL-6R expressing cells are immobilized. The antigen-binding molecule bound to the cells is then detected using an enzyme-labeled antibody that recognizes the antigen-binding molecule of interest. Alternatively, when FACS is used, a dilution series of the antigen binding molecule of interest is prepared and the binding titer of the antibody to IL-6R expressing cells can be determined to compare the binding activity of the antigen binding molecule of interest to IL-6R expressing cells.

The binding of an antigen-binding molecule of interest to an antigen expressed on the surface of cells suspended in buffer or similar can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices:

FACSCanto™ II

FACSAria™

FACSArray™

FACSVantage™ SE

FACSCalibur™ (all are trade names of BD Biosciences)

EPICS ALTRA HyPerSort

Cytomics FC 500

EPICS XL-MCL ADC EPICS XL ADC

Cell Lab Quanta/Cell Lab Quanta SC (all are Beckman Coulter trade names).

Preferred methods for assessing the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to an antigen include, for example, the following method. First, IL-6R expressing cells are reacted with the antigen-binding molecule of interest, and then they are stained with a FITC-labeled secondary antibody that recognizes the antigen-binding molecule. The antigen-binding molecule to be tested is appropriately diluted with an appropriate buffer to obtain the molecule at the desired concentration. For example, the molecule can be used at a concentration in the range of 10 µg/mL to 10 ng/mL. Then determine the intensity of fluorescence and the number of cells using FACSCalibur (BD). Fluorescence intensity obtained by analysis using the CELL QUEST (BD) software, i.e. the geometric mean reflects the amount of antibody bound to the cells. Therefore, the binding activity of an antigen-binding molecule under investigation, which reflects the amount of bound antigen-binding molecule under investigation, can be determined by determining the geometric mean.

Whether an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R shares a common epitope with another antigen-binding molecule can be assessed based on competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected using cross-blocking assays and the like. For example, a preferred cross-blocking assay is a competitive ELISA.

Specifically, in a cross-blocking assay, IL-6R protein immobilized on the wells of a microtiter plate is pre-incubated with or without a candidate competitive antigen-binding molecule, and then the antigen-binding molecule is added thereto. The amount of an antigen-binding molecule of interest bound to the IL-6R protein in the wells is indirectly correlated with the binding capacity of a candidate competitive antigen-binding molecule that competes for binding to the same epitope. Therefore, the higher the affinity of a competitive antigen-binding molecule for the same epitope, the lower is the binding activity of the tested antigen-binding molecule to IL-6R protein-coated wells.

The amount of antigen-binding molecule of interest bound to the wells via IL-6R can be readily determined by prelabeling the antigen-binding molecule. For example, a biotin labeled antigen binding molecule is detected using an avidin/peroxidase conjugate and an appropriate substrate. In particular, a cross-blocking assay that uses enzyme labels such as peroxidase is referred to as a "competitive ELISA assay". The antigen binding molecule can also be labeled with other labeling agents that provide detection or measurement. In particular, radioactive labels, fluorescent labels, and the like are known.

When the candidate competitive antigen-binding molecule can block binding of the antigen-binding molecule of interest containing the antigen-binding domain for IL-6R by at least 20%, preferably by at least 20-50%, and more preferably by at least 50% compared to binding activity in a control experiment conducted in the absence of a competitive antigen-binding molecule, it is determined that the antigen-binding molecule under test substantially binds to the same epitope to which the competitive antigen-binding molecule binds or competes for binding to the same epitope.

When the structure of the epitope to which the test antigen-binding molecule binds, containing the antigen-binding domain for IL-6R, has already been identified, then whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of the two antigen-binding molecules to the peptide obtained by adding amino acid mutations in the peptide that forms the epitope.

To measure the above binding activity, for example, the binding activity of test and control antigen-binding molecules against a linear peptide in which a mutation has been introduced is compared in the ELISA format described above. In addition to ELISA methods, binding activity to a mutant peptide bound to a column can be determined by passing test and control antigen-binding molecules through the column and then quantifying the antigen-binding molecule eluted in the elution solution. Methods for adsorbing a mutant peptide onto a column, for example in the form of a fusion peptide to GST, are known.

Alternatively, when the identified epitope is a conformational epitope, then whether test and control antigen-binding molecules share a common epitope can be assessed in the following manner. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced in the epitope are obtained. Test and control antigen-binding molecules are added to a cell suspension obtained by suspending the cells in an appropriate buffer such as PBS. The cell suspensions are then properly washed with buffer and a FITC-labeled antibody that recognizes the test and control antigen-binding molecules is added to them. Fluorescence intensity and the number of cells stained with labeled antibody are determined using FACSCalibur (BD). Test and control antigen-binding molecules are appropriately diluted with an appropriate buffer and used at desired concentrations. For example, they can be used at a concentration in the range of 10 μg/ml to 10 ng/ml. Fluorescence intensity determined by analysis using CELL QUEST (BD) software, i.e. geometric mean, reflects the amount of labeled antibody bound to the cells. Therefore, the binding activity of test and control antigen-binding molecules, as reflected by the amount of bound labeled antibody, can be determined by measuring the geometric mean.

In the method described above, that the antigen-binding molecule "substantially does not bind to cells expressing the mutant IL-6R" can be assessed, for example, by the following method. First, test and control antigen-binding molecules bound to cells expressing the mutant IL-6R are stained with labeled antibody. The fluorescence intensity of the cells is then determined. When using the FACSCalibur to detect fluorescence by flow cytometry, the determined fluorescence intensity can be analyzed using the CELL QUEST software. From the geometric averages in the presence and absence of the antigen-binding molecule, a comparative value (ΔGeo-Mean) can be calculated using the following formula to determine the ratio of the increase in fluorescence intensity due to binding by the antigen-binding molecule.

ΔGeo-Mean = Geo-Mean (in the presence of an antigen-binding molecule)/Geo-Mean (in the absence of an antigen-binding molecule)

The geometric mean comparative value (ΔGeo-Mean value for the mutant IL-6R molecule) determined by the assay described above, which reflects the amount of the antigen-binding molecule of interest bound to cells expressing the mutant IL-6R, is compared with the comparative ΔGeo-Mean value, which reflects the amount of the antigen-binding molecule of interest bound to IL-6R expressing cells. In this case, it is particularly preferred that the concentrations of the antigen-binding molecule of interest used to determine the comparative ΔGeo-Mean values for IL-6R expressing cells and cells expressing mutant IL-6R are adjusted to be equal or substantially equal. As a control antigen-binding molecule, an antigen-binding molecule that has been confirmed to recognize an epitope in IL-6R is used.

If the comparative ΔGeo-Mean of the test antigen-binding molecule for cells expressing the mutant IL-6R is less than the comparative ΔGeo-Mean of the test antigen-binding molecule for IL-6R-expressing cells by at least 80%, preferably by 50%, more preferably by 30 %, and particularly preferably 15%, then the antigen-binding molecule under investigation "substantially does not bind to cells expressing the mutant IL-6R". The formula for determining the Geo-Mean value (geometric mean) is described in the CELL QUEST (BD biosciences) software user manual. If the comparison shows that the comparative values are essentially equivalent, then it can be determined that the epitope for the test and control antigen-binding molecules is the same.

Antigen binding domain

Within the scope of the present invention, an "antigen-binding domain" may be any structure, provided that it binds to the antigen of interest. Such domains preferably include, for example:

variable regions of heavy chains and light chains of antibodies;

a module of approximately 35 amino acids called the A domain, which is contained in the cell membrane protein avimer in vivo (WO 2004/044011, WO 2005/040229);

adnectin containing the 10Fn3 domain, which binds to the protein part of fibronectin, a glycoprotein expressed on the cell membrane (WO 2002/032925);

an affibody which consists of a three-stranded bundle of 58-amino acids based on the backbone of the IgG binding domain of protein A (WO 1995/001937);

Modeled ankyrin repeat proteins (DARPin), which is a region exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a subunit consisting of a turn containing 33 amino acid residues, two antiparallel helices, and loops repeatedly stacked (WO 2002/020565);

anticalins, and the like, which are four-loop domains that support one side of a barrel structure of eight circularly arranged anti-parallel strands that are highly conserved among lipocalin molecules such as gelatin-associated neutrophil lipocalin (NGAL ) (WO 2003/029462); and

a concave region formed by a parallel layer structure within a horseshoe-shaped structure formed by stacked repeats of the leucine-rich repeat (LRR) module of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is used in the adaptive immune system in jawless vertebrates such as like lamprey and hagfish (WO 2008/016854). Preferred antigen-binding domains of the present invention include, for example, antigen-binding domains having antibody heavy chain and light chain variable regions. Preferred examples of antigen binding domains include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv2)", "Fab" and "F(ab')2".

The antigen-binding domains of the antigen-binding molecules of the present invention can bind to an identical epitope. Such an epitope may be present, for example, in a protein containing the amino acid sequence of SEQ ID NO: 1. Alternatively, an epitope may be present in a protein containing amino acids at positions 20-365 of the amino acid sequence of SEQ ID NO: 1. Alternatively, each of the antigen-binding domains of the antigen-binding molecules of the present invention may bind to a different epitope. Within the scope of the present invention, a different epitope may be present, for example, in a protein containing the amino acid sequence of SEQ ID NO: 1. Alternatively, an epitope may be present in a protein containing amino acids at positions 20-365 of the amino acid sequence of SEQ ID NO: 1.

Specificity

"Specific" means that one of the molecules that specifically binds does not show any significant binding to molecules other than one or more binding partner molecules. Moreover, "specific" is also used when the antigen binding domain is specific for a particular epitope among a plurality of epitopes in the antigen. When an epitope bound by an antigen-binding domain is contained in several different antigens, antigen-binding molecules containing the antigen-binding domain can bind to different antigens that have that epitope.

Antibodies

In the context of the present invention, "antibody" refers to a natural immunoglobulin or an immunoglobulin obtained by partial or complete synthesis. Antibodies can be isolated from natural sources such as natural plasma and serum, or from culture supernatants of antibody-producing hybridomas. Alternatively, antibodies can be partially or completely synthesized using methods such as genetic recombination. Preferred antibodies include, for example, antibodies of the immunoglobulin B isotype or subclass thereof. Known human immunoglobulins include antibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Of these isotypes, the antibodies of the present invention include IgG1, IgG2, IgG3 and IgG4. IgG constant regions include naturally occurring mutants. A number of allotypic sequences due to genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242 for human IgG1, human IgG2, human IgG3 and human IgG4 antibody constant regions, and any of these can be used in the present invention. In particular, for the sequence of human IgG1, the amino acid sequence of positions 356-358 (EU numbering) can be either DEL or EEM.

Methods for producing an antibody with the desired binding activity are known to those skilled in the art. The following is an example that describes a method for producing an antibody that binds to IL-6R (anti-IL-6R antibody). Antibodies that bind to an antigen other than IL-6R can also be prepared according to the example described below.

Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Anti-IL-6R antibodies produced are preferably mammalian-derived monoclonal antibodies. Such mammalian-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector carrying the antibody gene by genetic engineering techniques. Monoclonal antibodies of the present invention include "humanized antibodies" or "chimeric antibodies".

Monoclonal antibody-producing hybridomas can be obtained using known methods, for example, as described below. In particular, mammals are immunized by conventional immunization methods using the IL-6R protein as the sensitizing antigen. The resulting immune cells are fused with known parental cells by conventional cell fusion techniques. Anti-IL-6R antibody producing hybridomas can then be selected by screening monoclonal antibody producing cells using conventional screening methods.

In particular, monoclonal antibodies are prepared as described below. First, an IL-6R gene whose nucleotide sequence is shown in SEQ ID NO: 2 can be expressed to produce an IL-6R protein shown in SEQ ID NO: 1, which will be used as a sensitizing antigen for producing an antibody. To do this, the sequence of the gene encoding IL-6R is inserted into a known expression vector, and suitable host cells are transformed with this vector. The desired human IL-6R protein is purified from host cells or their culture supernatants by known methods. To obtain soluble IL-6R from culture supernatants, for example, a protein consisting of amino acids at positions 1-357 in the IL-6R polypeptide sequence of SEQ ID NO: 1, such as described in Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968) express as soluble IL-6R instead of IL-6R protein SEQ ID NO: 1. Purified natural IL-6R protein can also be used as sensitizing antigen.

The purified IL-6R protein can be used as a sensitizing antigen for mammalian immunization. Also, a partial IL-6R peptide can be used as a sensitizing antigen. In this case, the partial peptide can be produced by chemical synthesis based on the amino acid sequence of human IL-6R, or by inserting the partial IL-6R gene into an expression vector. Alternatively, the incomplete peptide can be generated by degradation of the IL-6R protein with a protease. The length and region of the partial IL-6R peptide is not limited to specific embodiments. The preferred region can be arbitrarily selected from the amino acid sequence at amino acid positions 20-357 of the amino acid sequence of SEQ ID NO: 1. The number of amino acids forming the peptide to be used as the sensitizing antigen is preferably at least five or more, six or more, or seven or more. More specifically, a peptide of 8-50 residues, more preferably 10-30 residues, can be used as the sensitizing antigen.

Alternatively, a fusion protein obtained by fusing the desired incomplete polypeptide or peptide of the IL-6R protein with a different polypeptide can be used for the sensitizing antigen. For example, antibody Fc fragments and peptide tags are preferably used as sensitizing antigens. Vectors for the expression of such fusion proteins can be constructed by in-frame fusion of genes encoding two or more desired polypeptide fragments and insertion of the fusion gene into an expression vector as described above. Methods for making fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. Press). Methods for producing IL-6R for use as a sensitizing antigen and methods for immunization using IL-6R are specifically described in WO 2003/000883, WO 2004/022754, WO 2006/006693 and the like.

There is no particular limitation on the mammals to be immunized with the sensitizing antigen. However, it is preferred to select mammals based on their compatibility with the parental cells used for cell fusion. Generally, rodents such as mice, rats and hamsters, rabbits and monkeys are preferably used.

The above animals are immunized with the sensitizing antigen by known methods. As a rule, performed methods of immunization include, for example, intraperitoneal or subcutaneous injection of the sensitizing antigen in mammals. In particular, the sensitizing antigen is suitably diluted with PBS (phosphate buffered saline), physiological saline or the like. If desired, a conventional adjuvant such as Freund's complete adjuvant is mixed with the antigen and the mixture is emulsified. The sensitizing antigen is then administered to the mammal several times at intervals of 4-21 days. When immunized with a sensitizing antigen, suitable carriers may be used. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it is sometimes desirable to couple the sensitizing antigenic peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

Alternatively, hybridomas producing the desired antibody can be obtained using DNA immunization methods as described below. DNA immunization is an immunization method that stimulates the immune system by expressing a sensitizing antigen in an animal immunized by introducing a vector DNA designed to cause the gene encoding the antigenic protein to be expressed in the animal. Compared to conventional immunization methods in which a protein antigen is administered to the animals to be immunized, DNA immunization is superior in that:

- can be provided with stimulation of the immune system while maintaining the structure of the membrane protein, such as IL-6R; and

- there is no need to purify the antigen for immunization.

To obtain a monoclonal antibody of the present invention using 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 resulting DNA is inserted into an appropriate expression vector, and then it is administered to the animal to be immunized. Preferably used expression vectors include, for example, commercially available expression vectors such as pcDNA3.1. Vectors can be introduced into the body using conventional methods. For example, DNA immunization is carried out using a gene gun to introduce expression vector-coated gold particles into the body cells of the animal to be immunized. Antibodies that recognize IL-6R. can also be obtained by the methods described in WO 2003/104453.

After immunizing the mammal as described above, an increase in serum IL-6R binding antibody titer was confirmed. The immune cells are then harvested from the mammal and then subjected to cell fusion. In particular, splenocytes are preferably used as immune cells.

As the cell to be fused with the above-mentioned immune cells, mammalian myeloma cells are used. The myeloma cells preferably contain a suitable selectable marker for screening. A selectable marker gives the cells characteristics for their survival (or death) under specific culture conditions. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selectable markers. HGPRT or TK deficient cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-responsive cells cannot synthesize DNA in HAT selective media and thus die. However, when cells are fused with normal cells, they can continue to synthesize DNA using the normal cell rescue pathway, and thus they can grow in HAT selective media.

Selection of cells deficient in HGPRT and TK can be carried out in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG) or 5'-bromodeoxyuridine, respectively. Normal cells die as they incorporate pyrimidine analogs into their DNA. Meanwhile, cells that are deficient in these enzymes can survive in a selective medium because they cannot incorporate these pyrimidine analogs. In addition, a selectable marker called G418 resistance conferred by the neomycin resistance gene confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogues). Various types of myeloma cells are known that are suitable for cell fusion.

For example, preferably myeloma cells can be used, including the following cells:

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);

3194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusion between immunocytes and myeloma cells is essentially carried out using known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion agent. Cell fusion agents include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, an auxiliary such as dimethyl sulfoxide can also be added to improve the efficiency of the fusion.

The ratio of immune cells to myeloma cells can be determined independently, preferably, for example, it is one myeloma cell for every one to ten immune cells. Culture media used for cell fusion include, for example, media that are suitable for growing cell lines, such as RPMI1640 media and MEM media, and other conventional culture media used for this type of cell culture. Additionally, whey supplements such as fetal calf serum (FCS) can preferably be added to the culture medium.

For cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed in the culture medium described above. Then add a solution of PEG (for example, the average molecular weight is from about 1000 to 6000), preheated to about 37°C, at a concentration typically from 30% to 60% (wt./vol.). This mixture is then gently mixed to obtain the desired confluent cells (hybridoma). Then, the appropriate culture medium mentioned above is gradually added to the cells, and the mixture is repeatedly centrifuged to remove the supernatant. Thus, cell fusion agents and the like that are unfavorable for hybridoma growth can be removed.

Selection of hybridomas thus obtained can be carried out by culturing using a conventional selection medium, for example HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culturing in the HAT medium described above for a sufficient period of time. As a rule, the period is from several days to several weeks. Then, hybridomas producing the desired antibody are screened and cloned individually by conventional limiting dilution techniques.

Selection of hybridomas thus obtained can be carried out using a selection medium based on the selectable marker possessed by the myeloma used for cell fusion. For example, selection of cells deficient in HGPRT or TK can be carried out by culturing using HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). In particular, when HAT-responsive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively proliferate in HAT media. Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culturing in the HAT medium described above for a sufficient period of time. In particular, the selection of desired hybridomas can be carried out by culturing, typically for several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned by conventional limiting dilution techniques.

The desired antibodies can preferably be selected and individually cloned by screening methods based on a known antigen/antibody reaction. For example, an IL-6R-binding monoclonal antibody can bind to IL-6R expressed on a cell surface. Such a monoclonal antibody can be screened using fluorescence activated cell sorting (FACS). FACS is a system that evaluates antibody cell surface binding by analyzing cells in contact with a fluorescent antibody using a laser beam and measuring the fluorescence emitted by individual cells.

To screen for hybridomas that produce the monoclonal antibody of the present invention by FACS, IL-6R expressing cells are first obtained. Cells preferably used for screening are mammalian cells in which IL-6R is not naturally expressed. As a control, selective detection of antibody activity that binds to cell surface IL-6R can be performed using untransformed mammalian cells as host cells. In particular, hybridomas producing an anti-IL-6R monoclonal antibody can be isolated by selecting hybridomas that produce an antibody that binds to cells that express IL-6R in an unnatural manner, but not to host cells.

Alternatively, antibody binding activity to immobilized IL-6R expressing cells can be assessed based on the ELISA principle. For example, IL-6R expressing cells are immobilized on the wells of an ELISA plate. The hybridoma culture supernatants are contacted with the immobilized cells in the wells and detect antibodies that bind to the immobilized cells. When the monoclonal antibodies are mouse-derived, the cell-bound antibodies can be detected using an anti-mouse immunoglobulin antibody. Selection of hybridomas producing the desired antibody having an antigen-binding ability is carried out by the above-described screening, and they can be cloned by the limiting dilution method and the like.

Monoclonal antibody-producing hybridomas thus obtained can be passaged in conventional culture media and stored in liquid nitrogen for an extended period of time.

The hybridomas described above are cultured in a conventional manner, and the desired monoclonal antibodies can be obtained from culture supernatants. Alternatively, hybridomas are administered to and grown in compatible mammals and monoclonal antibodies are obtained from ascitic fluid. The first of these methods is suitable for obtaining antibodies with high purity.

Preferably, antibodies encoded by antibody genes that are cloned from antibody-producing cells, such as the hybridomas described above, can also be used. The cloned antibody gene is inserted into a suitable vector and introduced into a host cell to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting genes into vectors, and transforming host cells have already been developed, 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.

For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells expressing an anti-IL-6R antibody. For this purpose, total RNA is extracted from hybridomas first. Methods used to extract mRNA from cells include, for example:

- guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299), and

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

The extracted mRNAs can be purified using mRNA Purification Kit (GE Healthcare Bioscience) and the like. Alternatively, kits for extracting total mRNA directly from cells are also commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bioscience). mRNA can be obtained from hybridomas using such kits. cDNAs encoding the V region of an antibody can be synthesized from the resulting mRNAs using reverse transcriptase. cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) and the like. Moreover, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85 (23) can be appropriately used for cDNA synthesis and amplification. , 8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932). In such a method for synthesizing cDNA, suitable restriction enzyme sites can be inserted at both ends of the cDNA, as described below.

The cDNA fragment of interest is purified from the resulting PCR product and then ligated into vector DNA. Thus, a recombinant vector is constructed and introduced into E. coli and the like. After colony selection, the desired recombinant vector can be obtained from the colony-forming E. coli. It is then examined whether the recombinant vector has a cDNA nucleotide sequence of interest by a known method, such as a dideoxynucleotide chain termination method.

The 5'-RACE method, which uses primers to amplify a variable region gene, is conveniently used to isolate a gene encoding a variable region. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template. A commercially available kit, such as the SMART RACE cDNA amplification kit, is appropriately used to synthesize the 5'-RACE cDNA library.

The antibody gene is amplified by a PCR method using the resulting 5'-RACE cDNA library as a template. Primers for amplifying a mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary, depending on the immunoglobulin's subclass. Thus, it is preferred that the subclass be determined in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

In particular, for example, primers that allow amplification of genes encoding γ1, γ2a, γ2b and γ3 heavy chains and κ and λ light chains are used to isolate genes encoding mouse IgG. Typically, to amplify the gG variable region gene, a primer that hybridizes to a constant region region near the variable region is used as the 3' primer. Meanwhile, the primer supplied with the 5'-RACE cDNA library construction kit is used as the 5' primer.

The PCR products amplified in this way are used to reformulate immunoglobulin into combinations of heavy and light chains. Selection of the desired antibody can be carried out using the IL-6R binding activity of the reshaped immunoglobulin as an indicator. For example, when the goal is to isolate an antibody against IL-6R, it is more preferred that the binding of the antibody to IL-6R be specific. Screening for an IL-6R binding antibody can be performed, for example, using the following steps:

(1) contacting an IL-6R expressing cell with an antibody containing the V region encoded by cDNA isolated from the hybridoma;

(2) detection of antibody binding to an IL-6R expressing cell; and

(3) selecting an antibody that binds to an IL-6R expressing cell.

Methods for detecting antibody binding to IL-6R expressing cells are known. In particular, antibody binding to IL-6R expressing cells can be detected using the methods described above, such as FACS. Immobilized samples of IL-6R expressing cells are appropriately used to assess antibody binding activity.

Preferred antibody screening methods that use binding activity as an indicator also include panning methods using phage vectors. Screening methods using phage vectors are advantageous when antibody genes are isolated from libraries of heavy chain and light chain subclasses from a population of cells expressing a polyclonal antibody. Genes encoding heavy chain and light chain variable regions can be linked with an appropriate linker sequence to generate a single chain Fv (scFv). Phages displaying scFv on their surface can be obtained by inserting the gene encoding the scFv into a phage vector. The phages are contacted with the antigen of interest. DNA encoding scFvs having binding activity of interest can then be isolated by harvesting antigen-bound phages. This process can be repeated as needed to increase the content of scFvs having binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-IL-6R antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs at low frequency in the nucleotide sequence of an antibody gene. Moreover, preferably, a restriction site for the enzyme that forms the sticky end is introduced into the vector of interest to insert one copy of the cleaved fragment in the correct orientation. The cDNA coding region of the anti-IL-6R antibody V region is digested as described above and inserted into an appropriate expression vector to construct an expression vector for the antibody. In this case, if the gene encoding the constant region of the antibody (C-region) and the gene encoding the above-described V-region are fused in-frame, a chimeric antibody will be obtained. In the context of the present invention, "chimeric antibody" means that the origin of the constant region is different from the variable region. Thus, in addition to heterochimeric mouse/human antibodies, the chimeric antibodies of the present invention include human/human allochimeric antibodies. An expression vector for a chimeric antibody can be constructed by inserting the V region gene into an expression vector that already has a constant region. In particular, for example, a sequence recognized by the first restriction enzyme that cleaves upstream of the V region gene can be appropriately placed at the 5' end of an expression vector containing DNA encoding the desired constant region (C region) of an antibody. An expression vector for a chimeric antibody is constructed by in-frame fusion of two genes cleaved with the same combination of restriction enzymes.

To obtain an anti-IL-6R monoclonal antibody, the antibody genes are inserted into an expression vector such that the genes are expressed under the control of an expression regulating region. The expression control region for antibody expression includes, for example, enhancers and promoters. Moreover, a suitable signal sequence can be attached to the N-terminus so that the expressed antibody is secreted to the outside of the cells. In the examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as a signal sequence. Meanwhile, other suitable signal sequences can be attached. The expressed polypeptide is cleaved at the C-terminus of the sequence described above and the resulting polypeptide is secreted from the cells as the mature polypeptide. Suitable host cells are then transformed with an expression vector to produce recombinant cells expressing the DNA encoding the IL-6R antibody.

The DNA encoding the heavy chain (H chain) and the light chain (L chain) of an antibody are separately inserted into various expression vectors to express the antibody gene. An antibody molecule having H and L chains can be expressed by co-transfection of the same host cell with vectors into which the H chain and L chain genes are respectively inserted. Alternatively, host cells can be transformed with a single erexpression vector into which DNA encoding the H and L chains is inserted (see WO 1994/011523).

There are various known host cell/expression vector combinations for producing antibodies by introducing isolated antibody genes into appropriate hosts. All of these expression systems are suitable for isolating the antigen binding domains of the present invention. Suitable eukaryotic cells for use as host cells include animal cells, plant cells and fungal cells. In particular, animal cells include, for example, the following cells.

(1) mammalian cells: CHO, COS, myeloma cells, Chinese hamster kidney (BHK), HeLa, Vero human embryonic kidney (HEK) 293 cells, Freestyle™293, etc.;

(2) amphibian cells: Xenopus oocytes and the like; and

(3) insect cells: sf9, sf21, Tn5, etc.

In addition, as a plant cell, an expression system for antibody genes using cells derived from the genus Nicotiana such as Nicotiana tabacum is known. Cultured callus cells can be appropriately used to transform plant cells.

Moreover, the following cells can be used as fungal cells:

yeast: the genus Saccharomyces, such as Saccharomyces serevisiae, and the genus Pichia, such as Pichia pastoris; and

- filamentous fungi: the genus Aspergillus, such as Aspergillus niger.

Moreover, expression systems for antibody genes are also known which use prokaryotic cells. For example, when using bacterial cells, E. coli cells, Bacillus subtilis cells, and the like can be suitably used within the scope of the present invention. Expression vectors carrying the antibody genes of interest are introduced into cells by transfection. The transfected cells are cultured in vitro and the desired antibody can be obtained from the culture of the transformed cells.

In addition to the host cells described above, transgenic animals can also be used to produce a recombinant antibody. Therefore, an antibody can be obtained from an animal that has been introduced with a gene encoding the antibody of interest. For example, an antibody gene can be constructed as a fusion gene by inserting in frame into a gene that codes for a protein specifically produced in milk. As the protein secreted in milk, for example, goat β-casein and the like can be used. The DNA fragments containing the fusion gene into which the antibody gene is inserted are injected into a goat embryo, and then the embryo is implanted into a female goat. The desired antibodies can be obtained as a milk fusion protein from the milk produced by a transgenic goat born from a goat that is the recipient of the embryo (or its offspring). In addition, hormones can be administered to the transgenic goat to increase the volume of milk containing the desired antibody produced by the transgenic goat, Ebert, K.M. et al., Bio/Technology (1994) 12 (7), 699-702).

When the antigen-binding molecule described herein is administered to a human, an antigen-binding domain derived from a genetically recombinant antibody that is artificially modified to reduce heterologous antigenicity against a human or the like can be suitably used as the antigen-binding domain of the molecule. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably prepared by known methods.

The variable region of an antibody used to derive the antigen-binding domain of the antigen-binding molecule described herein is typically formed by three complementarity-determining regions (CDRs) that are separated by four framework regions (FRs). The CDR is a region that essentially determines the binding specificity of an antibody. The amino acid sequences of the CDRs are highly diverse. On the other hand, FR-forming amino acid sequences often have high identity, even among antibodies with differing binding specificities. Thus, in general, the binding specificity of a particular antibody can be introduced into another antibody by CDR grafting.

A humanized antibody is also referred to as a reshaped human antibody. In particular, humanized antibodies are known that are obtained by grafting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody, or the like. Also known are standard methods of genetic engineering for the production of humanized antibodies. In particular, for example, overlapped extension PCR is known as a method for transplanting mouse antibody CDRs into human FRs. In overlapped PCR extension, the nucleotide sequence encoding the CDR of the mouse antibody to be transplanted is added to the primers for the synthesis of the FR of the human antibody. Primers are prepared for each of the four FRs. It is generally accepted that when transplanting a mouse CDR into a human FR, selecting a human FR that has a high identity of the mouse FR is advantageous for maintaining CDR function. Thus, it is generally preferred to use a human FR containing an amino acid sequence that has a high identity with the amino acid sequence of the FR adjacent to the CDR of the mouse to be transplanted.

The nucleotide sequences to be ligated are designed to be joined to each other in reading frame. Human FRs are synthesized separately using the appropriate primers. As a result, products are obtained in which the DNA encoding mouse CDRs is linked to individual DNAs encoding FRs. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Complementary chain synthesis is then carried out to hybridize the overlapping CDR products synthesized using the human antibody gene as a template. With this reaction, human FRs are ligated through mouse CDR sequences.

The full-length V-region gene, in which three CDRs and four FRs are eventually ligated, is amplified using primers that hybridize to its 5' or 3' end, to which the appropriate sequences are added that are recognized by the restriction enzyme. An expression vector for a humanized antibody can be obtained by inserting the DNA prepared as described above and the DNA that encodes the C region of a human antibody into an expression vector such that they are ligated in-frame. After transfection with a recombinant host vector to obtain recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed to obtain a humanized antibody in cell culture (see European Patent Publication No. EP 239400 and International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively determining and evaluating the antigen-binding activity of the humanized antibody prepared as described above, human FR antibodies can be suitably selected that allow the CDR to form a suitable antigen-binding site when ligated through the CDR. The amino acid residues in the FR can be changed as needed so that the CDRs of the rearranged human antibody form the appropriate antigen binding site. For example, amino acid sequence mutations can be introduced into FR using the PCR method that is used to transplant mouse CDRs into human FRs. More specifically, partial mutations in the nucleotide sequence can be introduced into primers that hybridize to FR. Nucleotide sequence mutations are introduced into FRs synthesized using such primers. Mutant FR sequences having the desired characteristics can be selected by measuring and evaluating the antigen binding activity of the amino acid mutant antibody using the method mentioned above (Cancer Res. (1993) 53: 851-856).

Alternatively, the desired human antibodies can be obtained by immunizing transgenic animals having the full complement of human antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO 1996/033735) by DNA immunization.

Moreover, methods for producing human antibodies by panning using human antibody libraries are also known. For example, the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of a phage by a phage display method. You can select phage expressing scFv that bind to the antigen. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of selected phages. The scFv DNA sequence that binds to the antigen is determined. An expression vector is prepared by fusing the in-frame V region sequence to the C region sequence of the desired human antibody and inserting it into an appropriate expression vector. The expression vector is introduced into cells suitable for expression, such as those described above. A human antibody can be produced by expressing a gene encoding a human antibody in cells. These methods are already known (see WO 1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO 1995/015388).

In addition to the methods described above, B cell cloning methods (identification of each antibody coding sequence, cloning and isolation of each sequence; use in the construction of an expression vector to obtain each antibody (IgG1, IgG2, IgG3 or IgG4 , in particular), etc.), as described, for example, Bernasconi et al. (Science (2002) 298: 2199-2202) or WO 2008/081008.

EU numbering system and Kabat numbering system

In accordance with the methods used in the framework of the present invention, the amino acid positions assigned to the CDRs and FRs of antibodies are indicated according to Kabat numbering (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)) . For the purposes of the present invention, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids of the variable region are indicated according to the Kabat numbering system, while the amino acids of the constant region are indicated according to the EU numbering system based on the Kabat amino acid positions.

Ion Concentration Conditions

Conditions for the concentration of metal ions

In one embodiment of the present invention, the ion concentration refers to the concentration of metal ions.

"Metal ions" refers to ions of Group I elements other than hydrogen, such as alkali metals and copper group elements, ions of Group II elements such as alkaline earth metals and zinc group elements, ions of Group III elements other than boron, ions of Group III elements IV, with the exception of carbon and silicon, ions of group VIII elements such as iron group elements and platinum group elements, ions of elements belonging to subgroup A of groups V, VI and VII, and metal elements such as antimony, bismuth and polonium. Metal atoms have the property of releasing valence electrons so that they become cations. This is called the ionization trend. Metals with a high tendency to ionize are considered reactive.

In the context of the present invention, preferred metal ions include, for example, calcium ion. The calcium ion is involved in the modulation of a variety of biological phenomena, including muscle contraction such as skeletal, smooth, and cardiac muscles; activation of movement, phagocytosis, etc. leukocytes; activation of shape change, secretion, etc. platelets; activation of lymphocytes; mast cell activation, including histamine secretion; cellular responses mediated by the catecholamine a receptor or the acetylcholine receptor; exocytosis; the release of substances-transmitters from the endings of neurons; and flow of axoplasm in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and the myosin light chain, which have multiple calcium ion binding sites and are believed to have a common origin in terms of molecular evolution. There are also many known calcium-binding motifs. Such well-known motifs include, for example, cadherin domains, calmodulin EF-hand, protein kinase C C2 domain, coagulation factor IX protein Gla domain, acyaroglycoprotein receptor and mannose-binding receptor C-type lectins, LDL receptor A domains, annexations , thrombospondin type 3 domain, and EGF-like domains.

In the context of the present invention, when the metal ion is a calcium ion, the calcium ion concentration conditions include low calcium ion concentrations and high calcium ion concentrations. "Binding activity varies depending on calcium ion concentrations" means that the antigen-binding activity of an antigen-binding molecule varies due to differences between low and high calcium ion conditions. For example, the antigen-binding activity of an antigen-binding molecule may be higher at a high concentration of calcium ions than at a low concentration of calcium ions. Alternatively, the antigen-binding activity of an antigen-binding molecule may be higher at a low calcium ion concentration than at a high calcium ion concentration.

Within the scope of the present invention, the high concentration of calcium ions is not particularly limited to a specific value; however, the concentration may preferably be selected from values ranging from 100 μM to 10 mM. In another embodiment, the concentration may be selected from values ranging from 200 μM to 5 mm. In an alternative embodiment, the concentration may be selected from values ranging from 500 μM to 2.5 mm. In another embodiment, the concentration can be selected from values ranging from 200 μm to 2 mm. Moreover, the concentration can be selected from values ranging from 400 μM to 1.5 mM. In particular, a concentration selected from values ranging from 500 μM to 2.5 mm, which is close to the concentration of calcium ions in plasma (blood) in vivo, is preferred.

Within the scope of the present invention, the low concentration of calcium ions is not particularly limited to a specific value; however, the concentration may preferably be selected from values ranging from 0.1 μM to 30 μM. In another embodiment, the concentration may be selected from values ranging from 0.2 μM to 20 μM. In another embodiment, the concentration may be selected from values ranging from 0.5 μM to 10 μM. In an alternative embodiment, the concentration may be selected from values ranging from 1 μM to 5 μM. Moreover, the concentration can be selected from values ranging from 2 μM to 4 μM. In particular, a concentration selected from values ranging from 1 μM to 5 μM, which is close to the concentration of ionized calcium in early endosomes in vivo, is preferred.

In the context of the present invention, "antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration" means that the antigen-binding activity of an antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 0.1 μM to 30 μM than at a calcium ion concentration selected from values ranging from 100 μM to 10 mM. Preferably, this means that the antigen-binding activity of the antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 0.5 μM to 10 μM than at a calcium ion concentration selected from values ranging from 200 μM to 5 mM. Particularly preferably, this means that the antigen-binding activity at the calcium ion concentration in the early endosome in vivo is weaker than at the plasma calcium ion concentration in vivo; and in particular, this means that the antigen-binding activity of the antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 1 μM to 5 μM than at a calcium ion concentration selected from values ranging from 500 μM to 2, 5 mm.

Whether the antigen-binding activity of an antigen-binding molecule changes with metal ion concentration can be determined, for example, using known measurement methods such as those described in the "Binding Activity" section above. For example, to confirm that the antigen-binding activity of an antigen-binding molecule becomes higher at a high calcium ion concentration than at a low calcium ion concentration, the antigen-binding activity of an antigen-binding molecule at low and high calcium ion concentrations is compared.

Within the scope of the present invention, the expression "antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration" can also be expressed as "the antigen-binding activity of an antigen-binding molecule is higher at a high calcium ion concentration than at a low calcium ion concentration" . In the present description, "antigen-binding activity is lower at low calcium ion concentration than at high calcium ion concentration" is sometimes represented as "antigen-binding capacity is weaker at low calcium ion concentration than at high calcium ion concentration". Also, "the antigen-binding activity at a low calcium ion concentration is reduced so that it is lower than the antigen-binding activity at a high calcium ion concentration" can be represented as "the antigen-binding ability at a low calcium ion concentration is weakened compared to the antigen-binding ability at a high calcium ion concentration" .

When determining antigen-binding activity, conditions other than calcium ion concentration can be appropriately selected by those skilled in the art, and they are not specifically limited. For example, activity can be determined at 37° C. in HEPES buffer. For example, Biacore (GE Healthcare) and the like can be used to determine. When the antigen is a soluble antigen, the antigen-binding activity of the antigen-binding molecule can be assessed by passing the antigen as an analyte over a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of the antigen-binding molecule to the membrane antigen can be assessed by passing the antigen-binding molecule as an analyte over a chip on which the antigen is immobilized.

Provided that the antigen-binding activity of the antigen-binding molecule of the present invention at low calcium ion concentration is weaker than at high calcium ion concentration, the ratio of antigen-binding activity between antigen-binding activity between low calcium ion concentration and high calcium ion concentration is not specifically limited; and the KD (Ca 3 μM)/KD (Ca 2 mM) value, which is the ratio of the dissociation constant (KD) of an antigen at low calcium ion concentration and KD at high calcium ion concentration, is preferably 2 or more; more preferably, the value of KD (Ca 3 μm)/KD (Ca 2 mm) is 10 or more; and even more preferably, the value of KD (Ca 3 μm)/KD (Ca 2 mm) is 40 or more. The upper limit of the value of KD (Ca 3 μM)/KD (Ca 2 mM) is not particularly limited, and it can be any value such as 400, 1000 or 10000, provided that the molecule can be produced by methods known to those skilled in the art. . Alternatively, the KD value (Ca 3 μM)/KD (Ca 1.2 mM) is indicated. Therefore, the value of KD (Ca 3 μm)/KD (Ca 1.2 mm) is 2 or more; more preferably, the value of KD (Ca 3 μm)/KD (Ca 1.2 mm) is 10 or more; and even more preferably, the value of KD (Ca 3 μm)/KD (Ca 1.2 mm) is 40 or more. The upper limit of the value of KD (Ca 3 μM)/KD (Ca 1.2 mM) is not particularly limited, and it can be any value such as 400, 1000 or 10000, provided that the molecule can be produced by methods known to those skilled in the art. .

When the antigen is a soluble antigen, KD (dissociation constant) can be used to display antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used to display the activity. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), a Scatchard plot, or a flow cytometer.

Alternatively, for example, the dissociation rate constant (kd) can preferably also be used as an index to display the ratio of the antigen-binding activity of the antigen-binding molecule of the present invention at low and high calcium concentrations. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index to display the ratio of binding activities, the ratio of dissociation rate constants (kd) at low and high calcium concentrations, i. the value of kd (low calcium concentration)/kd (high calcium concentration) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of Kd (low calcium concentration)/kd (high calcium concentration) is not particularly limited, and may be any value such as 50, 100, or 200, as long as the molecule can be obtained by methods known to those skilled in the art.

When the antigen is a soluble antigen, kd (dissociation rate constant) can be used to display antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent kd (apparent dissociation rate constant) can be used to display the antigen-binding activity. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be determined by methods known to those skilled in the art, for example using Biacore (GE healthcare) or a flow cytometer. Within the scope of the present invention, when the antigen-binding activity of an antigen-binding molecule is determined at different concentrations of calcium ions, it is preferable to use the same conditions except for the calcium concentrations.

For example, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, including the steps:

(a) determining the antigen-binding activity of the antigen-binding domain or antibody at a low calcium concentration;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody at a high calcium concentration; and

(c) selecting an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.

Moreover, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than 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, including stages:

(a) contacting the antigen with an antigen-binding domain or antibody or library thereof at a high calcium concentration;

(b) incubation at a low calcium concentration of the antigen-binding domain or antibody that has bound to the antigen in step (a); and

(c) isolating the antigen-binding domain or antibody dissociated in step (b).

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than 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, including the steps :

(a) contacting the antigen with a library of antigen-binding domains or antibodies at a low calcium concentration;

(b) selecting an antigen-binding domain or antibody that did not bind to the antigen in step (a);

(c) allowing the antigen-binding domain or antibody selected in step (b) to bind to the antigen at a high calcium concentration; and

(d) isolating the antigen-binding domain or antibody that has bound to the antigen in step (c).

In addition, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening process comprising the steps of:

(a) contacting, at a high calcium concentration, a library of antigen-binding domains or antibodies with a column on which the antigen is immobilized;

(b) eluting the antigen-binding domain or antibody that has bound to the column in step (a) from the column at a low calcium concentration; and

(c) isolating the antigen-binding domain or antibody eluted in step (b).

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening comprising the steps of:

(a) allowing the library of antigen-binding domains or antibodies at low calcium concentration to pass through the column on which the antigen is immobilized;

(b) collecting the antigen-binding domain or antibody that eluted without being bound to the column in step (a);

(c) allowing the antigen-binding domain or antibody assembled in step (b) to bind to the antigen at a high calcium concentration; and

(d) isolating the antigen-binding domain or antibody that has bound to the antigen in step (c).

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening comprising the steps of:

(a) contacting the antigen with a library of antigen-binding domains or antibodies at a high calcium concentration;

(b) obtaining an antigen-binding domain or an antibody that has bound to the antigen in step (a);

(c) low calcium incubation of the antigen-binding domain or antibody obtained in step (b); and

(d) isolating an antigen-binding domain or an antibody whose antigen-binding activity in step (c) is weaker than the selection criterion in step (b).

The steps described above may be repeated two or more times. Thus, the present invention relates to antigen-binding domains or antibodies, the antigen-binding activity of which is lower at a low concentration of calcium ions than at a high concentration of calcium ions, which are obtained by screening methods that further include the step of repeating two or more times of steps (a) - (c) or (a)-(d) in the screening methods described above. The number of cycles of steps (a)-(c) or (a)-(d) is not particularly limited, but is usually 10 or less.

In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at a low calcium concentration is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 0.1 μM to 30 μM, but preferably it is an antigen-binding activity at concentration of ionized calcium from 0.5 μM to 10 μM. More preferably, it is an antigen-binding activity at a concentration of ionized calcium in the early endosome in vivo, in particular from 1 μM to 5 μM. Meanwhile, the antigen-binding activity of the antigen-binding domain or antibody at a high calcium concentration is not particularly limited as long as it is an antigen-binding activity at an ionized calcium concentration of 100 μM to 10 mM, but preferably it is an antigen-binding activity at an ionized calcium concentration of 200 μM. up to 5 mm. More preferably, it is an antigen-binding activity at an in vivo plasma concentration of ionized calcium, in particular from 0.5 mM to 2.5 mM.

The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Conditions other than ionized calcium concentration can be determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be measured as dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate constant (kd), apparent dissociation constant (apparent kd), and the like. They can be determined by methods known to those skilled in the art, such as using Biacore (GE healthcare), Scatchard plot, or FACS.

Within the scope of the present invention, the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration is identical to the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium concentration than at high concentration of calcium.

As long as the antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration, the difference in antigen-binding activity between the high and low concentrations is not particularly limited; however, the antigen-binding activity at a high calcium concentration is preferably two times or more, more preferably 10 times or more, and even more preferably 40 times or more than a low calcium concentration.

The antigen-binding domains or antibodies of the present invention to be screened by the screening methods described above can be any of the antigen-binding domains and antibodies. For example, the antigen-binding domains or antibodies described above can be screened. For example, you can screen for antigen-binding domains or antibodies having natural sequences or sequences with substituted amino acids.

Libraries

In one embodiment, an antigen-binding domain or an antibody of the present invention can be obtained from a library that primarily consists of a plurality of antigen-binding molecules whose sequences differ from each other and whose antigen-binding domains have at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecules in depending on the concentration of ions. The ion concentrations preferably include, for example, a metal ion concentration and a hydrogen ion concentration.

In the context of the present invention, a "library" refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules or nucleic acids or polynucleotides encoding their sequences. The sequences of the plurality of antigen-binding molecules or the plurality of antigen-binding molecule-containing fusion polypeptides in the library are not identical, but differ from each other.

In the context of the present invention, the expression "sequences differ from each other" in the expression "a plurality of antigen-binding molecules whose sequences differ from each other" means that the sequences of antigen-binding molecules in the library differ from each other. In particular, in a library, the number of sequences that differ from each other reflects the number of independent clones with different sequences, and may also be referred to as "library size". The library size for a typical phage display library ranges from 10 ⁶ to 10 ¹² . The library size can be increased up to 10 ¹⁴ using known methods such as ribosome display. However, the actual number of phage particles used in panning selection of a phage library is typically 10-10,000 times the size of the library. This excess multiplicity is also referred to as "library equivalents" and means that there are between 10 and 10,000 individual clones that share the same amino acid sequence. Thus, within the scope of the present invention, the expression "sequences differ from each other" means that the sequences of the independent antigen-binding molecules in the library, excluding library equivalents, differ from each other. More specifically, the above means that there are 10 ⁶ to 10 ¹⁴ antigen-binding molecules whose sequences differ from each other, preferably 10 ⁷ to 10 ¹² molecules, more preferably 10 ⁸ to 10 ¹¹ molecules, and particularly preferably 10 ⁸ up to 10 ¹² molecules, the sequences of which differ from each other.

Within the scope of the present invention, the expression "multiple" in the expression "a library mainly consists of a plurality of antigen-binding molecules" in the case of, for example, antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors or viruses of the present invention mainly refers to a group of two or more types of substance. For example, when two or more substances differ from each other in a particular characteristic, this means that there are two or more types of substance. Such examples may include, for example, mutant amino acids seen at specific amino acid positions in the amino acid sequence. For example, when there are two or more antigen-binding molecules of the present invention whose sequences are substantially the same or preferably the same except for non-strict residues or except for specific mutant amino acids at surface-exposed hypervariable positions, there are a plurality of antigen-binding molecules of the present invention. In another example, when there are two or more polynucleotide molecules whose sequences are substantially the same or preferably the same, with the exception of nucleotides encoding non-strict residues or nucleotides encoding mutant amino acids of surface-exposed hypervariable positions, there are a plurality of polynucleotide molecules of the present invention. .

In addition, in the context of the present invention, the expression "mainly consists of" in the expression "a library mainly consists of a plurality of antigen-binding molecules" reflects the number of antigen-binding molecules whose antigen-binding activity varies, depending on the concentration of ions, among independent clones with different sequences in the library . It is particularly preferred that there be at least 10 ⁴ antigen-binding molecules having such binding activity in the library. More preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁵ antigen-binding molecules having such binding activity. Even more preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁶ antigen-binding molecules having such binding activity. Particularly preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁷ antigen-binding molecules having such binding activity. Even more preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁸ antigen-binding molecules having such binding activity. Alternatively, it can also preferably be expressed as the ratio of the number of antigen-binding molecules whose antigen-binding activity varies depending on the concentration of ions and the number of independent clones having different sequences in the library. In particular, the antigen-binding domains of the present invention can be obtained from a library in which the antigen-binding molecules having such binding activity are from 0.1% to 80%, preferably from 0.5% to 60%, more preferably from 1% to 40%. %, even more preferably 2% to 20%, and particularly preferably 4% to 10% independent clones with different sequences in the library. In the case of fused polypeptides, polynucleotide molecules, or vectors, similar expressions may be possible using numbers of molecules or a ratio to the total number of molecules. In the case of viruses, similar expressions using the number of virions or a ratio to the total number of virions may also be possible.

Amino acids that change the antigen-binding activity of antigen-binding domains depending on the concentration of calcium ions

The antigen-binding domains or antibodies of the present invention to be screened by the screening methods described above can be obtained in any way. For example, when the metal ion is a calcium ion, pre-existing antibodies, pre-existing libraries (phage library, etc.), antibodies or libraries derived from hybridomas obtained by immunizing animals or from B cells of immunized animals can be used, antibodies or libraries obtained by introducing calcium-chelating amino acids (such as aspartic acid and glutamic acid) or non-natural amino acid mutations into the antibodies or libraries described above (calcium-chelating amino acids (such as aspartic acid and glutamic acid), libraries with an increased content of non-natural amino acids, libraries obtained by introducing calcium-chelating amino acids (such as aspartic acid and glutamic acid) or unnatural amino acid mutations at specific positions, and the like.

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

Moreover, as amino acids that change the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations, for example, amino acids having a metal chelating activity can be preferably used. Examples of such metal-chelating amino acids include, for example, serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)) and glutamic acid (Glu(E)).

Positions in the antigen-binding domains containing the amino acids described above are not specifically limited to specific positions, and may be any positions in the heavy chain variable region or light chain variable region that form an antigen-binding domain, provided that they alter the antigen-binding activity of the antigen-binding molecules depending on the concentration of calcium ions. In particular, the antigen-binding domains of the present invention can be derived from a library primarily composed of antigen-binding molecules whose sequences differ from one another and whose heavy chain antigen-binding domains contain amino acids that alter the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations. In another non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose heavy chain CDR3 domains contain the amino acids mentioned above. In another non-limiting embodiment, the antigen-binding domains of the present invention can be derived from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose heavy chain CDR3 domains contain the above amino acids at positions 95, 96, 100a and/or 101 , as specified according to the Kabat numbering system.

Meanwhile, in a non-limiting embodiment of the present invention, the antigen-binding domains of the present invention can be obtained from a library mainly consisting of antigen-binding molecules whose sequences differ from each other and whose light chain antigen-binding domains contain amino acids that change the antigen-binding activity of the antigen-binding molecules depending on concentrations of calcium ions. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR1 domains contain the amino acids mentioned above. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR1 domains contain the above amino acids at positions 30, 31 and/or 32, as indicated in accordance with the Kabat system numbering.

In another non-limiting embodiment, the antigen-binding domains of the present invention can be derived from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR2 domains contain the amino acid residues mentioned above. In another embodiment, the present invention relates to libraries primarily consisting of antigen-binding molecules whose sequences are different from each other and whose light chain CDR2 domains contain the above amino acid residues at position 50, as indicated according to the Kabat numbering system.

In another non-limiting embodiment of the present invention, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR3 domains contain the amino acid residues mentioned above. In an alternative embodiment, the antigen-binding domains of the present invention may be derived from a library primarily composed of antigen-binding molecules whose sequences differ from one another and whose light chain CDR3 domains contain the above-mentioned amino acid residues at position 92, as indicated by system numbering. Kabat.

Moreover, in various embodiments of the present invention, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and in which two or three CDRs selected from the CDR1, CDR2 and CDR3 described above are light chains containing the amino acid residues mentioned above. Moreover, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chains contain the amino acid residues mentioned above at any one or more of positions 30, 31, 32, 50 and /or 92, as indicated in accordance with the Kabat system numbering.

In a particularly preferred embodiment, the light chain and/or heavy chain variable region framework sequences of the antigen binding molecule preferably comprise human germline framework sequences. Thus, in one embodiment of the present invention, when the framework sequences are fully human sequences, when such an antigen-binding molecule of the present invention is administered to a human (e.g., for the treatment of diseases), it is expected to induce little or no immunogenic response. In the above context, the expression "comprising a germline sequence" in the context of the present invention means that a portion of the framework sequences of the present invention is identical to a portion of any human germline framework sequences. For example, when the heavy chain FR2 sequence of an antigen binding molecule of the present invention is a combination of heavy chain FR2 sequences of different human germline framework sequences, such molecule is also an antigen binding molecule of the present invention "comprising a germline sequence".

Preferred examples of frameworks include, for example, fully human human framework sequences currently known, which are included on the V-Base website (http://vbase.mrc-cpe.cam.ac.uk/) or others. These framework sequences can be suitably used as the germline sequence contained in the antigen-binding molecule of the present invention. Germline sequences can be categorized according to their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798); Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al. (Nat. Genetics (1994) 7, 162-168)). Appropriate germline sequences can be selected from Vκ, which fall into seven subgroups; Vλ, which are subdivided into ten subgroups; and VH, which are subdivided into seven subgroups.

Fully human VH sequences preferably include, but are not limited to, for example, VH sequences:

VH1 subgroups (eg, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58 and VH1-69);

subgroups of VH2 (for example, VH2-5, VH2-26 and VH2-70);

VH3 subgroups (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35 , VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73 and VH3-74);

VH4 subgroups (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59 and VH4-61);

subgroups VH5 (VH5-51);

subgroups VH6 (VH6-1); and

subgroups of VH7 (VH7-4 and VH7-81).

They are also described in known documents (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)) and the like, and thus, those skilled in the art can appropriately model antigen-binding molecules according to of the present invention based on the knowledge of these sequences. It is also preferred to use other fully human frameworks or subframes.

Fully human VK sequences preferably include, but are not limited to, for example:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14 and O18 allocated to the Vk1 subgroup;

A1, A2, A3, A5, A7, A17, A18, A19, A23, O1 and O11 allocated to the Vk2 subgroup;

A11, A27, L2, L6, L10, L16, L20 and L25 allocated to the Vk3 subgroup;

B3 allocated to the Vk4 subgroup;

B2 (also referred to as Vk5-2), allocated to the Vk5 subgroup; and

A10, A14 and A26 allocated to the VK6 subgroup.

(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022); Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human VL sequences preferably include, but are not limited to, for example:

V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1- 20 and V1-22 allocated to the VL1 subgroup;

V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17 and V2-19 subgrouped VL1;

V3-2, V3-3 and V3-4 allocated to the VL3 subgroup;

V4-1, V4-2, V4-3, V4-4 and V4-6 allocated to the VL4 subgroup; and

V5-1, V5-2, V5-4 and V5-6 subgrouped VL5 (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).

Typically, these framework sequences differ from one another by one or more amino acid residues. These framework sequences can be used in combination with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations" of the present invention. Other examples of fully human frameworks used in combination with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations" of the present invention include, but are not limited to, e.g., KOL, NEWM, REI, EU , TUR, TEI, LAY, and POM (eg, Kabat et al. (1991), supra, Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

Without wishing to be bound by a particular theory, it is believed that one reason to expect that the use of germline sequences will prevent adverse immune responses in most individuals is as follows. As a result of the affinity maturation process during normal immune responses, somatic mutations often occur in the variable regions of immunoglobulins. Such mutations most often occur in the CDR region, the sequences of which are hypervariable, but also affect the remnants of the frame regions. Such framework region mutations do not exist in germline genes, but are also less likely to be immunogenic in patients. This is a consequence of the fact that most of the framework sequences expressed from germline genes are present in the normal human population and as a result of immune tolerance, these germline framework regions are expected to have low or no immunogenicity in patients. To maximize the potential for immune tolerance, genes encoding the variable region can be selected from a group of frequently occurring functional germline genes.

To obtain the antigen-binding molecules of the present invention, in which the framework sequences described above contain amino acids that change the antigen-binding activity of the antigen-binding molecules depending on the concentration of calcium ions, known methods such as site-directed mutagenesis (Kunkel et al. (Proc Natl Acad Sci USA (1985) 82, 488-492)) and overlapping PCR.

For example, a library that contains a plurality of antigen-binding molecules of the present invention, the sequences of which differ from each other, can be constructed by combining heavy chain variable regions obtained as a randomized library of variable region sequences, where the light chain variable region is selected as a framework sequence, initially containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. As a non-limiting example, when the ion concentration is calcium ion concentration, such preferred libraries include, for example, libraries constructed by combining a light chain variable region sequence belonging to the Vk5-2 family corresponding to SEQ ID NO: 62 (Vk5-2), and the heavy chain variable region generated as a randomized library of variable region sequences.

Alternatively, the light chain variable region sequence selected as the framework region, initially containing at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule as mentioned above, can be designed to contain different amino acid residues other than the amino acid residues mentioned above. . In the present description, such residues are called non-strict residues. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on the ion concentrations. In particular, the heavy chain and/or light chain CDR sequences and/or FR sequences may contain one or more non-strict residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of non-strict residues to be introduced into the light chain variable region sequence of SEQ ID NO: 62 (Vk5-2) include the amino acid residues shown in Tables 1 or 2.

As used herein, non-strict residues refer to the variation in amino acid residues present at hypervariable positions at which several different amino acids are present in the light chain and heavy chain variable regions when the amino acid sequences of known and/or native antibodies or antigen-binding domains are compared. Hypervariable positions are usually located in the CDR. In one embodiment, the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) are useful for determining hypervariable positions in known and/or native antibodies. Moreover, online databases (http://vbase.mrc-cp.cam.ac.uk/, http://www.bioinf.org.uk/abs/index.html) provide assembled sequences of many light chains and human heavy chains and their positions. Sequence and position information is useful for determining hypervariable positions within the scope of the present invention. In accordance with the present invention, when a particular amino acid position has preferably from about 2 to about 20 possible amino acid residues, preferably from about 3 to about 19, preferably from about 4 to about 18, preferably from 5 to 17, preferably from 6 to 16 , preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, and preferably 10 to 12 possible variants of amino acid residues, this position is hypervariable. In some embodiments, a particular amino acid position may preferably have at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, and preferably about 12 amino acid residue variants.

Alternatively, a library containing a plurality of antigen-binding molecules of the present invention whose sequences are different from each other can be constructed by combining heavy chain variable regions obtained as a randomized variable region sequence library with light chain variable regions in which at least one amino acid is introduced. a residue that changes the antigen-binding activity of antigen-binding molecules depending on ion concentrations, as mentioned above. When the ion concentration is a calcium ion concentration, non-limiting examples of such libraries preferably include, for example, libraries in which heavy chain variable regions generated as a randomized library of variable region sequences are combined with light chain variable region sequences in which a particular residue (and ) in a germline sequence 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 by at least one amino acid residue , which changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. Non-limiting examples of such amino acid residues include amino acid residues in the light chain CDR1. Moreover, non-limiting examples of such amino acid residues include amino acid residues in the light chain CDR2. In addition, other non-limiting examples of such amino acid residues also include amino acid residues in the light chain CDR3.

Non-limiting examples of such amino acid residues contained in the light chain CDR1 include residues at positions 30, 31 and/or 32 in the light chain variable region CDR1, as indicated by Kabat numbering. Moreover, non-limiting examples of such amino acid residues contained in the light chain CDR2 include the amino acid residue at position 50 in the light chain variable region CDR2, as indicated by Kabat numbering. Moreover, non-limiting examples of such amino acid residues contained in the light chain CDR3 include the amino acid residue at position 92 in the light chain variable region CDR3, as indicated by Kabat numbering. These amino acid residues may be present alone or in combination, provided that they form a calcium-binding motif and/or provided that the antigen-binding activity of the antigen-binding molecule varies depending on the concentration of calcium ions. Meanwhile, since troponin C, calmodulin, parvalbumin, and myosin light chain are known to have multiple calcium ion binding sites and are believed to have a common origin in terms of molecular evolution, it is possible to construct light chain CDR1, CDR2 and/or CDR3, so that they have their binding motives. For example, domains of cadherin, EF-hand of calmodulin, C2 domain of protein kinase C, Gla domain of blood coagulation factor IX protein, lectins of the C-type acyaroglycoprotein receptor and mannose-binding receptor, A-domains of LDL receptors, annexin, thrombospondin 3 domains can be used. type, and EGF-like domains as appropriate for the purposes described above.

When heavy chain variable regions obtained as a randomized library of variable region sequences and light chain variable regions in which at least one amino acid residue is introduced that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of ions, the sequences are combined as described above. light chain variable regions can be designed to contain non-strict residues in a manner similar to that described above. The number and position of such non-strict residues is not specifically limited to specific embodiments, provided that the antigen-binding activity of the antigen-binding molecules of the present invention varies depending on ion concentrations. In particular, the heavy chain and/or light chain CDR sequences and/or FR sequences may contain one or more non-strict residues. When the ion concentration is the calcium ion concentration, non-limiting examples of non-strict residues introduced into the light chain variable region sequence include the amino acid residues shown in Tables 1 and 2.

Preferred heavy chain variable regions to be combined include, for example, randomized variable region libraries. To obtain a randomized library of the variable region, known methods are combined in an appropriate way. In a non-limiting embodiment of the present invention, as a randomized variable region library, it is preferable to use an immune library constructed on the basis of antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infections, individuals with an increased titer of antibodies in the blood as a result of vaccination, patients with cancer or patients with an autoimmune disease.

In another non-limiting embodiment of the present invention, a synthetic library obtained by replacing the CDR sequences of V genes in genomic DNA or functional reshaped V genes with a set of synthetic oligonucleotides containing sequences encoding codon sets of the appropriate length can also preferably be used as a randomized variable region library. . In this case, since diversity is observed in the heavy chain CDR3 sequence, only the CDR3 sequence can also be replaced. A criterion for the occurrence of amino acid diversity in the variable region of an antigen-binding molecule is that diversity occurs at amino acid residues at surface-exposed positions of the antigen-binding molecule. Surface-exposed position refers to a position that is considered capable of being exposed to a surface and/or contacting an antigen based on the structure, group of structures, and/or modeled structure of the antigen-binding molecule. Typically, these provisions are CDRs. Preferably, surface exposed positions are determined using coordinates from a 3D model of the antigen binding molecule using a computer program such as the InsightII program (Accelrys). Surface exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Surface exposed positions can be determined using software suitable for protein modeling and the 3D structure information obtained from the antibody. Software that can be used for this purpose preferably includes SYBYL Biopolymer Module software (Tripos Associates). Generally, or preferably, when the algorithm requires the user to enter a size parameter, the "size" of the sample used in the calculation is set to a radius of about 1.4 Angstroms or less. Moreover, methods for determining surface-exposed areas and areas using personal computer software are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46- 53).

In another non-limiting embodiment of the present invention, as a randomized variable region library, it is also particularly preferred to use a naive library that is constructed from antibody genes derived from lymphocytes of healthy individuals, and whose gene set consists of naive sequences that are antibody sequences with no preference ( Gejima et al (Human Antibodies (2002) 11, 121-129); Cardoso et al (Scand. J. Immunol. (2000) 51, 337-344)). In the present description, an amino acid sequence containing a naive sequence refers to an amino acid sequence obtained from such a naive library.

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, the sequences of which differ from each other, obtained by combining the variable regions of the light chain, designed as a randomized variable region library, where the variable region heavy chain is selected as a backbone sequence that initially contains "at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule as a function of ion concentrations." When the ion concentration is the calcium ion concentration, non-limiting examples of such libraries preferably include libraries constructed by combining light chain variable regions constructed as a randomized variable region library with the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1). Alternatively, such a library can be constructed by selecting appropriate light chain variable regions from light chain variable regions that have germline sequences, instead of light chain variable regions constructed as a randomized variable region sequence library. Such preferred libraries include, for example, libraries in which the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) is combined with light chain variable regions, having a germline sequence.

Alternatively, the heavy chain variable region sequence selected as the framework sequence, which initially contains "at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule", as mentioned above, can be designed to contain non-strict residues. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on the ion concentrations. In particular, the heavy chain and/or light chain CDR and/or FR sequences may contain one or more non-strict residues. When the ion concentration is the calcium ion concentration, non-limiting examples of non-strict residues introduced into the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) include all heavy chain CDR1 and CDR2 amino acid residues and heavy chain CDR3 amino acid residues, except for residues at positions 95, 96 and/or 100a. Alternative non-limiting examples of non-strict residues introduced into the heavy chain variable region sequence of SEQ ID NO: 10 (6KC4-1#85-IgG1) include all amino acid residues of heavy chain CDR1 and CDR2 and amino acid residues of heavy chain CDR3, excluding residues at amino acid positions 95 and/or 101.

Alternatively, a library containing a plurality of antigen-binding molecules whose sequences differ from one another can be constructed by combining light chain variable regions designed as a randomized library of variable region sequences, or light chain variable regions having germline sequences, with heavy chain variable regions, wherein "at least one amino acid residue responsible for an ion concentration-dependent change in the antigen-binding activity of the antigen-binding molecule" has been introduced. When the ion concentration is a calcium ion concentration, non-limiting examples of such libraries preferably include libraries in which light chain variable regions constructed as a randomized library of variable region sequences or light chain variable regions having germline sequences are combined with a heavy variable region sequence. a chain in which the specific residue(s) is replaced by at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. Non-limiting examples of such amino acid residues include heavy chain CDR1 amino acid residues. The following non-limiting examples of such amino acid residues include heavy chain CDR2 amino acid residues. In addition, non-limiting examples of such amino acid residues also include heavy chain CDR3 amino acid residues. Non-limiting examples of such heavy chain CDR3 amino acid residues include amino acids at positions 95, 96, 100a and/or 101 in the heavy chain variable region CDR3, as indicated by Kabat numbering. Moreover, these amino acid residues may be present alone or in combination, provided that they form a calcium-binding motif and/or the antigen-binding activity of the antigen-binding molecule varies depending on the concentration of calcium ions.

When light chain variable regions designed as a randomized library of variable region sequences, or light chain variable regions having a germline sequence, are combined with a heavy chain variable region in which at least one amino acid residue is inserted that alters the antigen-binding activity of the antigen-binding molecule to depending on the concentration of ions, as mentioned above, the sequence of the heavy chain variable region can also be designed to contain non-strict residues, in a similar way as described above. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on ion concentrations. In particular, the heavy chain CDR and/or FR sequences may contain one or more non-strict residues. Moreover, randomized variable region libraries can preferably be used as amino acid sequences of the heavy chain variable region CDR1, CDR2 and/or CDR3 other than the amino acid residues that alter the antigen-binding activity of the antigen-binding molecule. When germline sequences are used as light chain variable regions, non-limiting examples of such sequences include SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), and SEQ ID NO: 8 (Vk4).

Preferably, any of the amino acids described above that alter the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentrations can be used, provided that they form a calcium-binding motif. In particular, such amino acids include electron donor amino acids. Preferred examples of such electron donor amino acids include serine, threonine, asparagine, glutamic acid, aspartic acid, and glutamic acid.

Conditions for the concentration of hydrogen ions

In one embodiment of the present invention, the ion concentration conditions refer to hydrogen ion concentration conditions or pH conditions. Within the scope of the present invention, the proton concentration, i.e. the nucleus of a hydrogen atom, is used as a synonym for pH (pH). When the activity of hydrogen ions in an aqueous solution is displayed as aH+, the pH is defined as -log10aH+. When the ionic strength of the aqueous solution is low (eg, below 10 ^-3 ), aH+ is substantially equal to the ionic strength of hydrogen. For example, the ionic product of water at 25°C and 1 atmosphere is Kw=aH+aOH=10 ^-14 , and thus, in pure water aH+=aOH=10 ^-7 . In this case, pH=7 is neutral; an aqueous solution whose pH is below 7 is acidic, or whose pH is greater than 7 is alkaline.

In the context of the present invention, when pH conditions are used as ion concentration conditions, the pH conditions include high hydrogen ion concentrations or low pH values, i. acidic pH range, and low hydrogen ion concentrations or high pH values, i.e. range of neutral pH values. "Binding activity varies with pH conditions" means that the antigen-binding activity of an antigen-binding molecule varies due to differences in conditions of high hydrogen ion concentration or low pH values (acidic pH range) and low hydrogen ion concentration or high pH values (neutral pH range). ). This includes, for example, the case where the antigen-binding activity of the antigen-binding molecule is higher in the neutral pH range than in the acidic pH range, and the case where the antigen-binding activity of the antigen-binding molecule is higher in the acidic pH range than in the neutral pH range. pH.

In the present description, the range of neutral pH values is not limited to a particular value, and is preferably selected from the range of pH6.7 to pH10.0. In another embodiment, the pH value may be selected from the range of pH6.7 to pH9.5. In another embodiment, the pH value may be selected from the range of pH7.0 to pH9.0. In another embodiment, the pH value may be selected from the range of pH7.0 to pH8.0. A particularly preferred pH value includes a pH of 7.4, which is close to the in vivo plasma (blood) pH.

In the present description, the range of acidic pH values is not limited to a particular value, and is preferably selected from the range of pH4.0 to pH6.5. In another embodiment, the pH value may be selected from the range of pH4.5 to pH6.5. In another embodiment, the pH value may be selected from the range of pH5.0 to pH6.5. In another embodiment, the pH may be selected from the range of pH5.5 to pH6.5. A particularly preferred pH includes a pH of 5.8, which is close to the ionized calcium concentration in the early endosome in vivo.

In the context of the present invention, "the antigen-binding activity of an antigen-binding molecule at high hydrogen ion concentration or low pH (acidic pH range) is lower than at low hydrogen ion concentration or high pH (neutral pH range)" means that the antigen-binding activity an antigen-binding molecule at a pH value selected from the range of pH4.0 to pH6.5 is weaker than at a pH value selected from the range of pH6.7 to pH10.0; preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH4.5 to pH6.5 is weaker than at a pH selected from the range of pH6.7 to pH9.5; more preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH5.0 to pH6.5 is weaker than at a pH value selected from the range of pH7.0 to pH9.0; even more preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH5.5 to pH6.5 is weaker than at a pH value selected from the range of pH7.0 to pH8.0; particularly preferably, this means that the antigen-binding activity at the pH of the early endosome in vivo is weaker than the antigen-binding activity at the pH of the plasma in vivo; and specifically, this means that the antigen-binding activity of the antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.

Determining whether the antigen-binding activity of an antigen-binding molecule changes with pH conditions can be done, for example, using known measurement methods such as those described in the "Binding Activity" section above. In particular, binding activity is measured under various pH conditions using the measurement methods described above. For example, the antigen-binding activity of an antigen-binding molecule is compared under acidic pH range and neutral pH range conditions to confirm that the antigen-binding activity of the antigen-binding molecule changes such that it is higher under neutral pH range conditions than under acidic pH range conditions.

Moreover, within the scope of the present invention, the expression "antigen-binding activity at high hydrogen ion concentration or low pH value, i.e. in the acidic pH range, is lower than at low hydrogen ion concentration or high pH value, i.e. . in the neutral pH range" can also be expressed as "the antigen-binding activity of the antigen-binding molecule at low hydrogen ion concentration or high pH, i.e. in the neutral pH range, is higher than at high hydrogen ion concentration or low pH , i.e. in the range of acidic pH values". Within the scope of the present invention, "antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the acidic pH range, is lower than at low hydrogen ion concentration or high pH, i.e. in the neutral range pH" can be described as "antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the acidic pH range, is weaker than antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the neutral pH range". Alternatively, "antigen-binding activity at high hydrogen ion concentration or low pH, i.e., in the acidic pH range, is reduced to be lower than at low hydrogen ion concentration or high pH, i.e., in the range neutral pH values" can be described as "antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the acidic pH range, is reduced so that it is weaker than antigen-binding activity at low hydrogen ion concentration or high values pH, i.e. in the range of neutral pH values".

Conditions other than hydrogen ion concentration or pH for measurement and antigen-binding activity can be appropriately selected by those skilled in the art and are not specifically limited. The measurement can be carried out, for example, at 37°C using HEPES buffer. The measurement can be carried out, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the antigen-binding activity of the antigen-binding molecule can be determined by evaluating the binding activity of the soluble antigen by applying the antigen as an analyte to a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of the membrane antigen can be assessed by applying the antigen-binding molecule as an analyte to a chip on which the antigen is immobilized.

Provided that the antigen-binding activity of the antigen-binding molecule of the present invention at a high concentration of hydrogen ions or a low pH, i. e. in the range of acidic pH values, is weaker than at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range, the ratio of antigen-binding activity to antigen-binding activity at high hydrogen ion concentration or low pH, i. e. in the range of acidic pH values, and at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range is not particularly limited, and the KD (pH 5.8)/KD (pH 7.4) value, which is the ratio of the dissociation constant (KD) for an antigen at a high hydrogen ion concentration or a low pH, i.e. e. in the range of acidic pH values, and KD at low concentration of hydrogen ions or high pH value, i.e. in the neutral pH range, preferably 2 or more; more preferably, the value of KD (pH 5.8)/KD (pH 7.4) is 10 or more; and even more preferably, the value of KD (pH 5.8)/KD (pH 7.4) is 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 the molecule can be produced by methods known to those skilled in the art. .

When the antigen is a soluble antigen, the dissociation constant (KD) can be used as a measure of the antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. Dissociation constant (KD) and apparent dissociation constant (KD) can be measured by methods known to those skilled in the art, and Biacore (GE healthcare), Scatchard plot, flow cytometer, and the like can be used.

Alternatively, for example, the dissociation rate constant (kd) can be appropriately used as an indication of the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention to activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, and at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values. When kd (dissociation rate constant) is used as an indicator indicating the ratio of binding activities instead of KD (dissociation constant), the value of kd (in the range of acidic pH values)/kd (in the range of neutral pH values), which is the ratio of kd (constant dissociation rate) in relation to the antigen at a high concentration of hydrogen ions or a low pH value, i.e. in the range of acidic values, pH and kd (dissociation rate constant) at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range, preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of kd (in the acidic pH range)/kd (in the neutral pH range) is not particularly limited, and may be any value such as 50, 100, or 200, as long as the molecule can be produced by methods well-known specialists in this field.

When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the amount of antigen-binding activity, and when the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. Dissociation rate constant (kd) and apparent dissociation rate constant (kd) can be determined by methods known to those skilled in the art, and Biacore (GE healthcare), a flow cytometer, and the like can be used. Within the scope of the present invention, when the antigen-binding activity of an antigen-binding molecule is measured at different concentrations of hydrogen ions, i.e. pH, conditions other than hydrogen ion concentration, i.e. The pH is preferably the same.

For example, an antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which are one of the embodiments contemplated by the present invention, can be obtained by screening antigen-binding domains or antibodies, including the following steps (a)-(c):

(a) obtaining a value of the antigen-binding activity of the antigen-binding domain or antibody in the range of acidic pH values;

(b) obtaining a value of the antigen-binding activity of the antigen-binding domain or antibody in the range of neutral pH values; and

(c) selecting an antigen-binding domain or antibody whose antigen-binding activity is lower in an acidic pH range than in a neutral pH range.

Alternatively, an antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which are one of the embodiments contemplated by the present invention, can be obtained by screening antigen-binding domains or antibodies or their library, including the following steps (a)-(c):

(a) contacting the antigen-binding domain, or antibody, or library thereof, in the neutral pH range, with the antigen;

(b) placing the antigen-binding domain or antibody bound to the antigen in step (a) in an acidic pH range; and

(c) isolating the antigen-binding domain or antibody dissociated in step (b).

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment provided by the present invention, can be obtained by screening antigen-binding domains, or antibodies, or their library, including the following steps (a)-(d):

(a) contacting in the range of acidic pH values of the antigen with a library of antigen-binding domains or antibodies;

(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 the antigen in the neutral pH range; and

(d) isolating the antigen-binding domain or antibody bound to the antigen in step (c).

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(c):

(a) contacting in the range of neutral pH values of the library of antigen-binding domains or antibodies with a column on which the antigen is immobilized;

(b) eluting in the acidic pH range from the column of the antigen-binding domain or antibody bound to the column in step (a); and

(c) isolating the antigen-binding domain or antibody eluted in step (b).

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(d):

(a) allowing, in an acidic pH range, a library of antigen-binding domains or antibodies to pass through the column onto which the antigen is immobilized;

(b) collecting the antigen-binding domain or antibody eluted without binding to the column in step (a);

(c) allowing the antigen-binding domain or antibody assembled in step (b) to bind to the antigen in the neutral pH range; and

(d) isolating the antigen-binding domain or antibody bound to the antigen in step (c).

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(d):

(a) contacting the antigen with a library of antigen-binding domains or antibodies in the neutral pH range;

(b) obtaining an antigen-binding domain or antibody bound to the antigen in step (a);

(c) placing the antigen-binding domain or antibody obtained in step (b) in an acidic pH range; and

(d) isolating an antigen-binding domain or an antibody whose antigen-binding activity in step (c) is weaker than the standard selected in step (b).

The steps described above may be repeated two or more times. Thus, the present invention relates to antigen-binding domains and antibodies, the antigen-binding activity of which in the range of acidic pH values is lower than in the range of neutral pH values, which are obtained by a screening method, which further comprises the steps of repeating two or more times of steps (a) - (c) or (a)-(d) in the screening methods described above. The number of repetitions of steps (a)-(c) or (a)-(d) is not particularly limited; however, usually the number is 10 or less.

In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at high hydrogen ion concentration or low pH, i.e. acidic pH range, is not particularly limited as long as it is antigen-binding activity at pH 4.0 to 6.5, and includes antigen-binding activity at pH 4.5 to 6.6 as preferred pH values. Antigen-binding activity also includes antigen-binding activity at pH 5.0 to 6.5, and antigen-binding activity at pH 5.5 to 6.5 as other preferred pH values. Antigen-binding activity also includes antigen-binding activity at pH in the early endosome in vivo as a more preferred pH, and in particular, antigen-binding activity at pH 5.8. Meanwhile, the antigen-binding activity of an antigen-binding domain or an antibody at low hydrogen ion concentration or high pH, i.e. in the neutral pH range, is not particularly limited, as long as it is antigen-binding activity at pH 6.7 to 10, and includes antigen-binding activity at pH 6.7 to 9.5 as preferred pH values. Antigen binding activity also includes antigen binding activity at pH 7.0 to 9.5 and antigen binding activity at pH 7.0 to 8.0 as other preferred pH values. Antigen-binding activity also includes antigen-binding activity at plasma pH in vivo as more preferred pH values, and in particular antigen-binding activity at pH 7.4.

The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Those skilled in the art can appropriately define conditions other than ionized calcium concentration. The antigen-binding activity of an antigen-binding domain or antibody can be estimated based on dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), apparent dissociation rate constant (kd), and the like. They can be determined by methods known to those skilled in the art, such as using Biacore (GE healthcare), Scatchard plot, or FACS.

Within the scope of the present invention, the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values, is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, is synonymous with the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values.

Provided that the antigen-binding activity at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, the difference between antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values, and antigen-binding activity at a high concentration of hydrogen ions or a low pH value, i.e. in the range of acidic pH values, not specifically limited; however, antigen-binding activity at low hydrogen ion concentration or high pH, ie. in the neutral pH range, preferably two times or more, more preferably 10 times or more, and even more preferably 40 times or more, the antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values.

The antigen-binding domain or antibody of the present invention screened by the screening methods described above may be any antigen-binding domain or antibody, and the above-mentioned antigen-binding domain or antibody can be screened. For example, an antigen-binding domain or antibody having a native sequence can be screened, and an antigen-binding domain or antibody can be screened in which their amino acid sequences have been changed.

The antigen-binding domain or antibody of the present invention to be screened by the screening methods described above can be obtained by any method. For example, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries derived from B-cells of immunized animals or hybridomas obtained by immunizing animals, antibodies or libraries (libraries with increased amino acid content with pKa side chain 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acids, libraries containing amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acid mutations at specific positions, etc.) obtained by introducing amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acid mutations in the antibodies or libraries described above.

Methods for producing an antigen-binding domain or antibody having antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values, is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, from antigen-binding domains or antibodies derived from hybridomas obtained by immunization of animals or from B-cells of immunized animals, preferably include, for example, an antigen-binding molecule or antibody in which at least one of the amino acids of the antigen-binding domain or antibody is replaced an amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or a mutation to a non-natural amino acid or antigen-binding domain or antibody into which an amino acid with a side chain pKa of 4.0-8.0 is inserted (for example, histidine and glutamic acid) or a non-natural amino acid such as described in WO 2009/125825.

Mutation sites for amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids are not particularly limited, and may be any position, as long as the antigen-binding activity in the acidic pH range becomes weaker than in the neutral pH range (KD (in the acidic pH range)/KD (in the neutral pH range) or kd (in the acidic pH range)/kd (in the neutral pH range) increases) compared to with the value before replacement or insertion. For example, when the antigen binding molecule is an antibody, the variable region and CDR of the antibody are useful. Those skilled in the art can appropriately determine the number of amino acids to be replaced, or the number of amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids to be inserted. Substitution can be made with one amino acid having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or one non-natural amino acid; insertion of one amino acid having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or one non-natural amino acid can be carried out; a substitution can be made with two or more amino acids having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or with two or more unnatural amino acids; and two or more amino acids having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or two or more unnatural amino acids can be inserted. Alternatively, other amino acids can be deleted, added, inserted, and/or substituted in parallel, in addition to 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 insertions of amino acids having a side chain pKa chains 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acids. Substitution or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids can be performed randomly by methods such as histidine scanning, in which alanine in the alanine scanning method known to those skilled in the art areas, replace with histidine. Antigen-binding molecules exhibiting a higher KD (acidic pH range)/KD (neutral pH range) or kd (acidic pH range)/kd (neutral pH range) value than before the mutation can be choose from antigen-binding domains or antibodies that are randomly inserted or mutated to amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids.

Preferred examples of antigen-binding molecules containing a mutation to amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids as described above, and whose antigen-binding activity in the acidic pH range is lower than in the neutral pH range, include antigen-binding molecules whose antigen-binding activity in the neutral pH range after mutation to amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or non-natural amino acids is comparable to the antigen-binding activity before mutation on amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acids. Within the scope of the present invention, "an antigen-binding molecule, after mutating to amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid), or non-natural amino acids, has an antigen-binding activity comparable to that before mutating to amino acids having a pKa side chain 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids" means that, upon taking the antigen-binding activity of the antigen-binding molecule prior to mutating to amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid), or non-natural amino acids as 100%, the antigen-binding activity of the antigen-binding molecule after mutation to amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid), or non-natural amino acids is at least 10% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. Antigen binding activity after mutation to amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids at pH 7.4 may be higher than before mutation to amino acids with a side chain pKa of 4.0 -8.0 (eg, histidine and glutamic acid) or unnatural amino acids at pH 7.4. If the antigen-binding activity of an antigen-binding molecule is reduced due to insertion or substitution with amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids, the antigen-binding activity can be made comparable to the antigen-binding activity prior to insertion or substitution with amino acids with side chain pKa 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids by substitution, deletion, insertion and/or insertion of one or more amino acids of the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity is adjusted to be comparable by substitution, deletion, insertion and/or insertion of one or more amino acids after substitution or insertion of an amino acid with a side chain pKa of 4.0-8.0 ( for example, histidine and glutamic acid) or non-natural amino acids.

Meanwhile, when the antigen-binding molecule is a substance containing an antibody constant region, preferred embodiments of antigen-binding molecules whose antigen-binding activity is lower in an acidic pH range than in a neutral pH range include methods in which the antibody constant regions contained in antigen-binding molecules, modified. Specific examples of modified antibody constant regions preferably include the constant regions of SEQ ID NOs: 11, 12, 13 and 14.

Amino acids that alter the antigen-binding activity of the antigen-binding domain depending on hydrogen ion concentration conditions

The antigen binding domains or antibodies of the present invention to be screened by the screening methods described above can be obtained by any method. For example, when the ion concentration conditions are hydrogen ion concentration conditions or pH conditions, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries derived from B cells of immunized animals or from hybridomas obtained by immunization can be used. animals, antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or non-natural amino acids, libraries that have been mutated for amino acids with a side chain pKa of 4.0-8.0 8.0 (e.g. histidine and glutamic acid) or unnatural amino acids at specific positions, etc.) obtained by mutating amino acids with a side chain pKa of 4.0-8.0 (e.g. histidine and glutamic acid) or unnatural amino acids in the antibodies or libraries described above.

In one non-limiting embodiment of the present invention, a library containing a plurality of antigen-binding molecules of the present invention, the sequences of which differ from each other, can also be constructed by combining the heavy chain variable regions obtained as a randomized variable region sequence library with light chain variable regions, in which introduced "at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions."

Such amino acid residues include, but are not limited to, for example, the amino acid residues contained in the light chain CDR1. Amino acid residues also include, but are not limited to, for example, amino acid residues contained in the light chain CDR2. Amino acid residues also include, but are not limited to, for example, amino acid residues contained in the light chain CDR3.

The above-described amino acid residues contained in the light chain CDR1 include, but are not limited to, for example, amino acid residues at positions 24, 27, 28, 31, 32 and/or 34 according to Kabat numbering in the light chain variable region CDR1. Meanwhile, the amino acid residues contained in the light chain CDR2 include, but are not limited to, for example, the amino acid residues at positions 50, 51, 52, 53, 54, 55 and/or 56 according to Kabat numbering in the light chain variable region CDR2. Moreover, amino acid residues in the light chain CDR3 include, but are not limited to, for example, amino acid residues at positions 89, 90, 91, 92, 93, 94 and/or 95A according to Kabat numbering, in the CDR3 of the light chain variable region. Moreover, the amino acid residues may be contained alone, or they may be contained in a combination of two or more amino acids, provided that they allow the antigen-binding activity of the antigen-binding molecule to be varied depending on the concentration of hydrogen ions.

Even when the heavy chain variable region obtained as a randomized variable region sequence library is combined with the above-described light chain variable region in which "at least one amino acid residue is introduced that changes the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions" , it can be designed so that non-strict residues are contained in the sequence of the variable region of the light chain in the same way as described above. The number and position of non-strict residues is not specifically limited to a specific embodiment, as long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the hydrogen ion concentration conditions. In particular, the heavy chain and/or light chain CDR and/or FR sequences may contain one or more non-strict residues. For example, the non-strict residues to be introduced in the light chain variable region sequences include, but are not limited to, for example, the amino acid residues shown in Tables 3 and 4. Meanwhile, the amino acid sequences of the light chain variable regions other than the non-strict residues and the amino acid residues that change the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions, respectively include, but are not limited to, 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).

(position indicated according to Kabat numbering)

(position indicated according to Kabat numbering)

Any amino acid residue can be suitably used as the above-described amino acid residues that change the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions. In particular, such amino acid residues include amino acids with a side chain pKa of 4.0-8.0. Such electron-donating amino acids preferably include, for example, naturally occurring amino acids such as histidine and glutamic acid, as well as non-natural amino acids such as histidine analogs (US 20090035836), 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 preferred amino acid residues include, for example, amino acids with a side chain pKa of 6.0-7.0. Such electron-donating amino acid residues preferably include, for example, histidine.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap PCR can be appropriately used to modify the amino acids of antigen-binding domains. Moreover, various known methods can also be used as an amino acid modification method to replace amino acids with amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci U.S.A. (2003) 100(11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which an amber suppressor tRNA that is complementary to a UAG codon (an amber codon) which is a stop codon can be appropriately used is linked to a non-natural amino acid.

Preferred heavy chain variable regions that are used in combination include, for example, randomized variable region libraries. Known methods are suitably combined as a method for obtaining a randomized variable region library. In a non-limiting embodiment of the present invention, as a randomized variable region library, an immune library constructed from antibody genes derived from animals immunized with specific antigens, patients with an infection, or individuals with an increased antibody titer in the blood as a result of vaccination, patients with malignant tumor or lymphocytes from patients with immune diseases.

In another non-limiting embodiment of the present invention, similarly as described above, a synthetic library obtained by replacing the CDR sequences of V genes in genomic DNA or functional reshaped V genes with a set of synthetic oligonucleotides can also be suitably used as a randomized variable region library, containing sequences encoding sets of codons of the appropriate length. In this case, since diversity is observed in the heavy chain CDR3 sequence, only the CDR3 sequence can also be replaced. The basis for obtaining variants of amino acids in the variable region of the antigen-binding molecule is to obtain variants of amino acid residues exposed on the surface of the provisions of the antigen-binding molecule. Surface exposed position refers to a position that is considered capable of surface exposure and/or contact with an antigen based on the structure, group of structures and/or modeled structure of the antigen binding molecule, and typically such positions are CDRs. Preferably, surface exposed positions are determined using coordinates from a 3D model of the antigen binding molecule using a computer program such as the InsightII program (Accelrys). Surface exposed positions can be determined using algorithms known in the art (e.g. Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface exposed positions can be made based on 3D structure information using software suitable for protein modeling. Software that can be used for this purpose preferably includes SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires the user to enter a size parameter, the "size" of the sample used in the calculation is set to a radius of approximately 1.4 Angstroms or less. Moreover, methods for determining surface-exposed areas and areas using personal computer software are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46- 53).

In another non-limiting embodiment of the present invention, as a randomized variable region library, it is also particularly useful to use a naive library that is constructed from antibody genes derived from lymphocytes of healthy individuals, and whose gene set consists of naive sequences that are antibody sequences with no preference ( Gejima et al (Human Antibodies (2002) 11, 121-129); Cardoso et al (Scand. J. Immunol. (2000) 51, 337-344)).

Neutralizing activity

A non-limiting embodiment of the present invention relates to an antigen-binding molecule having a human FcRn-binding activity in the acidic pH range, comprising an antigen-binding domain and an Fcγ receptor-binding domain, and having a neutralizing activity against an antigen, wherein the antigen-binding domain has an antigen-binding activity that varies depending on ion concentration conditions, and the Fcγ receptor binding domain has higher Fcγ receptor binding activity under neutral pH range conditions than the Fc region of native human IgG, in which the sugar chain linked at position 297 (EU numbering) is a containing fucose chain sugars; and a pharmaceutical composition containing an antigen-binding molecule. Mainly, the neutralizing activity refers to the activity of inhibiting the biological activity of a ligand such as viruses and toxins having a biological activity against cells. Thus, substances having neutralizing activity refer to substances that bind to the ligand or receptor to which the ligand binds and inhibit the binding between the ligand and the receptor. Receptors whose binding to a ligand is blocked due to neutralizing activity are unable to exhibit biological activity through that receptor. When the antigen-binding molecule is an antibody, such an antibody having neutralizing activity is generally referred to as a neutralizing antibody. The neutralizing activity of an analyte can be measured by comparing biological activity in the presence of a ligand between situations where the analyte is present and absent.

For example, the main candidate ligands for the IL-6 receptor preferably include IL-6 as shown in SEQ ID NO: 15. The IL-6 receptor, which is a type I membrane protein whose N-terminus forms an extracellular domain, forms a heterotetramer with the receptor gp130, which is induced to dimerize by IL-6 (Heinrich et al. (Biochem. J. (1998) 334, 297-314)). Formation of the heterotetramer activates Jak, which is associated with the gp130 receptor. Jak undergoes autophosphorylation and phosphorylates the receptor. The receptor and Jak phosphorylation site serves as a binding site for SH2 containing molecules belonging to the Stat family such as Stat3; MAP kinase; PI3/Act; and other SH2 containing proteins and adapters. Further, Stat bound to the gp130 receptor is phosphorylated by Jak. Phosphorylated Stat dimerizes and translocates to the nucleus and regulates the transcription of target genes. Jak or Stat may also be involved in signal transduction cascades through other classes of receptors. Dysregulation of IL-6 signaling cascades is observed in inflammatory and pathological conditions of autoimmune diseases and malignancies such as prostate cancer and multiple myeloma. Stat3, which can act as an oncogene, is activated constitutively in many cancers. In prostate cancer and multiple myeloma, there is a cross-talk between the signaling cascade through the IL-6 receptor and the signaling cascade through members of the epithelial growth factor receptor (EGFR) family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

Such intracellular signal transduction cascades differ for each cell type; thus, appropriate target molecules can be determined for each target cell of interest and are not limited to the factors mentioned above. Neutralization activity can be assessed by measuring the activation of signal transduction in vivo. Moreover, in vivo signal transduction activation can be detected using transcription induction of a target gene that is located downstream of the in vivo signal transduction cascade as an indicator of action. A change in the transcriptional activity of a target gene can be detected by the principle of reporter assays. Specifically, a reporter gene such as green fluorescent protein (GFP) or luciferase is placed downstream of a promoter region or a transcription factor of a target gene, its reporter activity is measured, and thus a change in transcription activity as a reporter activity can be measured. Commercially available in vivo signal transduction activation kits (eg, Mercury Pathway Profiling Luciferase System (Clontech)) may be used appropriately.

Moreover, for methods of measuring the neutralization activity of EGF receptor family receptors/ligands and the like, which typically affects signal transduction cascades that act to promote cell proliferation, the neutralization activity of neutralizing antibodies can be assessed by measuring the proliferation activity of target cells. For example, when cell proliferation is stimulated by EGF family growth factors such as HB-EGF, an inhibitory effect on the proliferation of such cells, based on the neutralizing activity of an anti-HB-EGF antibody, can be suitably assessed or measured by the following methods: to assess or measure proliferation inhibition activity cells in vitro use the method of measuring the incorporation of [ ³ H]-labeled thymidine added to the environment, viable cells as an indicator of the ability to replicate DNA. As more convenient methods, a dye exclusion method, which measures the ability of cells to exclude a dye such as trypan blue from a cell under a microscope, and the MTT method are used. The last of these methods exploits the ability of living cells to convert MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which is a tetrazolium salt, into the blue formazan product. More specifically, the test antibody is added together with the ligand to the culture solution of the cells to be tested, and after a certain period of time, MTT is added to the culture solution and left to stand for a period of time to incorporate the MTT into the cell. As a result, MTT, which is a yellow compound, is converted to a blue compound by the action of succinate dehydrogenase in the mitochondria of the cells. After dissolving this blue staining product, its absorbance is measured and used as an indicator of the number of living cells. In addition to MTT, reagents such as MTS, XTT, WST-1 and WST-8 (Nacalai Tesque and the like) are also commercially available and can be suitably used. For activity measurement, a binding antibody that is an antibody of the same isotype as an anti-HB-EGF antibody but does not have cell proliferation inhibiting activity can be used as a control antibody in the same way as an anti-HB-EGF antibody, and the activity can be determine when an anti-HB-EGF antibody exhibits a higher cell proliferation inhibition activity than a control antibody.

Cells that can preferably be used for activity evaluation include, for example, cells whose proliferation is stimulated by HB-EGF, such as the RMG-1 ovarian cancer cell line, and Ba/F3 mouse cells transformed with a vector to express the gene encoding hEGFR/mG -CSFR, which is a fusion protein in which the extracellular domain of human EGFR is fused in-frame to the intracellular domain of the mouse GCSF receptor. Thus, those skilled in the art can appropriately select cells to use for activity evaluation and use them to measure cell proliferation activity, as mentioned above.

Since the antigen-binding molecule of the present invention can eliminate antigens from plasma, the antigen-binding molecule does not need to have neutralizing activity by itself. However, it is more convenient to block the function of an antigen present in plasma by exerting neutralizing activity against the antigen until the antigen is taken up with an antigen binding molecule into Fcγ receptor expressing cells via Fcγ receptor mediated endocytosis.

Moreover, since the antigen-binding molecule provided by the present invention can accelerate the intracellular dissociation of an antigen that may be extracellularly associated with the antigen-binding molecule from the antigen-binding molecule, the antigen that has dissociated from the antigen-binding molecule inside the cell is degraded in the lysosome. Thus, the antigen-binding molecule itself need not have neutralizing activity. However, it is more convenient to block the function of an antigen present in plasma by neutralizing activity against the antigen until the antigen is taken up with an antigen-binding molecule into cells expressing the Fcγ receptor through Fcγ receptor-mediated endocytosis.

Moreover, since the antigen-binding molecule of the present invention may reduce the total antigen concentration or the plasma concentration of free antigen, the antigen-binding molecule itself need not have neutralizing activity. However, it is more convenient to block the function of an antigen present in plasma through neutralizing activity against the antigen until the antigen is taken up with an antigen binding molecule into Fcγ receptor expressing cells via Fcγ receptor mediated endocytosis.

Fcγ receptor

Fcγ receptor refers to a receptor capable of binding to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody and includes all members of the family of proteins essentially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), including the isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotype H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the FcγRIIIa isoform (including the V158 and F158 allotype) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotype); as well as unidentified human FcγRs, FcγR isoforms and their allotypes. However, the Fcγ receptor is not limited to these examples. Without limitation, FcγR includes human, mouse, rat, rabbit and monkey FcγR. FcγR can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as unidentified mouse FcγRs, FcγR isoforms, and allotypes thereof. Such preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16) and/or FcγRIIIb (CD16). The polynucleotide sequence and amino acid sequence of FcγRI are shown in SEQ ID NOs: 16 (NM_00056.3) and 17 (NP_000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIa (allotype H131) are shown in SEQ ID NOs: 18 (BC020823.1) and 19 (AAH20823.1) (allotype R131 is the sequence in which the amino acid at position 166 of SEQ ID NO: 19 is replaced by Arg ), respectively; the polynucleotide sequence and amino acid sequence of FcγIIB are shown in SEQ ID NOs: 20 (BC146678.1) and 21 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIa are shown in SEQ ID NOs: 22 (BC033678.1) and 23 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIb are shown in SEQ ID NOs: 24 (BC128562.1) and 25 (AAI28563.1), respectively (RefSeq accession number is shown in parentheses in each case). Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be assessed by ALPHA screening (Luminescence Proximity Enhanced Assay), BIACORE method based on Surface Plasma Resonance (SPR), and others (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to the FACS and ELISA formats described above.

Meanwhile, "Fc ligand" or "effector ligand" refers to a molecule, and preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. A molecule can originate from any organism. Binding of an Fc ligand to an Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, FcγR, FcγR, FcγR, FcRn, C1q and C3, mannan-binding lectin, mannose receptor, Staphylococcus A protein, Staphylococcus G protein, and viral FcγRs. Fc ligands also include the Fc receptor (FcRH) homologues (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to FcγR. Fc ligands also include unidentified molecules that bind to Fc.

In FcγRI (CD64), including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including the V158 and F158 allotypes) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotypes), the α-chain that binds with the Fc part of IgG, is associated with a common γ-chain with ITAM responsible for the transmission of an intracellular activating signal. Meanwhile, the cytoplasmic domain of FcγRII (CD32), including FcγRIIa isoforms (including H131 and R131 allotypes) and FcγRIIc, contains ITAM. These receptors are expressed on many cells such as macrophages, mast cells and antigen presenting cells. The activating signal transmitted when these receptors bind to the Fc portion of IgG leads to an increase in the phagocytic activity of macrophages, the production of inflammatory cytokines, degranulation of mast cells, and an increase in the function of antigen-presenting cells. Fcγ receptors having the ability to transmit an activating signal as described above are also referred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM responsible for inhibitory signaling. Binding between FcγRIIb and the B cell receptor (BCR) on B cells suppresses the activating signal from the BCR, resulting in suppression of antibody production via the BCR. Binding of FcγRIII and FcγRIIb on macrophages inhibits phagocytic activity and production of inflammatory cytokines. Fcγ receptors having the ability to transmit an inhibitory signal as described above are also referred to as inhibitory Fcγ receptor.

Fcγ receptor binding activity

The binding activity of the FcγR binding domain, which is included in the antigen-binding molecule of the present invention, to any of the human Fcγ receptors, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and/or FcγRIIIb, can be confirmed using the FACS and ELISA format described above, as well as the ALPHA screening (proximity-enhanced luminescence analysis), the BIACORE method, which is based on the surface plasma resonance (SPR) phenomenon, and the like. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The extracellular domain of the human Fcγ receptor can be used as the soluble antigen in these assays.

ALPHA screening is carried out using ALPHA technology based on the principle described below, where two types of beads are used: donor and acceptor. The luminescent signal is only detected when the molecules bound to the donor beads biologically interact with the molecules bound to the acceptor beads and the two types of beads are close to each other. Excited by a laser beam, the photosensitizer in the donor granules converts the oxygen around the granule into excited singlet oxygen. When singlet oxygen propagates among donor beads and reaches nearby acceptor beads, a chemiluminescent reaction is induced in the acceptor beads. This eventually results in the emission of light. When the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, the singlet oxygen produced by the donor beads does not reach the acceptor beads and the chemiluminescent reaction does not occur.

For example, a biotin-labeled antigen-binding molecule containing an Fc region is immobilized on donor beads, and an Fcγ receptor labeled with glutathione S-transferase (GST) is immobilized on acceptor beads. In the absence of an antigen-binding molecule containing a competing Fc region variant, the Fcγ receptor interacts with an antigen-binding molecule containing a native Fc region, resulting in signal induction in the 520-620 nm region. An antigen-binding molecule having an unlabeled variant Fc region competes with an antigen-binding molecule containing a native Fc region to interact with the Fcγ receptor. Relative binding affinity can be determined by quantifying the reduction in fluorescence due to competition. Methods for biotinylating antigen-binding molecules such as antibodies using sulfo-NHS-biotin and the like are known. Appropriate methods for adding a GST tag to the Fcγ receptor include methods that involve fusing polypeptides encoding Fcγ and GST in frame, expressing the fused gene using cells into which a vector to which the gene is operably linked is introduced, and then purifying using a column. with glutathione. The induced signal can preferably be analyzed, for example, by fitting it to a one-way competition model based on non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

When observing their interaction, one of the substances is immobilized as a ligand on a thin gold film on a sensor chip. When light is incident on the rear surface of the sensor chip, so that the overall reflection occurs on the contact surface between the thin gold film and the glass, the intensity of the reflected light is partially reduced in a certain area (SPR signal). The other of the substances, when an interaction is detected, is injected as an analyte onto the surface of the sensor chip. When an analyte binds to a ligand, the mass of the immobilized ligand molecule increases. A change in the refractive index causes a shift in the position of the SPR signal (on the other hand, dissociation causes the signal to shift back to its original position). In the Biacore system, the shift amount mentioned above (ie, mass change on the surface of the sensor chip) is plotted on the vertical axis, and thus the mass change over time is shown as measurement data (sensogram). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensogram curves, and affinity (KD) is determined from the ratio of these two constants. The BIACORE method preferably uses an inhibition assay. Examples of such an inhibition assay are described in Proc. Natl. Acad. Sci USA (2006) 103(11): 4005-4010.

Fcγ receptor binding domain

An Fcγ receptor binding domain having a higher Fcγ receptor binding activity than the Fc region of native human IgG, in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, can be obtained by modifying the native amino acid Fc regions of human IgG. Moreover, for the Fcγ receptor binding domain, any antigen binding domain structure previously described that is characterized in that it binds to the Fcγ receptor can be used. In such a case, the Fcγ receptor binding domain can be generated without the need for amino acid changes, or the affinity for the Fcγ receptor can be increased by making additional changes. Examples of such an Fcγ receptor binding domain include the Fab fragment of an antibody that binds 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, camelid-derived single domain antibody and single chain Fv antibody and FcγRI binding cyclic peptide described in FASEB J. 2009 Feb; 23(2):575-85. Whether the FcγR binding activity of the Fcγ receptor binding domain is higher than that of the Fc region of native human IgG in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain can be determined appropriately from using the method described in the binding activity section mentioned above.

Within the scope of the present invention, the human IgG Fc region is a useful example of a starting material Fcγ receptor binding domain. Within the scope of the present invention, "amino acid change" or "amino acid change" of an Fc region includes changing the amino acid sequence of a parental Fc region to a different amino acid sequence. As long as the modified version of the parent Fc region can bind to the human Fcγ receptor in the neutral pH range, any Fc region can be used as the parent Fc region. Moreover, the Fc region obtained by further modification of the already modified Fc region used as the original Fc region can also be preferably used as the Fc region of the present invention. "Original Fc region" may refer to the polypeptide itself, the composition containing the parent Fc region, or the amino acid sequence encoding the parent Fc region. The original Fc-region may contain the Fc-region of a known IgG antibody, obtained by recombination, briefly described in the section "Antibodies". The source of the original Fc regions is not limited, and they can be obtained from a human or any non-human organisms. Such organisms preferably include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, bovines, horses, camels, and organisms selected from non-human primates. In another embodiment, the original Fcγ receptor binding domains can also be derived from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. The parent Fc regions can preferably be derived from human IgG1; however, they are not limited to any particular IgG class. This means that a human IgG1, IgG2, IgG3 or IgG4 Fc region can be appropriately used as the parent Fc region, and within the scope of the present invention, it also means that an Fc region of an arbitrary class can preferably be used as the parent Fc region. or an IgG subclass derived from any of the organisms described above. Examples of naturally occurring variants or modified forms of IgG are described in published documents (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, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338); however, they are not limited to these examples.

Examples of changes include changes with one or more mutations, such as mutations by substituting different amino acid residues for the amino acids of the original Fc regions, by inserting one or more amino acid residues into the original Fc regions, or by deleting one or more amino acids from the original Fc region. Preferably, the amino acid sequences of the altered Fc regions include at least a portion of the amino acid sequence of the non-native Fc region. Such variants necessarily have less than 100% sequence identity or similarity to their parent Fc region. In a preferred embodiment, the variants have an amino acid sequence identity or similarity of from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to less than 100%, even more preferably, from about 90% to less than 100%, and even more preferably from about 95% to less than 100% with the amino acid sequence of their parent Fc region. In a non-limiting embodiment of the present invention, at least one amino acid differs between the modified Fc region of the present invention and its original Fc region. The amino acid difference between the modified Fc region of the present invention and its original Fc region can also preferably be indicated based on the amino acid differences at the specific amino acid positions described above, according to the EU numbering system.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap PCR can be appropriately used to modify the amino acids of Fc regions. Moreover, various known methods can also be used as an amino acid modification method to replace amino acids with amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci U.S.A. (2003) 100(11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which an amber suppressor tRNA that is complementary to a UAG codon (an amber codon) which is a stop codon can be appropriately used is linked to a non-natural amino acid.

The Fc region having Fcγ receptor binding activity in the neutral pH range contained in the antigen-binding molecules of the present invention can be obtained by any method, however, in particular, the Fc region having Fcγ receptor binding activity in the neutral pH range , can be obtained by altering the amino acids of the human IgG immunoglobulin used as the starting Fc region. Preferred IgG immunoglobulin Fc regions to be modified include, for example, human IgG Fc regions (IgG1, IgG2, IgG3 or IgG4 and variants thereof). Fc regions of IgG include mutants naturally formed from them. A number of allotypic sequences due to genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, for the Fc regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of them can be used within the scope of this inventions. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356-358 (EU numbering) can be either DEL or EEM.

Amino acids at any positions can be changed to other amino acids, provided that the Fc region has Fcγ receptor binding activity in the neutral pH range, or its Fcγ receptor binding activity in the neutral range can be enhanced. When the antigen binding molecule contains a human IgG1 Fc region, it is preferable to make changes that result in increased Fcγ receptor binding in the neutral pH range compared to the binding activity of the original human IgG1 Fc region. Amino acid changes to enhance Fcγ receptor binding activity in the neutral pH range are described, for example, in WO 2007/024249, WO 2007/021841, WO 2006/031370, WO 2000/042072, WO 2004/029207, WO 2004/099249, WO 2006/105338, WO 2007/041635, WO 2008/092117, WO 2005/070963, WO 2006/020114, WO 2006/116260 and WO 2006/023403.

Examples of such amino acids that may be modified include at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266 , 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294 , 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329 , 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 in accordance with EU numbering. Altering these amino acids enhances binding of the Fcγ receptor to the Fc region of IgG immunoglobulin in the neutral pH range.

Particularly preferred modifications for use in the present invention include the following modifications:

amino acid at position 221 on either Lys or Tyr;

amino acid at position 222 on any of Phe, Trp, Glu, and Tyr;

amino acid at position 223 on any of Phe, Trp, Glu and Lys;

amino acid at position 224 on any of Phe, Trp, Glu, and Tyr;

amino acid at position 225 on any of Glu, Lys and Trp;

amino acid at position 227 on any of Glu, Gly, Lys and Tyr;

amino acid at position 228 on any of Glu, Gly, Lys and Tyr;

amino acid at position 230 on any of Ala, Glu, Gly and Tyr;

amino acid at position 231 on any of Glu, Gly, Lys, Pro and Tyr;

amino acid at position 232 on any of Glu, Gly, Lys and Tyr;

amino acid at position 233 on any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 234 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 235 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 236 on any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 237 on any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 238 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 239 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp and Tyr;

amino acid at position 240 on any of Ala, Ile, Met and Thr;

amino acid at position 241 on any of Asp, Glu, Leu, Arg, Trp and Tyr;

amino acid at position 243 on any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr;

amino acid at position 244 on His;

amino acid at position 245 on Ala;

amino acid at position 246 on any of Asp, Glu, His, and Tyr;

amino acid at position 247 on any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr;

amino acid at position 249 on any of Glu, His, Gln and Tyr;

amino acid at position 250 on either Glu or Gln;

amino acid at position 251 on Phe;

amino acid at position 254 on any of Phe, Met and Tyr;

amino acid at position 255 on any of Glu, Leu and Tyr;

amino acid at position 256 on any of Ala, Met and Pro;

amino acid at position 258 on any of Asp, Glu, His, Ser, and Tyr;

amino acid at position 260 on any of Asp, Glu, His, and Tyr;

amino acid at position 262 on any of Ala, Glu, Phe, Ile, and Thr;

amino acid at position 263 on any of Ala, Ile, Met and Thr;

amino acid at position 264 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr;

amino acid at position 265 on any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp and Tyr;

amino acid at position 266 on any of Ala, Ile, Met and Thr;

amino acid at position 267 on any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr;

amino acid at position 268 on any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val and Trp;

amino acid at position 269 on any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 270 on any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr;

amino acid at position 271 on any of Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 272 on any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 273 on either Phe or Ile;

amino acid at position 274 on any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 275 on either Leu or Trp;

amino acid at position 276 on any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 278 on any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp;

amino acid at position 279 on Ala;

amino acid at position 280 on any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr;

amino acid at position 281 on any of Asp, Lys, Pro and Tyr;

amino acid at position 282 on any of Glu, Gly, Lys, Pro and Tyr;

amino acid at position 283 on any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr;

amino acid at position 284 on any of Asp, Glu, Leu, Asn, Thr and Tyr;

amino acid at position 285 on any of Asp, Glu, Lys, Gln, Trp and Tyr;

amino acid at position 286 on any of Glu, Gly, Pro and Tyr;

amino acid at position 288 on any of Asn, Asp, Glu and Tyr;

amino acid at position 290 on any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr;

amino acid at position 291 on any of Asp, Glu, Gly, His, Ile, Gln and Thr;

amino acid at position 292 on any of Ala, Asp, Glu, Pro, Thr and Tyr;

amino acid at position 293 on any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 294 on any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 295 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;

amino acid at position 296 on any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val;

amino acid at position 297 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 298 on any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr;

amino acid at position 299 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr;

amino acid at position 300 on any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp;

amino acid at position 301 on any of Asp, Glu, His, and Tyr;

amino acid at position 302 on Ile;

amino acid at position 303 on any of Asp, Gly and Tyr;

amino acid at position 304 on any of Asp, His, Leu, Asn, and Thr;

amino acid at position 305 on any of Glu, Ile, Thr and Tyr;

amino acid at position 311 on any of Ala, Asp, Asn, Thr, Val and Tyr;

amino acid at position 313 on Phe;

amino acid at position 315 on Leu;

amino acid at position 317 on either Glu or Gln;

amino acid at position 318 on any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr;

amino acid at position 320 on any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr;

amino acid at position 322 on any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr;

amino acid at position 323 on Ile;

amino acid at position 324 on any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr;

amino acid at position 325 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 326 on any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr;

amino acid at position 327 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr;

amino acid at position 328 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 329 on any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 330 on any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr;

amino acid at position 331 on any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr;

amino acid at position 332 on 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 on any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr;

amino acid at position 334 on any of Ala, Glu, Phe, Ile, Leu, Pro, and Thr;

amino acid at position 335 on any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr;

amino acid at position 336 on any of Glu, Lys and Tyr;

amino acid at position 337 on any of Glu, His and Asn;

amino acid at position 339 on any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr;

amino acid at position 376 on either Ala or Val;

amino acid at position 377 on either Gly or Lys;

amino acid at position 378 on Asp;

amino acid at position 379 on Asn;

amino acid at position 380 on any of Ala, Asn and Ser;

amino acid at position 382 on either Ala or Ile;

amino acid at position 385 on Glu;

amino acid at position 392 on Thr;

amino acid at position 396 on Leu;

amino acid at position 421 on Lys;

amino acid at position 427 on Asn;

amino acid at position 428 on either Phe or Leu;

amino acid at position 429 on Met;

amino acid at position 434 on Trp;

amino acid at position 436 on Ile; and

amino acid at position 440 on any of Gly, His, Ile, Leu and Tyr,

according to the EU numbering in the Fc region.

The number of amino acids that are changed is not specifically limited. An amino acid may be changed at only one position, or amino acids may be changed at two or more positions. Examples of combinations of amino acid changes at two or more positions include those shown in Table 5 (Tables 5-1 to 5-3).

For the pH conditions for measuring the binding activity of the Fcγ receptor binding domain contained in the antigen binding molecule of the present invention and the Fcγ receptor, conditions in the acidic pH range or in the neutral pH range can be suitably used. The neutral pH range as conditions for measuring the binding activity of the Fcγ receptor binding domain contained in the antigen-binding molecule of the present invention and the Fcγ receptor is generally indicated as pH 6.7 to pH 10.0. Preferably, it is a range indicated by arbitrary pH values from pH 7.0 to pH 8.0; and preferably it is selected from pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 and pH 8.0; and especially preferably it is a pH value of 7.4, which is close to the plasma (blood) pH in vivo. Within the scope of the present invention, an acidic pH range as a condition for the binding activity of the Fcγ receptor binding domain contained in the antigen binding molecule of the present invention and the Fcγ receptor is generally indicated as pH 4.0 to pH 6.5. Preferably, it is indicated as pH 5.5 to pH 6.5, and particularly preferably it is indicated as pH 5.8 to pH 6.0, which is close to the pH value in the early endosome in vivo. With regard to the temperature used as measurement conditions, the binding affinity between the Fcγ receptor binding domain and the human Fcγ receptor can be assessed at any temperature from 10°C to 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between the human Fcγ receptor binding domain and the Fcγ receptor. More preferably, any temperature between 20° C. and 35° C., such as any of 20° C., 21° C., 22° C., 23° C. C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C. The temperature of 25°C is a non-limiting example in one embodiment of the present invention.

In the context of the present invention, "the binding activity of the Fcγ receptor binding domain for an Fcγ receptor is higher than the binding activity of the native Fc region for an activating Fcγ receptor" means that the binding activity of the Fcγ receptor binding domain for any of Fc(human) receptors of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and/or FcγRIIIb is higher than the binding activity of the native Fcγ receptor binding domain for these human Fcγ receptors.

For example, it refers to a binding activity of an antigen-binding molecule comprising an Fcγ receptor binding domain that is 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more. more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more more, 185% or more, 190% or more, 195% or more, twice or more, 2.5 times or more, three times or more, 3.5 times or more, four times or more more, 4.5 times or more, five times or more, 7.5 times or more, ten times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more the binding activity of an antigen-binding molecule comprising the Fc region of native human IgG, which is used as a control, based on the above method of analysis. For a native Fcγ receptor-binding domain, an Fcγ receptor-binding domain as a starting material can be used, and a native antibody Fcγ receptor-binding domain belonging to the same subclass can also be used.

Within the scope of the present invention, in particular, a native human IgG Fc region in which the sugar chain linked to the amino acid at position 297 (EU numbering) is a fucose-containing sugar chain is suitably used as a native human IgG Fc region to be use as a control. Whether the sugar chain linked to the amino acid at position 297 (EU numbering) of a fucose-containing sugar chain can be determined using the method described in Non-Patent Document 24. For example, such a method as described below provides a determination of whether the chain is sugars linked to a native human IgG Fc region containing fucose by a sugar chain. Through the reaction of N-glycosidase F (Roche diagnostics) with the native human IgG to be tested, the sugar chain is dissociated from the native human IgG to be tested (Weitzhandler et al. (J. Pharma. Sciences (1994) 83, 12, 1670-1675) ). Next, the concentrated thickened material of the reaction solution from which proteins have been removed by reaction with ethanol (Schenk et al. (J. Clin. Investigation (2001) 108 (11) 1687-1695)) is fluorescently labeled with 2-aminopyridine (Bigge et al. (Anal. Biochem. (1995) 230 (2) 229-238)). The fluorescently labeled 2-AB modified sugar chain obtained by removing the reagent by solid phase extraction using a cellulose cassette is analyzed by normal phase chromatography. Observation of a detectable chromatogram peak provides a determination of whether the sugar chain associated with native human IgG Fc region is a fucose-containing sugar chain. Non-limiting examples of such a native human IgG Fc region in which the sugar chain linked to the amino acid at position 297 (EU numbering) is a fucose-containing sugar chain include Fc regions incorporated into antibodies produced by expression in CHO cells such as CHO-K1 (American Type Culture Collection, ATCC No. CRL-61) or DXB11 (American Type Culture Collection, ATCC No. CRL-11397), genes encoding antibodies containing a native human IgG Fc region. Using the Fcγ receptor binding activity of the Fc regions included in such antibodies as controls, the Fcγ receptor binding activity of the Fc regions of the present invention was compared. This provides a determination of whether the Fcγ receptor binding domain of the present invention has a higher Fcγ receptor binding activity than the native human IgG Fc region in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain. . Moreover, the amount of fucose associated with the sugar chain included compared to the Fc regions can be compared by determining the amount of fucose in the chain associated with the Fc region included in these antibodies and the amount of fucose in the sugar chain associated with Fc- areas of the present invention using such methods as mentioned above.

As an antigen-binding molecule containing an Fc region of a native antibody of the same subclass as used as a control, antigen-binding molecules having an Fc region of a monoclonal IgG antibody can be suitably used. The structures of these Fc regions are shown in SEQ ID NO: 11 (A added to the N-terminus of RefSeq accession number AAC82527.1), SEQ ID NO: 12 (A added to the N-terminus of RefSeq accession number AAB59393.1) , SEQ ID NO: 13 (RefSeq accession number CAA27268.1) and SEQ ID NO: 14 (A added at the N-terminus of RefSeq accession number AAB59394.1). Moreover, when an antigen-binding molecule having an Fc region obtained by modifying an antibody of a particular isotype used as the original Fc region is used as a test substance, an antigen-binding molecule having an Fc region of an IgG monoclonal antibody of that isotype is used as a control for confirming the effects on the activity of binding to Fcγ receptors of an antigen-binding molecule having an altered Fc region. Thus, antigen-binding molecules containing an Fc region confirmed to have high Fcγ receptor binding activity as described above are selected accordingly.

Examples of Fcγ receptor binding domains suitable for use in the present invention include Fcγ receptor binding domains with the property of having higher binding activity for certain Fcγ receptors than other Fcγ receptors (Fcγ receptor binding domains with selective binding activity). Fcγ receptor). When an antibody is used as an antigen binding molecule (when an Fc region is used as an Fcγ receptor binding domain), since one antibody molecule can only bind to one Fcγ receptor, one antigen binding molecule that has bound to an inhibitory Fcγ receptor cannot bind with another activating FcγR, and one antigen-binding molecule that has bound to an activating Fcγ receptor cannot bind to either another activating Fcγ receptor or an inhibitory Fcγ receptor.

As described above, suitable examples of an activating Fcγ receptor are FcγRI (CD64), including FcγRIa, FcγRIb and FcγRIc and FcγRIII (CD16), including the FcγRIIIa isoforms (including the V158 and F158 allotypes) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotypes). ). FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) is a useful example of an inhibitory Fcγ receptor.

FcγR binding domain with selective Fcγ receptor binding activity

Whether the FcγR binding domain of the present invention has selective binding activity can be confirmed by comparing the binding activity to the corresponding Fcγ receptors as determined by the method described in the Fcγ receptor binding activity section above. An FcγR binding domain with higher binding activity to inhibitory Fcγ receptors than to activating Fcγ receptors can be used as a selective FcγR binding domain included in an antigen binding molecule of the present invention. In one non-limiting embodiment, an FcγR binding domain with higher binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) than to an activating Fcγ receptor selected from the group consisting of FcγRI(CD64) including FcγRIa, FcγRIb, FcγRIc , FcγRIII(CD16), including the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) and FcγRII (CD32), including isoforms FcγRIIa and FcγRIIc (including allotypes H131 and R131), can be used as a selective FcγR-binding domain included in the antigen-binding molecule of the present invention. Moreover, in a non-limiting embodiment of the present invention, an FcγR binding domain with higher binding activity to FcγRIIb-1 and/or FcγRIIb-2 than to FcγRIa, FcγRIb and FcγRIc, FcγRIIIa, including the V158 allotype, FcγRIIIa, including the F158 allotype, FcγRIIIb , including the FcγRIIIb-NA1 allotype, FcγRIIIb, including the FcγRIIIb-NA2 allotype, FcγRIIa, including the H131 allotype, FcγRIIa, including the R131 and/or FcγRIIc allotype, can be used as a selective FcγR-binding domain included in the antigen-binding molecule provided herein inventions. Whether the FcγR binding domain to be investigated has selective binding activity to Fcγ receptors can be determined by comparing the value (ratio) obtained by dividing the KD values of the FcγR binding domain with respect to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including the V158 allotype , FcγRIIIa, including the F158 allotype, FcγRIIIb, including the FcγRIIIb-NA1 allotype, FcγRIIIb, including the FcγRIIIb-NA2 allotype, FcγRIIa, including the H131 allotype, FcγRIIa, including the R131 and/or FcγRIIc allotype, by KD values for FcγRIIb-1 and/or FcγRIIb -2, where the KD values are determined by the method described in the Fcγ receptor binding activity section mentioned above or, more specifically, by comparing the FcγR selectivity values presented in Equation 1.

[Equation 1]

Selectivity index FcγR = KD value for activating FcγR/KD value for inhibitory FcγR

In Equation 1 above, "activating FcγR" refers to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb-NA1 allotype, FcγRIIIb including the FcγRIIIb-NA2 allotype, FcγRIIa including allotype H131, FcγRIIa, including allotype R131 and/or FcγRIIc and inhibitory FcγR refers to FcγRIIb-1 and/or FcγRIIb-2. Although the activating FcγR and inhibitory FcγR used to measure the KD value may be selected from any combination, in a non-limiting embodiment, the value (ratio) obtained by dividing the KD value for FcγRIIa, including the H131 allotype, by the KD value for FcγRIIb-1 and /or FcγRIIb-2.

For example, FcγR selectivity values are 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1 ,9 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more , 95 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more , 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 42 0 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 520 or more, 540 or more, 560 or more, 580 or more more, 600 or more, 620 or more, 640 or more, 660 or more, 680 or more, 700 or more, 720 or more, 740 or more, 760 or more, 780 or more, 800 or more, 820 or more, 840 or more, 860 or more, 880 or more, 900 or more, 920 or more, 940 or more, 960 or more, 980 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, or 100000 or more.

A non-limiting embodiment of a selective FcγR binding domain in an antigen binding molecule of the present invention includes, for example, Fc regions obtained by modifying an FcγR binding domain included in an Fc region (an IgG class Fc region refers to the region from the cysteine at position 226 ( EU numbering) up to the C-terminus or from the proline at position 230 (EU numbering) to the C-terminus, but not limited to), which is part of the constant region presented as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), human IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17). An example of a method for obtaining modified Fc regions includes the method described in the section on amino acid changes mentioned above. Examples of such altered Fc regions include the Fc region in which the amino acid at position 238 (EU numbering) is Asp, or the Fc region in which the amino acid at position 328 (EU numbering) is Glu in human IgG (IgG1, IgG2 , IgG3 or IgG4). the Fc region in which the amino acid at position 238 (EU numbering) is Asp, or the Fc region in which the amino acid at position 328 (EU numbering) is Glu in human IgG (IgG1, IgG2, IgG3 or IgG4), and antigen-binding molecules containing such an Fc region exhibit higher binding activity to FcγRIIb-1 and/or FcγRIIb-2 than to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including the V158 allotype, FcγRIIIa, including the F158 allotype, FcγRIIIb, including the FcγRIIIb allotype -NA1, FcγRIIIb, including the FcγRIIIb allotype-NA2, FcγRIIa, including the H131 allotype, FcγRIIa, including the R131 and/or FcγRIIc allotype.

The constant regions containing a selective FcγR binding domain that are included in the antigen binding molecules of the present invention and the antigen binding molecules containing such a constant region may also be Fc regions and antigen binding molecules containing an Fc region that retains or exhibits a reduced activating FcγR binding activity (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including the V158 allotype, FcγRIIIa, including the F158 allotype, FcγRIIIb, including the FcγRIIIb-NA1 allotype, FcγRIIIb, including the FcγRIIIb-NA2 allotype, FcγRIIa, including the H131 allotype, FcγRIIa, including the H131 allotype, R131 and/or FcγRIIc) compared to the Fc region (IgG class Fc region refers to the region from cysteine at position 226 (EU numbering) to the C-terminus or from proline at position 230 (EU numbering) to the C-terminus, but not limited to) constituting a part of human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17) (hereinafter referred to as Fc- wild-type region) and an antigen-binding molecule containing such a wild-type Fc region.

Compared with the wild-type Fc region and the antigen-binding molecule containing the wild-type Fc region, the degree of the above-mentioned reduction in the activity of binding to the activating FcγR of the Fc region containing the selective FcγR-binding domain included in the antigen-binding molecule of the present invention and the antigen-binding molecule containing such an Fc region is, for example, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less , 91% or less, 90% or less, 8 8% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less less than, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less less than, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less less, 5% or less, 4% or less, 3% or m Less than, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05 % or less, 0.01% or less, or 0.005% or less.

Fc regions containing a selective FcγR binding domain and constant regions containing such an Fc region and antigen-binding molecules containing such a constant region, which are included in the antigen-binding molecules of the present invention, may also be Fc regions and antigen-binding molecules, containing such an Fc region that exhibit enhanced binding activity to an inhibitory FcγR (FcγRIIb-1 and/or FcγRIIb-2) compared to an Fc region (an IgG class Fc region refers to the region from cysteine at position 226 (EU numbering) to C-terminus or from the proline at position 230 (EU numbering) to the C-terminus), constituting part of the constant region present as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17) of a human (hereinafter referred to as a wild-type Fc region), and an antigen-binding molecule containing such a wild-type Fc region.

Compared with the wild-type Fc region and the antigen-binding molecule containing the wild-type Fc region, the degree of the above enhancement of the binding activity to the inhibitory FcγR of the Fc region containing the selective FcγR-binding domain included in the antigen-binding molecule of the present invention and the antigen-binding molecule containing such an Fc region is, for example, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more , 109% or more, 110% or more, 112% or more, 114% or more, 116% or more, 118% or more, 120% or more, 122% or more, 124% or more, 126% or more , 128% or more, 130% or more, 132% or more, 134% or more, 136% or more, 138% or more, 140% or more, 142% or more, 144% or more, 146% or more , 148% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more , one 95% or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1,000 times or more, 10,000 times or more, or 100,000 times or more.

Moreover, an Fc region containing a selective FcγR binding domain included in an antigen-binding molecule of the present invention and an antigen-binding molecule containing such an Fc region may be an Fc region and an antigen-binding molecule containing such an Fc region that retains or shows reduced binding activity to an activating FcγR (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including the V158 allotype, FcγRIIIa, including the F158 allotype, FcγRIIIb, including the FcγRIIIb-NA1 allotype, FcγRIIIb, including the FcγRIIIb-NA2 allotype, FcγRIIa, including the R131 allotype, FcγRIIa, including the H131 allotype, and/or FcγRIIc) compared to the Fc region (the Fc region of the IgG class refers, for example, to the region from cysteine at position 226 (EU numbering) to the C-terminus or from proline at position 230 (EU numbering) to the C-terminus , but not limited to) constituting part of a constant region present as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17)human (hereinafter referred to as a wild-type Fc region), and an antigen-binding molecule containing such a wild-type Fc region; and shows enhanced binding activity to an inhibitory FcγR (FcγRIIb-1 and/or FcγRIIb-2) compared to the Fc region (an IgG class Fc region refers, for example, to the region from cysteine at position 226 (EU numbering) to the C-terminus or from the proline at position 230 (EU numbering) to the C-terminus), constituting part of the constant region present as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17) of a human (hereinafter referred to as a wild-type Fc region), and an antigen-binding molecule containing such a wild-type Fc region.

Moreover, an Fc region containing a selective FcγR binding domain included in an antigen-binding molecule of the present invention and an antigen-binding molecule containing such an Fc region may be an Fc region and an antigen-binding molecule containing such as an Fc region with more a high degree of enhancement of binding activity to the inhibitory Fcγ receptor (FcγRIIb-1 and/or FcγRIIb-2) than to the activating Fcγ receptor (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb- allotype NA1, FcγRIIIb including the FcγRIIIb-NA2 allotype, FcγRIIa including the H131 allotype, FcγRIIa including the R131 allotype), compared to the Fc region (the Fc region of the IgG class refers, for example, to the region from cysteine at position 226 (EU numbering) to C- end or from the proline at position 230 (EU numbering) to the C-terminus), constituting part of the constant region present as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15 ), IgG3 (SEQ ID NO: 16) or IgG4 (SEQ ID NO: 17) of a human (hereinafter referred to as a wild-type Fc region) and an antigen-binding molecule containing such a wild-type Fc region.

Within the scope of the present invention, at least one other Fc region change can be added to the Fc region in which the amino acid at position 238 (EU numbering) is Asp and the Fc region in which the amino acid at position 328 (EU numbering) is is Glu according to the embodiments and the like described in the amino acid changes section mentioned above. In addition to these changes, additional changes can also be added. Additional changes can be selected from any substitutions, deletions and modifications of amino acids and combinations thereof. For example, changes can be added that increase FcγRIIb binding activity while maintaining or decreasing FcγRIIa (H-type) and FcγRIIa (R-type) binding activity. The addition of these changes increases the selectivity of binding to FcγRIIb relative to FcγRIIa.

Among them, changes that increase the selectivity for FcγRIIb over FcγRIIa (R-type) are favorable, and changes that increase the binding selectivity for FcγRIIb over FcγRIIa (H-type) are more favorable. Examples of preferred amino acid substitutions for such changes include: change by changing Gly at position 237 (EU numbering) to Trp; change by replacing Gly at position 237 (EU numbering) with Phe; change by replacing Pro in position 238 (EU numbering) with Phe; change by replacing Asn in position 325 (EU numbering) with Met; change by replacing Ser at position 267 (EU numbering) with Ile; change by replacing Leu in regulation 328 (EU numbering) with Asp; change by replacing Ser at position 267 (EU numbering) with Val; change by replacing Leu in regulation 328 (EU numbering) with Trp; change by replacing Ser at position 267 (EU numbering) with Gln; change by replacing Ser at position 267 (EU numbering) with Met; change by replacing Gly at position 236 (EU numbering) with Asp; change by replacing Ala in regulation 327 (EU numbering) with Asn; change by replacing Asn at position 325 (EU numbering) with Ser; change by replacing Leu in regulation 235 (EU numbering) with Tyr; change by replacing Val at position 266 (EU numbering) with Met; change by replacing Leu in regulation 328 (EU numbering) with Tyr; change by replacing Leu in regulation 235 (EU numbering) with Trp; change by replacing Leu at position 235 (EU numbering) with Phe; change by replacing Ser at position 239 (EU numbering) with Gly; change by replacing Ala at position 327 (EU numbering) with Glu; change by replacing Ala at position 327 (EU numbering) with Gly; change by replacing Pro in position 238 (EU numbering) with Leu; change by replacing Ser at position 239 (EU numbering) with Leu; change by replacing Leu at position 328 (EU numbering) with Thr; change by replacing Leu at position 328 (EU numbering) with Ser; change by replacing Leu in regulation 328 (EU numbering) with Met; change by replacing Pro in regulation 331 (EU numbering) with Trp; change by replacing Pro in regulation 331 (EU numbering) with Tyr; change by replacing Pro in regulation 331 (EU numbering) with Phe; change by replacing Ala in regulation 327 (EU numbering) with Asp; change by replacing Leu at position 328 (EU numbering) with Phe; change by replacing Pro in position 271 (EU numbering) with Leu; change by replacing Ser at position 267 (EU numbering) with Glu; change by replacing Leu in regulation 328 (EU numbering) with Ala; change by replacing Leu in regulation 328 (EU numbering) with Ile; change by replacing Leu in regulation 328 (EU numbering) with Gln; change by replacing Leu at position 328 (EU numbering) with Val; change by replacing Lys at position 326 (EU numbering) with Trp; change by replacing Lys at position 334 (EU numbering) with Arg; change by replacing His at position 268 (EU numbering) with Gly; change by replacing His at position 268 (EU numbering) with Asn; change by replacing Ser at position 324 (EU numbering) with Val; change by replacing Val at position 266 (EU numbering) with Leu; change by replacing Pro in position 271 (EU numbering) with Gly; change by replacing Ile at position 332 (EU numbering) with Phe; change by replacing Ser at position 324 (EU numbering) with Ile; change by replacing Glu at position 333 (EU numbering) with Pro; change by replacing Tyr at position 300 (EU numbering) with Asp; change by replacing Ser at position 337 (EU numbering) with Asp; change by replacing Tyr at position 300 (EU numbering) with Gln; change by replacing Thr at position 335 (EU numbering) with Asp; change by replacing Ser at position 239 (EU numbering) with Asn; change by replacing Lys at position 326 (EU numbering) with Leu; change by replacing Lys at position 326 (EU numbering) with Ile; change by replacing Ser at position 239 (EU numbering) with Glu; change by replacing Lys at position 326 (EU numbering) with Phe; change by replacing Lys at position 326 (EU numbering) with Val; change by replacing Lys at position 326 (EU numbering) with Tyr; change by replacing Ser at position 267 (EU numbering) with Asp; change by replacing Lys at position 326 (EU numbering) with Pro; change by replacing Lys at position 326 (EU numbering) with His; change by replacing Lys at position 334 (EU numbering) with Ala; change by replacing Lys at position 334 (EU numbering) with Trp; change by replacing His at position 268 (EU numbering) with Gln; change by replacing Lys at position 326 (EU numbering) with Gln; change by replacing Lys at position 326 (EU numbering) with Glu; change by replacing Lys at position 326 (EU numbering) with Met; change by replacing Val at position 266 (EU numbering) with Ile; change by replacing Lys at position 334 (EU numbering) with Glu; change by replacing Tyr at position 300 (EU numbering) with Glu; change by replacing Lys at position 334 (EU numbering) with Met; change by replacing Lys at position 334 (EU numbering) with Val; change by replacing Lys at position 334 (EU numbering) with Thr; change by replacing Lys at position 334 (EU numbering) with Ser; change by replacing Lys at position 334 (EU numbering) with His; change by replacing Lys at position 334 (EU numbering) with Phe; change by replacing Lys at position 334 (EU numbering) with Gln; change by replacing Lys at position 334 (EU numbering) with Pro; change by replacing Lys at position 334 (EU numbering) with Tyr; change by replacing Lys at position 334 (EU numbering) with Ile; change by replacing Gln in regulation 295 (EU numbering) with Leu; change by replacing Lys at position 334 (EU numbering) with Leu; change by replacing Lys at position 334 (EU numbering) with Asn; change by replacing His at position 268 (EU numbering) with Ala; change by replacing Ser at position 239 (EU numbering) with Asp; change by replacing Ser at position 267 (EU numbering) with Ala; change by replacing Leu in regulation 234 (EU numbering) with Trp; change by replacing Leu in regulation 234 (EU numbering) with Tyr; change by replacing Gly at position 237 (EU numbering) with Ala; change by replacing Gly at position 237 (EU numbering) with Asp; change by replacing Gly at position 237 (EU numbering) with Glu; change by replacing Gly at position 237 (EU numbering) with Leu; change by replacing Gly at position 237 (EU numbering) with Met; change by replacing Gly at position 237 (EU numbering) with Tyr; change by replacing Ala at position 330 (EU numbering) with Lys; change by replacing Ala at position 330 (EU numbering) with Arg; change by replacing Glu at position 233 (EU numbering) with Asp; change by replacing His at position 268 (EU numbering) with Asp; change by replacing His at position 268 (EU numbering) with Glu; change by replacing Lys at position 326 (EU numbering) with Asp; change by replacing Lys at position 326 (EU numbering) with Ser; change by replacing Lys at position 326 (EU numbering) with Thr; change by replacing Val at position 323 (EU numbering) with Ile; change by replacing Val at position 323 (EU numbering) with Leu; change by replacing Val at position 323 (EU numbering) with Met; change by replacing Tyr in regulation 296 (EU numbering) with Asp; change by replacing Lys at position 326 (EU numbering) with Ala; change by replacing Lys at position 326 (EU numbering) with Asn; and change by replacing Ala at position 330 (EU numbering) with Met.

Favorable amino acid substitutions among these changes are, for example, a change by changing Gly at position 237 (EU numbering) to Trp; change by replacing Gly at position 237 (EU numbering) with Phe; change by replacing Ser at position 267 (EU numbering) with Val; change by replacing Ser at position 267 (EU numbering) with Gln; change by replacing His at position 268 (EU numbering) with Asn; change by replacing Pro in position 271 (EU numbering) with Gly; change by replacing Lys at position 326 (EU numbering) with Leu; change by replacing Lys at position 326 (EU numbering) with Gin; change by replacing Lys at position 326 (EU numbering) with Glu; change by replacing Lys at position 326 (EU numbering) with Met; change by replacing Ser at position 239 (EU numbering) with Asp; change by replacing Ser at position 267 (EU numbering) with Ala; change by replacing Leu in regulation 234 (EU numbering) with Trp; change by replacing Leu in regulation 234 (EU numbering) with Tyr; change by replacing Gly at position 237 (EU numbering) with Ala; change by replacing Gly at position 237 (EU numbering) with Asp; change by replacing Gly at position 237 (EU numbering) with Glu; change by replacing Gly at position 237 (EU numbering) with Leu; change by replacing Gly at position 237 (EU numbering) with Met; change by replacing Gly at position 237 (EU numbering) with Tyr; change by replacing Ala at position 330 (EU numbering) with Lys; change by replacing Ala at position 330 (EU numbering) with Arg; change by replacing Glu at position 233 (EU numbering) with Asp; change by replacing His at position 268 (EU numbering) with Asp; change by replacing His at position 268 (EU numbering) with Glu; change by replacing Lys at position 326 (EU numbering) with Asp; change by replacing Lys at position 326 (EU numbering) with Ser; change by replacing Lys at position 326 (EU numbering) with Thr; change by replacing Val at position 323 (EU numbering) with Ile; change by replacing Val at position 323 (EU numbering) with Leu; change by replacing Val at position 323 (EU numbering) with Met; change by replacing Tyr in regulation 296 (EU numbering) with Asp; change by replacing Lys at position 326 (EU numbering) with Ala; change by replacing Lys at position 326 (EU numbering) with Asn; and change by replacing Ala at position 330 (EU numbering) with Met.

The change mentioned above may be a change at one position, or changes at two or more positions may be combined. Suitable examples of such changes are those described in tables 14-15, tables 17-24 and tables 26-28.

Fc region obtained by modifying the FcγR binding domain included in the Fc region represented as IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16) or IgG4 ( SEQ ID NO: 17) of a human may be exemplified by another non-limiting embodiment of the selective FcγR binding domain included in the antigen binding molecules of the present invention. The method for obtaining modified Fc regions is, for example, the method described in the section on amino acid changes mentioned above. Examples of such altered Fc regions include an Fc region in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly in human IgG (IgG1, IgG2, IgG3 or IgG4) . Fc region in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly in human IgG (IgG1, IgG2, IgG3 or IgG4), and antigen-binding molecules containing such Fc region exhibit higher binding activity to FcγRIIb-1 and/or FcγRIIb-2 than to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb-NAl allotype, FcγRIIIb including the FcγRIIIb- allotype NA2, FcγRIIa including H131 allotype, FcγRIIa including R131 and/or FcγRIIc allotype.

Within the scope of the present invention, at least one other Fc region change can be added to an Fc region in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly, by means of embodiments and the like described in the above section on amino acid changes. In addition to these changes, additional changes can also be added. Additional changes may be selected from any of the substitutions, deletions and modifications of amino acids and combinations thereof. For example, changes can be added that retain or reduce binding activity to activating Fcγ receptors (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb-NAl allotype, FcγRIIIb including the FcγRIIIb-NA2 allotype, FcγRIIIa including allotype H131, FcγRIIa including allotype R131). Modifications can be added that increase binding activity to inhibitory Fcγ receptors (FcγRIIb-1 and/or FcγRIIb-2) while maintaining or decreasing binding activity to FcγRIIa (H-type) and FcγRIIa (R-type). Moreover, it is also possible to add changes where the degree of enhancement of binding activity to inhibitory Fcγ receptors (FcγRIIb-1 and/or FcγRIIb-2) is higher than the degree of enhancement of binding activity to activating Fcγ receptors (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb-NA1 allotype, FcγRIIIb including the FcγRIIIb-NA2 allotype, FcγRIIa including the H131 allotype, FcγRIIa including the R131 allotype). The addition of these changes increases the selectivity of binding to FcγRIIb relative to FcγRIIa.

An example of a non-limiting embodiment of an altered Fc region containing a selective FcγR binding domain includes an altered Fc region in which one or more of positions 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296 , 326, 327, 330, 331, 332, 333 and 396 (EU numbering) are replaced in the Fc region in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly in Human IgG (IgG1, IgG2, IgG3 or IgG4).

In addition, an example of a non-limiting embodiment of an altered Fc region containing a selective FcγR binding domain is an altered Fc region containing any one or more of

Asp at amino acid position 233,

Tyr at amino acid position 234,

Asp at amino acid position 237,

Ile at amino acid position 264,

Glu at amino acid position 265,

any of Phe, Met and Leu at amino acid position 266,

any of Ala, Glu, Gly and Gln at amino acid position 267,

Asp or Glu at amino acid position 268,

Asp at amino acid position 269,

any of Asp, Phe, Ile, Met, Asn and Gln at amino acid position 272,

Asp at amino acid position 296,

Ala or Asp at amino acid position 326,

Gly at amino acid position 327,

Lys or Arg at amino acid position 330,

Ser at amino acid position 331,

Thr at amino acid position 332,

any of Thr, Lys and Arg at amino acid position 333,

any of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg and Tyr at amino acid position 396,

shown by EU numbering, in the Fc region in which the amino acid at position 238 is Asp and the amino acid at position 271 (EU numbering) is Gly in human IgG (IgG1, IgG2, IgG3 or IgG4).

Examples of a non-limiting embodiment of an Fc region that further comprises at least one additional Fc region change, and further comprises the additional changes mentioned above, include the Fc regions shown in Tables 6-1 through 6-7.

(Table 6-2 is a continuation of Table 6-1.)

(Table 6-3 is a continuation of Table 6-2.)

(Table 6-4 is a continuation of Table 6-3.)

(Table 6-5 is a continuation of Table 6-4.)

(Table 6-6 is a continuation of Table 6-5.)

(Table 6-7 is a continuation of Table 6-6.)

Four types of FcγR have been identified in mice: FcγRI, FcγRIIb, FcγRIII, and FcγRIV. In humans, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIa, and FcγRIIIb have also been identified as relevant FcγRs in humans. FcγRIIb, which is considered the only inhibitory type among these FcγRs, is conserved in both humans and mice. Other FcγRs, with the exception of FcγRIIIb, signal activation via an immunoreceptor tyrosine activating motif (ITAM), while FcγRIIb transmits inhibitory signals via an immunoreceptor tyrosine inhibitory motif (ITIM) present inside cells (Nat. Rev. Immunol. (2008) 8 , 34-47).

FcγRIIb1 and FcγRIIb2 are described as FcγRIIb splicing variants. In both humans and mice, FcγRIIb1 has a longer intracellular domain than FcγRIIb2. FcγRIIb1 was confirmed to be expressed in B cells, and FcγRIIb2 was confirmed to be expressed in macrophages, mast cells, dendritic cells, basophils, neutrophils and eosinophils (J. Clin. Immunol. (2005) 25 (1), 1-18 ).

So far, it has been described that in humans, dysfunction and reduced expression of FcγRIIb correlates with the occurrence of autoimmune diseases. For example, it has been described that in some patients with SLE, transcription activator binding is attenuated due to a polymorphism in the FcγRIIb expression promoter region, resulting in reduced FcγRIIb expression (Hum. Genet. (2005) 117, 220-227; J. Immunol. (2004 ) 172, 7192-7199 and J. Immunol (2004) 172, 7186-7191). Moreover, two types of allotypes have been described among SLE patients, where the amino acid at position 233 is Ile or Thr in FcγRIIb. This position is in the transmembrane region of FcγRIIb and reportedly FcγRIIb is less likely to exist in the lipid raft when the amino acid at position 233 is Thr compared to when the amino acid is Ile and as a result, FcγRIIb signaling function is reduced (Nat. Med. (2005) 11, 1056-1058; Hum. Mol. Genet., (2005) 14, 2881-2892). Similarly, in mice, knockout mice obtained by disrupting the FcγRIIb gene in C57BL/6 mice have been reported to have SLE-like symptoms such as autoantibody production and glomerulonephritis (Immunity 13 (2000) 277-285; J. Exp. Med. (2002) 195, 1167-1174). Moreover, a reduced expression level of FcγRIIb and the like has been described to date. in mice considered to be models of naturally occurring SLE (Immunogenetics (2000) 51, 429-435; Int. Immunol. (1999) 11, 1685-1691; Curr. Biol. (2000) 10, 227-230; J. Immunol (2002) 169, 4340-4346). From these reports, FcγRIIb is believed to regulate humoral immunity in mice, as in humans.

When an Fc-containing antibody of the present invention eliminates antigens through FcγRIIb, the endocytosis function of FcγRIIb is considered to be the most important contributor among FcγRIIb functions. As described above, FcγRIIb1 and FcγRIIb2 exist as splicing variants of FcγRIIb, however, the latter is described to be primarily involved in the endocytosis of the antibody-antigen immunocomplex (J. Immunol. (1994), 152 574-585; Science (1992) 256, 1808 -1812; Cell (1989) 58, 317-327). So far, mouse FcγRIIb2 has been reported to be incorporated into a clathrin-coated well and undergo endocytosis (Cell (1989) 58, 317-327). Moreover, the dileucine motif has been reported to be essential for FcγRIIb2 mediated endocytosis, and the dileucine motif is conserved in both humans and mice (EMBO J. (1994) 13 (13), 2963-2969). Based on this, FcγRIIb2 may have endocytotic activity in humans, as well as in mice.

On the other hand, unlike FcγRIIb2, it was reported that FcγRIIb1 does not induce endocytosis. FcγRIIb1 has an inserted sequence in its intracellular domain that is not found in FcγRIIb2. This sequence is believed to inhibit the uptake of FcγRIIb1 into the clathrin-coated recess and as a result, endocytosis is inhibited (J. Cell. Biol. (1992) 116, 875-888; J. Cell. Biol. (1989) 109, 3291-3302). Similarly, in humans, FcγRIIb1 has an insertion sequence in a region similar to that of FcγRIIb2, as in mice; thus, presumably, the difference in endocytosis capacity between FcγRIIb1 and FcγRIIb2 is mediated by a similar mechanism. Moreover, in both humans and mice, approximately 40% of cell surface immunocomplexes have been reported to be taken up into the cell within 20 minutes (Mol. Immunol. (2011) 49, 329-337; Science (1992) 256, 1808 -1812). Thus, similarly in humans, FcγRIIb2 is expected to capture immunocomplexes into cells at levels similar to those in mice.

Since FcγRIIb is the only one that has intracellular ITIM in both humans and mice among the FcγR family and the distribution of expressing cells is the same, it is presumed that their function in immune control is similar. Moreover, given the fact that immunocomplexes are taken up into cells at similar levels in humans and mice, the effects of antibodies on antigen clearance mediated by FcγRIIb in humans may be predictable using mice. The antigen-binding molecules mF44 and mF46 have properties of binding to soluble antigens in a pH-dependent manner and have enhanced affinity for mouse FcγRIIb and FcγRIII compared to mIgG1, which is an antigen-binding molecule with the property of binding soluble antigen in a pH-dependent manner. Indeed, Example 5 shows that antigen clearance increased when mF44 or mF46 was administered to normal mice compared to when mIgG1 was administered.

Moreover, in Example 6 below, a similar experiment was performed using Fc receptor γ-chain deficient mice. FcγRs other than FcγRIIb have been reported to be expressed only in the co-presence of the gamma chain in mice. Thus, only FcγRIIb is expressed in Fc receptor γ-chain deficient mice. Administration of mF44 or mF46, which are antigen-binding molecules having the property of binding to soluble antigens in a pH-dependent manner, to mice deficient in the Fc receptor γ-chain allows evaluation of the effects of accelerating antigen clearance when FcγRIIb binding is selectively enhanced. From the results of Example 6, when mF44 or mF46 (which are antigen-binding molecules having the property of binding soluble antigens in a pH dependent manner) was administered to Fc receptor γ-chain deficient mice, it was shown that antigen clearance was increased compared to when mice were injected with mIgG1 (which is an antigen-binding molecule having the property of binding to soluble antigens in a pH-dependent manner). Moreover, the results of Example 6 demonstrate that when administered to Fc receptor γ-chain deficient mice, mF44 or mF46 induces a similar degree of antigen clearance compared to normal mice.

In Example 6, a similar experiment was performed using FcγRIII deficient mice. Because mIgG1, mF44, and mF46 only bind to FcγRIIb and FcγRIII among mFcγRs, administration of antibodies to FcγRIII-deficient mice allows evaluation of the effects of accelerating antigen clearance when FcγRIIb binding is selectively enhanced. The results of Example 6 indicate that when mF44 or mF46 was administered to FcγRIII mice, antigen clearance was increased compared to that of mice treated with mIgG1. Moreover, the results of Example 6 showed that when administered to FcγRIII deficient mice, mF44 and mF46 provided similar levels of antigen elimination when compared to when administered to Fc receptor γ chain deficient mice and when administered to normal mice.

These results indicate that antigen clearance can be accelerated by enhancing selective binding to FcγRIIb alone without enhancing binding to active FcγRs.

In addition to the reported documents reviewed to date, based on the results of the mouse assessment mentioned above, it is believed that uptake of immunocomplexes into cells via FcγRIIb occurs in vivo in humans, as in mice, and results in antibodies that possess Fc with selectively enhanced binding to human FcγRIIb can accelerate the elimination of its antigens. Moreover, as discussed above, since the uptake of immunocomplexes into cells via FcγRIIb is believed to occur at similar rates in mice and humans, the effects of accelerating antigen clearance compared to the effects of antibodies having Fc with increased affinity for mouse FcγRIIb can be achieved in vivo in humans using an Fc in which the affinity for human FcγRIIb is enhanced to a similar extent.

As described in WO 2009/125825, Fv4-IgG1 is an antibody that results from conferring antigen binding activity to a humanized anti-IL-6 receptor antibody H54/L28-IgG1 in a pH dependent manner, i. altering the variable region so as to impart to it the property of binding to antigen at pH 7.4 and dissociating from antigen at pH 5.8. WO 2009/125825 shows that elimination of soluble human IL-6 receptor is significantly accelerated in mice co-administered with IgG1 Fv4 and soluble human IL-6 receptor as antigen compared to mice co-administered with H54/L28-IgG1 and antigen . In this case, the H54-IgG1 heavy chain and L28-CK light chain included in H54/L28-IgG1 are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively; and the VH3-IgG1 heavy chain and VL3-CK light chain included in Fv4-IgG1 are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

The soluble human IL-6 receptor bound to the H54/L28-IgG1 antibody, which binds to the soluble human IL-6 receptor, is recycled to plasma along with the antibody via FcRn. Meanwhile, the Fv4-IgG1 antibody that binds to the soluble human IL-6 receptor in a pH-dependent manner dissociates from the soluble human IL-6 receptor that has bound to the antibody under acidic conditions in the endosome. Because the dissociated soluble human IL-6 receptor is degraded in the lysosome, elimination of the soluble human IL-6 receptor can be greatly accelerated and the Fv4-IgG1 antibody, which binds to the soluble human IL-6 receptor in a pH dependent manner, is recycled to plasma after binding to FcRn in endosome. Because the recycled antibody can re-bind to the soluble human IL-6 receptor, binding to the antigen (soluble human IL-6 receptor) and recycling to plasma via FcRn is repeated. As a result, a single antibody molecule can repeatedly bind to the soluble human IL-6 receptor multiple times (WO 2009/125825).

On the other hand, as described within the scope of the present invention, it has been found that the plasma concentration of soluble antigen can be significantly reduced by administering an antigen-binding molecule with enhanced FcγR binding activity of an Fcγ receptor-binding domain incorporated into an antigen-binding molecule that contains an antigen-binding domain, antigen-binding the activity of which varies depending on ion concentration conditions, such as pH, an FcRn-binding domain having FcRn-binding activity under acidic pH range conditions, and an Fcγ receptor-binding domain.

Without wishing to be bound by a particular theory, the unexpected decrease in plasma soluble antigen concentration observed upon administration of an antigen-binding molecule with enhanced FcγR binding that contains an antigen-binding domain whose antigen-binding activity varies depending on ionic concentration conditions such as pH and the FcRn-binding domain, having FcRn binding activity under acidic pH range conditions can be explained as follows.

As described above, an antigen-binding molecule such as Fv4-IgG1 containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions may be able to repeatedly bind to an antigen many times, but the effect of dissociation of a soluble antigen in the endosome on accelerating antigen elimination from plasma may depend on the rate of uptake of the complex of antigen and antigen-binding molecule into the endosome. Antigen-binding molecules with enhanced binding activity to various FcγRs, which contain an antigen-binding domain in which the antigen-binding activity changes depending on ion concentration conditions, are actively taken up into cells by binding to various FcγRs expressed on the cell membrane, and can be recirculated in plasma through recycling through binding between the FcRn and the FcRn binding domain in a molecule having FcRn binding activity under acidic pH range conditions. More specifically, since the antigen-binding molecules mentioned above that have formed complexes with soluble antigens in plasma are actively taken up into cells via FcγR expressed on the cell membrane, the effect of accelerating the elimination of soluble antigens in plasma may be more pronounced than that of antigen-binding molecules whose binding activity with different FcγR is not enhanced.

The FcγR-binding activity of antibodies that bind to membrane antigens plays an important role in the cytotoxic activity of antibodies. Thus, when cytotoxic activity is necessary for an antibody to be used as a pharmaceutical agent, a human IgG1 isotype that has a high FcγR binding activity is used, and a method for enhancing the FcγR binding activity of antibodies is widely used to enhance the cytotoxic activity of the antibody. On the other hand, the role of the FcγR-binding activity of antibodies that bind to soluble antigens and which are used as pharmaceuticals is not known and differences in physiological effects on organisms administered with human IgG1 with high binding activity of human FcγR and IgG2 and human IgG4 with low FcγR binding activity, due to their differences in FcγR binding activity, has not been fully investigated to date. As described below in the "Examples" section, it was indeed confirmed that in the plasma of individuals administered with antibodies whose FcγR binding activity had been lost, there was no effect on changes in the concentration of soluble antigen. On the other hand, within the scope of the present invention, it has been found that the plasma concentration of soluble antigens is significantly reduced in individuals administered antigen-binding molecules having enhanced FcγR binding activity and containing an antigen-binding domain whose binding activity to soluble antigens varies depending on ion concentration conditions. . More specifically, by combining an FcRn-binding domain having FcRn-binding activity under acidic pH range conditions and an antigen-binding domain whose binding to soluble antigens varies depending on ion concentration conditions, which are domains included in antigen-binding molecules targeting soluble antigens, the advantage of increased binding to FcγR has been shown for the first time.

Antigen binding molecule

In the context of the present invention, the term "antigen binding molecule" is used in its broadest sense to refer to a molecule having human FcRn binding activity in an acidic pH range and comprising an antigen binding domain and an Fcγ receptor binding domain. In particular, antigen-binding molecules include various types of molecules, so long as they exhibit antigen-binding activity. Molecules in which an antigen binding domain is associated with an Fc region include, for example, antibodies. Antibodies may include single monoclonal antibodies (including agonist and antagonist antibodies), human antibodies, humanized antibodies, chimeric antibodies, and the like. Alternatively, when used as antibody fragments, they preferably include antigen-binding domains and antigen-binding fragments (eg, Fab, F(ab')2, scFv and Fv). Scaffold molecules, where three-dimensional structures, such as the already known stable α/β-barrel protein structure, are used as a scaffold and only some parts of the structures are converted into libraries for constructing antigen-binding domains, are also included in the antigen-binding molecules of the present invention.

The antigen binding molecule of the present invention may contain at least some portions of the Fc region that mediate binding to the FcRn and Fcγ receptor. In a non-limiting embodiment, the antigen-binding molecule includes, for example, antibodies and Fc fusion proteins. A fusion protein refers to a chimeric polypeptide containing a polypeptide having a first amino acid sequence that is linked to a polypeptide having a second amino acid sequence that is not naturally linked. For example, a fusion protein may comprise the amino acid sequence of at least a portion of an Fc region (e.g., a portion of an Fc region responsible for binding to an FcRn or a portion of an Fc region responsible for binding to an Fcγ receptor) and a non-immunoglobulin polypeptide containing , for example, the amino acid sequence of the ligand-binding domain of the receptor or the receptor-binding domain of the ligand. The amino acid sequences may be in separate proteins that are carried together in a fusion protein, or, as a rule, they may be in the same protein, but they are included in the fusion polypeptide in a new order. Fusion proteins can be generated, for example, by chemical synthesis or genetic recombination techniques to express a polynucleotide encoding the peptide regions in the desired order.

The respective domains of the present invention may be linked together via linkers or directly via polypeptide binding.

Linkers include arbitrary peptide linkers that can be introduced by genetic engineering, synthetic linkers, and linkers as described, for example, in Protein Engineering (1996) 9(3), 299-305. However, peptide linkers are preferred within the scope of the present invention. The length of the peptide linkers is not particularly limited, and may be appropriately selected by those skilled in the art depending on the intended use. The length is preferably five amino acids or more (without particular limitation, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and 15 amino acids is particularly preferred.

For example, such peptide linkers preferably include:

Ser

Gly⋅Ser

Gly⋅Gly⋅Ser

Ser⋅Gly⋅Gly

Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 26)

Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 27)

Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 28)

Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 29)

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 30)

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 31)

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 32)

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 33)

(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 28))n

(Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 29))n

where n is an integer equal to 1 or more. The length or sequence of the peptide linkers may be appropriately selected by those skilled in the art, depending on the intended use.

Typically, synthetic linkers (chemical crosslinkers) are used to crosslink peptides, and for example:

N-hydroxysuccinimide (NHS),

disuccinimidyl suberate (DSS),

bis(sulfosuccinimidyl)suberate (BS ³ ),

dithiobis(succinimidyl propionate) (DSP),

dithiobis (sulfosuccinimidyl propionate) (DTSSP),

ethylene glycol bis(succinimidyl succinate) (EGS),

ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS),

disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES),

and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

When multiple linkers are used to link respective domains, they may all be of the same type, or they may be of different types.

In addition to the linkers illustrated above, linkers with peptide tags such as His tag, HA tag, myc tag and FLAG tag can also be appropriately used. Moreover, hydrogen bonding, disulfide bonding, covalent bonding, ionic interaction, and bonding properties with each other as a result of their combination can also be appropriately used. For example, the affinity between CH1 and CL of an antibody can be used, and Fc regions derived from the bispecific antibodies described above can also be used to heteroassociate Fc regions. Moreover, disulfide bonds formed between domains can also be appropriately used.

To link the respective domains with a peptide bond, the polynucleotides encoding the domains are linked together in frame. Known methods for linking polynucleotides in frame include methods such as restriction fragment ligation, fusion PCR, and overlap PCR. Such methods may suitably be used alone or in combination to construct the antigen-binding molecules of the present invention. Within the scope of the present invention, the terms "linked" and "fused" or "linked" and "merged" are used interchangeably. These terms mean that two or more elements or components, such as polypeptides, are fused together to form a single structure by any means, including the methods of chemical bonding described above and the methods of genetic recombination. In-frame fusion means, when two or more elements or components are polypeptides, linking two or more in-frame elements to form a continuous longer reading frame while maintaining the correct reading frames of the polypeptides. When two Fab molecules are used as an antigen-binding domain, an antibody can be used as the preferred antigen-binding molecule of the present invention, which is an antigen-binding molecule of the present invention, wherein the antigen-binding domain is linked in-frame to an Fc region via a peptide bond without a linker.

FcRn

In contrast to the Fcγ receptor, which belongs to the immunoglobulin family, FcRn, in particular human FcRn, is structurally similar to major histocompatibility complex (MHC) class I polypeptides, showing 22%-29% sequence identity with MHC class I molecules (Ghetie et al. Immunol Today (1997) 18 (12): 592-598). FcRn is expressed as a heterodimer consisting of a soluble β- or light chain (β2-microglobulin) complexed with a transmembrane α- or heavy chain. Like the MHC, the FcRn α chain contains three extracellular domains (α1, α2 and α3) and its short cytoplasmic domain anchors the protein to the cell surface, the α1 and α2 domains interact with the FcRn binding domain of the antibody Fc region (Raghavan et al. , Immunity (1994) 1: 303-315).

FcRn is expressed in the maternal placenta and the mammalian yolk sac and is involved in the transfer of IgG from mother to fetus. Furthermore, in the neonatal rodent small intestine, where FcRn is expressed, FcRn is involved in the transfer of maternal IgG across the brush border epithelium from ingested colostrum or milk. FcRn is expressed in a variety of other tissues and endothelial cell systems of various types. FcRn is also expressed in the adult human endothelium, muscle blood vessels, and sinusoidal capillaries of the liver. FcRn is believed to be involved in maintaining plasma IgG concentration by mediating the recycling of IgG to serum upon binding to IgG. As a rule, FcRn binding to IgG molecules is strongly pH dependent. Optimal binding is observed in the acidic pH range below 7.0.

A human FcRn whose precursor is a polypeptide having the signal sequence of SEQ ID NO: 34 (the signal sequence polypeptide is shown in SEQ ID NO: 35) forms a complex with human β2-microglobulin in vivo. As shown in the Reference Examples described below, soluble human FcRn complexed with β2-microglobulin was produced using conventional recombinant expression methods. The FcRn binding domain of the present invention can be evaluated for its binding activity to such soluble human FcRn in complex with β2-microglobulin. For the purposes of the present invention, unless otherwise indicated, a human FcRn refers to a form capable of binding to the FcRn binding domain of the present invention. Examples include the complex between human FcRn and human β2-microglobulin.

The antigen binding molecules of the present invention have an FcRn binding domain. The FcRn-binding domain is not particularly limited as long as the antigen-binding molecules have FcRn-binding activity in the acidic pH range, and may be a domain that has direct or indirect FcRn-binding activity. Preferred examples of such a domain include an IgG immunoglobulin Fc region, albumin, albumin domain 3, an anti-FcRn antibody, an anti-FcRn peptide, an anti-FcRn scaffold molecule, and the like, which have the activity of direct binding to FcRn, or molecules that bind to IgG or albumin, which have the activity of indirect binding to FcRn. Within the scope of the present invention, a domain having FcRn binding activity in the acidic pH range and in the neutral pH range is preferred. When the domain already has FcRn binding activity in the acidic pH range, it can preferably be used as is. When the domain has no or little FcRn binding activity in the neutral pH range, the amino acids in the antigen binding molecule can be modified to impart FcRn binding activity. Alternatively, amino acids can be altered in a domain already having FcRn binding activity in the acidic pH range to increase FcRn binding activity. For an amino acid change in the FcRn binding domain, a change of interest can be identified by comparing FcRn binding activity in an acidic pH range before and after the amino acid change.

A preferred human FcRn binding domain is one that binds directly to FcRn. Such preferred FcRn binding domains include, for example, the Fc regions of antibodies. Meanwhile, regions capable of binding to a polypeptide such as albumin or IgG that has FcRn binding activity can indirectly bind to FcRn via albumin, IgG, or the like. Thus, for the FcRn binding region of the present invention, a region that binds to a polypeptide having an FcRn binding activity can preferably be used. The Fc region contains an amino acid sequence derived from the heavy chain constant region of an antibody. The Fc region is part of an antibody heavy chain constant region beginning at the N-terminus of the hinge region at the papain cleavage site, which is located approximately at amino acid position 216 according to EU numbering, and includes the hinge region, CH2 and CH3 domains.

The binding activity of the FcRn-binding domain to an FcRn, in particular a human FcRn, or an antigen-binding molecule containing the domain

The binding activity of the FcRn binding domain of the present invention to FcRn, in particular human FcRn, can be measured by methods known to those skilled in the art, as described in the "Binding Activity" section above. Those skilled in the art can define conditions other than pH as appropriate. The antigen-binding activity and the binding activity to the human FcRn of the antigen-binding molecule can be evaluated based on dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), apparent dissociation rate constant (kd), and the like. They can be measured by methods known to those skilled in the art. For example, a Biacore (GE healthcare), a Scatchard plot, or a flow cytometer can be used.

When the binding activity of the human FcRn of the Fc region of the present invention is measured, conditions other than pH are not specifically limited and can be appropriately selected by those skilled in the art. Measurements can be carried out, for example, at 37°C using MES buffer as described in WO 2009/125825. Alternatively, the human FcRn binding activity of the Fc region of the present invention can be measured by methods known to those skilled in the art and can be measured using, for example, Biacore (GE Healthcare) and the like. The binding activity of an FcRn-binding domain to human FcRn can be assessed by applying, as an analyte, a human FcRn, an FcRn-binding domain, or an antigen-binding molecule of the present invention containing an FcRn-binding domain to a chip onto which the FcRn-binding domain is immobilized, an antigen-binding molecule of the present invention containing an FcRn-binding domain, or human FcRn.

The range of acidic pH values indicated as conditions for the presence of binding activity between FcRn and FcRn-binding domain in the antigen-binding molecule of the present invention, as a rule, refers to pH 4.0-pH 6.5. Preferably it is pH 5.5-pH 6.5 and particularly preferably it is pH 5.8-pH 6.0, which is close to the pH in the early endosome in vivo. The neutral pH range indicated as conditions for the presence of binding activity between an FcRn and an antigen-binding molecule of the present invention, or an FcRn-binding domain included in such a molecule, generally refers to pH 6.7 to pH 10.0. Preferably, the neutral pH range is a range indicated by arbitrary pH values from pH 7.0 to pH 8.0, and is preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7 .4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, and especially preferably a pH of 7.4, which is close to the in vivo plasma (blood) pH value. When assessing the binding affinity between a human FcRn and a human FcRn-binding domain or an antigen-binding molecule containing this domain at pH 7.4 is difficult due to low binding affinity, pH 7.0 can be used instead of pH 7.4. As a temperature to be used in the assay conditions, the binding affinity between the FcRn binding domain and FcRn can be evaluated at any temperature from 10°C to 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between the FcRn binding domain and human FcRn. More preferably, any temperature from 20°C to 35°C, such as any of 20, 21, 22, 23, 24, 25, 26, 27 can equally be used to determine the binding affinity between the FcRn binding domain and FcRn , 28, 29, 30, 31, 32, 33, 34 and 35°С. A non-limiting example of an embodiment of the present invention is a temperature of 25°C.

According to Yeung et al. (J. Immunol. (2009) 182, 7663-7671), the binding activity of natural human IgG1 to human FcRn in the acidic pH range (pH 6.0) is KD 1.7 μM, while this activity is practically unaffected by detection in the range of neutral pH values. Thus, in a preferred embodiment, an antigen-binding molecule of the present invention having a human FcRn binding activity in an acidic pH range can be used, which includes antigen-binding molecules whose human FcRn binding activity under acidic pH range conditions is a KD of 20 μM or greater, and binding activity to human FcRn which, under conditions of the neutral pH range, is equivalent to the binding activity of human FcRn in human IgG. In a more preferred embodiment, an antigen-binding molecule of the present invention can be used, comprising antigen-binding molecules whose binding activity to human FcRn under conditions of an acidic pH range is KD 2.0 μM or greater. In an even more preferred embodiment, antigen-binding molecules having a human FcRn binding activity in the acidic pH range of a KD of 0.5 μM or greater can be used. The KD values mentioned above are determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen-binding molecule on a chip and loading a human FcRn as the test sample).

Within the scope of the present invention, an Fc region is preferred which has FcRn binding activity under conditions of an acidic pH range. If the domain is an Fc region already having FcRn binding activity under acidic pH range conditions, it can be used as is. If the domain has no or little FcRn binding activity under acidic pH range conditions, the amino acids in the antigen binding molecule can be altered to provide an Fc region having the desired FcRn binding activity. An Fc region having a desired FcRn binding activity or enhanced FcRn binding activity under acidic pH range conditions can also preferably be obtained by altering the amino acids in the Fc region. An amino acid change in the Fc region that confers such a desired binding activity can be identified by comparing FcRn binding activity under conditions of an acidic pH range before and after the amino acid change. Appropriate amino acid changes can be made by those skilled in the art using a well known method, similar to the above mentioned method used to change the Fcγ receptor binding activity.

The Fc region having FcRn binding activity under acidic pH range conditions, which is included in the antigen-binding molecule of the present invention, can be obtained by any method; but in particular, an FcRn-binding domain having an FcRn-binding activity or having an enhanced FcRn-binding activity under acidic pH range conditions can be obtained by altering the amino acids of a human IgG immunoglobulin used as the parent Fc region. Preferred IgG immunoglobulin Fc regions for alteration include human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof). To change to other amino acids, amino acids at any position can be changed, provided that the Fc region has FcRn binding activity under acidic pH range conditions, or that human FcRn binding activity under acidic range conditions can be increased. When the antigen-binding molecule includes a human IgG1 Fc region as an Fc region, it preferably includes a change that has the effect of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region. Preferred examples of such amino acids that can be changed include amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356 , 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447 (EU numbering) as described in WO 2000/042072 . Similarly, preferred examples of amino acids that can be changed also include amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436 (EU numbering) as described in WO 2002/060919. Moreover, such amino acids that can be changed also include the amino acids at positions 250, 314 and 428 (EU numbering) as described in WO 2004/092219. Such amino acids that can be changed further include the amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434 and/or 436 (EU numbering) as described in WO 2010/045193. These amino acid changes enhance FcRn binding under conditions of the acidic pH range of the IgG immunoglobulin Fc region.

Within the scope of the present invention, an Fc region is preferred which has FcRn binding activity under conditions of an acidic pH range. If the domain is an Fc region already having FcRn binding activity under acidic pH range conditions, it can be used as is. If the domain has no or little FcRn binding activity under acidic pH range conditions, the amino acids in the antigen binding molecule can be altered to provide an Fc region having the desired FcRn binding activity. An Fc region having a desired FcRn binding activity or enhanced FcRn binding activity under acidic pH range conditions can preferably be obtained by altering the amino acids in the Fc region. Fc region amino acid changes that confer such desired binding activity can be identified by comparing FcRn binding activity under acidic pH range conditions before and after the amino acid change. Appropriate amino acid changes can be made by those skilled in the art using a well known method similar to the above mentioned method used to change the Fcγ receptor binding activity.

The Fc region having FcRn binding activity under acidic pH range conditions, which is included in the antigen-binding molecule of the present invention, can be obtained by any method; but in particular, an FcRn-binding domain having a binding activity or having an enhanced binding activity to FcRn under acidic pH range conditions can be obtained by changing the amino acids of a human IgG immunoglobulin used as a starting Fc region. Preferred examples of IgG immunoglobulin Fc regions to be modified include human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof). For changes to other amino acids, amino acids at any position can be changed, as long as the Fc region has FcRn binding activity under acidic pH range conditions, or human FcRn binding activity under acidic pH range conditions can be increased. When the antigen-binding molecule contains a human IgG1 Fc region as the Fc region, it preferably includes a change that provides increased binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region. Examples of such amino acids that can be changed include amino acids at positions 252, 254, 256, 309, 311, 315, 433 and/or 434, as well as amino acids that can be combined with them, which are amino acids at positions 253, 310, 435 and/or 426 (EU numbering) as described in WO 1997/034631. Preferred amino acids are amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376 , 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447 (EU numbering) as described in WO 2000/042072. Similarly, preferred examples of such amino acids that can be changed also include the amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436 (EU numbering) as described in WO 2002/060919. Moreover, such amino acids that can be changed include the amino acids at positions 250, 314 and 428 (EU numbering) as described in WO 2004/092219. In addition, preferred examples of such amino acids that can be changed include amino acids at positions 238, 244, 245, 249, 252, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427, 430, 431, 434, 436, 438, 440 and /or 442 as described in WO 2006/020114. Other preferred examples of such amino acids that can be changed also include the amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434 and/or 436 (EU numbering) as described in WO 2010/045193. These amino acid changes enhance FcRn binding under conditions of the acidic pH range of the IgG immunoglobulin Fc region.

In a non-limiting embodiment, changes that cause the effect of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of a native human IgG1 Fc region when a human IgG1 Fc region is included as an Fc region, examples include at least one or more amino acid changes selected from the group consisting of:

either Arg or Leu at amino acid position 251;

any of Phe, Ser, Thr and Tyr at amino acid position 252;

either Ser or Thr at amino acid position 254;

any of Arg, Gly, Ile and Leu at amino acid position 255;

any of Ala, Arg, Asn, Asp, Gln, Glu and Thr at amino acid position 256;

either Ile or Thr at amino acid position 308;

Pro at amino acid position 309;

any of Glu, Leu and Ser at amino acid position 311;

either Ala or Asp at amino acid position 312;

either Ala or Leu at amino acid position 314;

any of Ala, Arg, Asp, Gly, His, Lys, Ser and Thr at amino acid position 385;

any of Arg, Asp, ILe, Lys, Met, Pro, Ser and Thr at amino acid position 386;

any of Ala, Arg, His, Pro, Ser and Thr at amino acid position 387;

any of Asn, Pro and Ser at amino acid position 389;

any of Leu, Met, Phe, Ser and Thr at amino acid position 428;

any of Arg, Gln, His, Ile, Lys, Pro and Ser at amino acid position 433;

any of His, Phe and Tyr at amino acid position 434; and

any of Arg, Asn, His, Lys, Met and Thr at amino acid position 436

according to EU numbering. The number of amino acids to be changed is not particularly limited, and an amino acid can only be changed at one position or at two or more positions.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including Ile at amino acid position 308, Pro at amino acid position 309 and/or Glu at amino acid position 311 according to EU numbering. In another non-limiting embodiment of the changes, the changes can be Thr at amino acid position 308, Pro at amino acid position 309, Leu at amino acid position 311, Ala at amino acid position 312, and/or Ala at amino acid position 314. In another non-limiting embodiment of the changes, changes can be Ile or Thr at amino acid position 308; Pro at amino acid position 309; Glu, Leu or Ser at amino acid position 311; Ala at amino acid position 312; and/or Ala or Leu at amino acid position 314. In another non-limiting embodiment of the changes, the changes can be Thr at amino acid position 308, Pro at amino acid position 309, Ser at amino acid position 311, Asp at amino acid position 312, and/or Leu at position amino acids 314.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , comprising Leu at amino acid position 251, Tyr at amino acid position 252, Ser or Thr at amino acid position 254, Arg at amino acid position 255 and/or Glu at amino acid position 256 according to EU numbering.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including any of Leu, Met, Phe, Ser and Thr at amino acid position 428; any of Arg, Gln, His, Ile, Lys, Pro and Ser at amino acid position 433; any of His, Phe and Tyr at amino acid position 434; and/or any of Arg, Asn, His, Lys, Met and Thr at amino acid position 436 as indicated by EU numbering. In another non-limiting embodiment of the change(s), the change(s) may include His or Met at amino acid position 428 and/or His or Met at amino acid position 434.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including Arg at amino acid position 385, Thr at amino acid position 386, Arg at amino acid position 387 and/or Pro at amino acid position 389 according to EU numbering. In another non-limiting embodiment of the changes, the change(s) may be Asp at amino acid position 385, Pro at amino acid position 386, and/or Ser at amino acid position 389.

Moreover, when a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region include changes in at least at least one or more amino acids selected from the group consisting of:

either Gln or Glu at amino acid position 250; and

either Leu or Phe at amino acid position 428

according to EU numbering. The number of amino acids to be changed is not particularly limited, and an amino acid may be changed at only one position or two or more positions.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including Gln at amino acid position 250 and/or either Leu or Phe at amino acid position 428 according to EU numbering. In another non-limiting embodiment of the changes, the changes can be changes including Glu at amino acid position 250 and/or either Leu or Phe at amino acid position 428.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of changes that produce effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region, examples include changes to at least two or more amino acids selected from the group consisting of:

either Asp or Glu at amino acid position 251;

Tyr at amino acid position 252;

Gln at amino acid position 307;

Pro at amino acid position 308;

Val at amino acid position 378;

Ala at amino acid position 380;

Leu at amino acid position 428;

either Ala or Lys at amino acid position 430;

any of Ala, His, Ser and Tyr at amino acid position 434; and

Ile at amino acid position 436

according to EU numbering. The number of amino acids to be changed is not particularly limited, and an amino acid can only be changed at two positions or at three or more positions.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including Gln at amino acid position 307 and either Ala or Ser at amino acid position 434 according to EU numbering. In another non-limiting embodiment of the changes, the changes can include Pro at amino acid position 308 and Ala at amino acid position 434. In another non-limiting embodiment of the changes, changes can include Tyr at amino acid position 252 and Ala at amino acid position 434. In another non-limiting embodiment of the changes, changes can include Val at amino acid position 378 and Ala at amino acid position 434. In another non-limiting embodiment of the changes, the changes can include Leu at amino acid position 428 and Ala at amino acid position 434. In another non-limiting embodiment of the changes, changes can include Ala at amino acid position 434 and Ile at amino acid position 436. Moreover, in another non-limiting embodiment of the changes, the changes may include Pro at amino acid position 308 and Tyr at amino acid position 434. Moreover, in another different non-limiting embodiment, For changes, changes may include Gln at amino acid position 307 and Ile at amino acid position 436.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be the change(s) , including Gln at amino acid position 307, Ala at amino acid position 380 and Ser at amino acid position 434 as according to EU numbering. In another non-limiting embodiment, the changes may include Gln at amino acid position 307, Ala at amino acid position 380, and Ala at amino acid position 434. In another non-limiting embodiment of the changes, changes may include Tyr at amino acid position 252, Pro at amino acid position 308, and Tyr at amino acid position 434. In another non-limiting embodiment of the alterations, the alterations may include Asp at amino acid position 251, Gln at amino acid position 307, and His at amino acid position 434.

When a human IgG1 Fc region is included as the Fc region, examples of a non-limiting embodiment of changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region include changes at least two or more amino acids selected from the group consisting of:

Leu at amino acid position 238;

Leu at amino acid position 244;

Arg at amino acid position 245;

Pro at amino acid position 249;

Tyr at amino acid position 252;

Pro at amino acid position 256;

any of Ala, Ile, Met, Asn, Ser and Val at amino acid position 257;

Asp at amino acid position 258;

Ser at amino acid position 260;

Leu at amino acid position 262;

Lys at amino acid position 270;

either Leu or Arg at amino acid position 272;

any of Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 279;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 283;

Asn at amino acid position 285;

Phe at amino acid position 286;

either Asn or Pro at amino acid position 288;

Val at amino acid position 293;

any of Ala, Glu and Met at amino acid position 307;

any of Ala, Ile, Lys, Leu, Met, Val and Trp at amino acid position 311;

Pro at amino acid position 312;

Lys at amino acid position 316;

Pro at amino acid position 317;

either Asn or Thr at amino acid position 318;

any of Phe, His, Lys, Leu, Met, Arg, Ser and Trp at amino acid position 332;

any of Asn, Thr and Trp at amino acid position 339;

Pro at amino acid position 341;

any of Glu, His, Lys, Gln, Arg, Thr and Tyr at amino acid position 343;

Arg at amino acid position 375;

any of Gly, Ile, Met, Pro, Thr and Val at amino acid position 376;

Lys at amino acid position 377;

either Asp or Asn at amino acid position 378;

any of Asn, Ser and Thr at amino acid position 380;

any of Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 382;

Asn at amino acid position 423;

Asn at amino acid position 427;

any of Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, and Tyr at amino acid position 430;

either His or Asn at amino acid position 431;

any of Phe, Gly, His, Trp and Tyr at amino acid position 434;

any of Ile. Leu and Thr at amino acid position 436;

any of Lys, Leu, Thr and Trp at amino acid position 438;

Lys at amino acid position 440; and

Lys at amino acid position 442;

according to EU numbering. The number of amino acids to be changed is not particularly limited, and an amino acid can only be changed at two positions or at three or more positions.

When a human IgG1 Fc region is included as an Fc region, a non-limiting embodiment of the changes that cause the effects of enhancing binding to FcRn under acidic pH range conditions compared to the binding activity of the original human IgG1 Fc region can be changes including Ile at amino acid position 257 and Ile at amino acid position 311 according to EU numbering. In another non-limiting embodiment of the alterations, the alterations may include Ile at amino acid position 257 and His at amino acid position 434. In another non-limiting embodiment of alterations, alterations may include Val at amino acid position 376 and His at amino acid position 434.

Moreover, as described below, for the FcRn-binding domain included in the antigen-binding molecule of the present invention, an Fc region having FcRn-binding activity under neutral pH range conditions can also be preferably used. Such an Fc region can be obtained by any method according to the above-mentioned method for obtaining Fc regions having FcRn binding activity under acidic pH range conditions, but in particular, an FcRn binding domain having FcRn binding activity or enhanced FcRn binding activity. under conditions of the neutral pH range, can be obtained by changing the amino acids of human IgG immunoglobulin used as the original Fc region. Preferred IgG immunoglobulin Fc regions for alteration include human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof). For changes to other amino acids, one can change the amino acid at any position, provided that the Fc region has FcRn binding activity under neutral pH range conditions or human FcRn binding activity under acidic pH range conditions can be increased. When the antigen-binding molecule includes a human IgG1 Fc region as an Fc region, it preferably includes a change that has the effects of enhancing binding to FcRn under neutral pH range conditions compared to the binding activity of the original human IgG1 Fc region. Preferred examples of such altered Fc regions include human FcRn binding domains wherein at least one or more amino acids selected from the group consisting of amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256 , 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382 , 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 in the region of the original Fc region according to EU numbering differ from the corresponding amino acids in the native Fc region.

Preferred examples of such altered Fc regions include Fc regions containing at least one or more amino acids selected from the group consisting of:

Met at amino acid position 237;

Ala at amino acid position 238;

Lys at amino acid position 239;

Ile at amino acid position 248;

any of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp and Tyr at amino acid position 250;

any of Phe, Trp and Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

any of Asp, Glu and Gln at amino acid position 256;

any of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr and Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Phe at amino acid position 270;

either Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Gly at amino acid position 298;

Ala at amino acid position 303;

Ala at amino acid position 305;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 307;

any of Ala, Phe, Ile, Leu, Met, Pro, Gln and Thr at amino acid position 308;

any of Ala, Asp, Glu, Pro and Arg at amino acid position 309;

any of Ala, His and Ile at amino acid position 311;

either Ala or His at amino acid position 312;

either Lys or Arg at amino acid position 314;

either Ala or His at amino acid position 315;

Ala at amino acid position 317;

Gly at amino acid position 325;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

either Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

either Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 428;

Lys at amino acid position 433;

any of Ala, Phe, His, Ser, Trp and Tyr at amino acid position 434; and

His at amino acid position 436;

in the Fc region according to EU numbering.

For example, using these amino acid changes alone or multiple changes in combination, binding of the IgG Fc region to FcRn in the acidic pH range and/or in the neutral pH range can be enhanced; however, the amino acid changes to be made are not particularly limited, and as long as the effect of increasing the plasma retention time is reported, any amino acid change can be made.

Pharmaceutical compositions

When a conventional neutralizing antibody against a soluble antigen is administered, plasma retention of the antigen is expected to be prolonged due to binding to the antibody. As a rule, antibodies have a long half-life (from one week to three weeks), while the half-life of an antigen is usually short (one day or less). Meanwhile, antibody-bound antigens have a significantly longer plasma half-life compared to when the antigens are present alone. For this reason, the introduction of an existing neutralizing antibody leads to an increase in the plasma concentration of the antigen. Such cases are described for various neutralizing antibodies that target soluble antigens, including, for example, IL-6 (J. Immunotoxicol. (2005) 3, 131-139), amyloid-beta (mAbs (2010) 2 (5), 1 -13), MCP-1 (ARTHRITIS & RHEUMATISM (2006) 54, 2387-2392), hepcidin (AAPS J. (2010) 4, 646-657) and sIL-6 receptor (Blood (2008) 112 (10), 3959-64). It is described that the introduction of existing neutralizing antibodies increases the total concentration of antigen in plasma approximately 10-1000 times (the level of increase varies, depending on the antigen) from the initial level. In the context of the present invention, the total plasma antigen concentration refers to the concentration as the total amount of antigen in the plasma, i.e. the sum of the concentrations of antibody-bound and non-antibody-bound antigen. An increase in total plasma antigen concentration is undesirable for such antibody based pharmaceuticals that target soluble antigen. The reason for this is that the antibody concentration must be higher than at least the total plasma concentration of the antigen in order to neutralize the soluble antigen. Specifically, "total plasma antigen concentration is increased 10-1000-fold" means that in order to neutralize the antigen, the plasma concentration of the antibody (i.e., the antibody dose) must be 10-1000 times higher than when the increase the total concentration of antigen in plasma does not occur. Conversely, if the total plasma concentration of the antigen can be reduced by 10-1000 times compared to the existing neutralizing antibody, the dose of the antibody can also be reduced to a similar extent. Thus, antibodies capable of reducing total plasma antigen concentration by eliminating soluble antigen from plasma are highly useful compared to existing neutralizing antibodies.

Although the present invention is not limited to a particular theory, the reason for the increase in the number of antigens that can bind to a single antigen-binding molecule and the reason for the increased decrease in plasma antigen concentration when the antigen-binding molecule is introduced into a living body, resulting in an increase in the uptake of the antigen-binding molecule into cells in vivo, wherein the antigen-binding molecule comprises an antigen-binding domain whose antigen-binding activity varies with ion concentration conditions such that the antigen-binding activity is lower under acidic pH range conditions than under neutral pH range conditions, and an FcRn-binding domain such as a constant region an antibody having human Fcγ receptor binding activity under neutral pH range conditions can be explained as follows.

For example, when an antibody that binds to a membrane antigen is administered in vivo, after binding to the antigen, the antibody in the antigen-bound state is incorporated into the endosome by intracellular internalization. The antibody is then transferred to the lysosome, remaining bound to the antigen, and degraded there along with the antigen. Internalization-mediated elimination from plasma is referred to as antigen-dependent elimination and has been described for many antibody molecules (Drug Discov Today (2006) 11(1-2), 81-88). When one IgG antibody molecule binds to antigens in a bivalent fashion, one antibody molecule is internalized while remaining bound to two antigens and degraded in the lysosome. In the case of typical antibodies, therefore, one IgG antibody molecule cannot bind to three or more antigen molecules. For example, one IgG antibody molecule with neutralizing activity cannot neutralize three or more antigen molecules.

The plasma retention time of the IgG molecule is relatively long (elimination is slow) because FcRn, which is known as the rescue receptor for the IgG molecule, functions. IgG molecules incorporated into endosomes by pinocytosis bind under the acidic conditions of the endosomes to FcRn expressed in the endosomes. IgG molecules that cannot bind to FcRn are transferred to and degraded by lysosomes. Meanwhile, FcRn-bound IgG molecules are transferred to the cell surface. IgG molecules dissociate from human FcRn under neutral conditions in plasma and are thus recycled back into plasma.

Alternatively, when the antigen-binding molecules are antibodies that bind to a soluble antigen, the antibodies administered in vivo bind to the antigens and then the antibodies are incorporated into cells while remaining bound to the antigens. Most antibodies incorporated into cells bind to FcRn in the endosome and then are transferred to the cell surface. Antibodies dissociate from FcRn under neutral plasma conditions and are released into the external environment of the cells. However, antibodies having typical antigen-binding domains, whose antigen-binding activity does not vary depending on ion concentration conditions such as pH, are released into the external environment of the cells, remaining bound to the molecules and thus they cannot bind again to the antigen. Thus, like antibodies that bind to membrane antigens, a single typical IgG antibody molecule, whose antigen-binding activity does not vary depending on ion concentration conditions such as pH, cannot bind three or more antigen molecules.

Antibodies that bind to antigens in a pH dependent manner that bind strongly to antigens under conditions of the neutral pH range and dissociate from antigens under conditions of the acidic pH range in the endosome (antibodies that bind under conditions of the neutral pH range and dissociate under conditions of the pH range acidic pH values), and antibodies that bind to antigens in a calcium ion concentration-dependent manner that bind strongly to antigens under conditions of high plasma calcium ions and dissociate from antigens under conditions of low endosome calcium ions (antibodies that bind to antigens under conditions of high concentration of calcium ions and dissociate under conditions of low concentration of calcium ions) can dissociate from the antigen in the endosome. Antibodies that bind to antigens in a pH-dependent or calcium ion concentration-dependent manner, when recycled to plasma via FcRn, can re-bind to the antigen after dissociation from the antigens. Thus, such a single antibody molecule can repeatedly bind to several antigen molecules. Meanwhile, the antigen bound to the antigen-binding molecules is dissociated from the antibody in the endosome and degraded in the lysosome without being recycled to the plasma. By administering such antigen-binding molecules in vivo, the uptake of antigen into cells is accelerated and it is possible to reduce the plasma concentration of antigen.

The uptake of antigens bound by antigen-binding molecules into cells is further enhanced by conferring or enhancing Fcγ receptor binding activity under conditions of the neutral pH range (pH 7.4) to antibodies that bind to antigens in a pH dependent manner that bind strongly to antigens under conditions of the pH range. neutral plasma pH and dissociate from antigens under acidic pH range conditions in the endosome (antibodies that bind to antigens under neutral pH range conditions and dissociate under acidic pH range conditions), and antibodies that bind to antigens in a concentration dependent manner calcium ions in a manner that binds strongly to antigens under conditions of high plasma calcium ion concentrations and dissociates from antigens under conditions of low endosome calcium ion concentrations (antibodies that bind to antigens under conditions of high calcium ion concentrations and dissociate under conditions of low ion concentrations) calcium). In particular, by administering such antigen-binding molecules in vivo, the elimination of the antigen is accelerated, and it is possible to lower the plasma concentration of the antigen. Exemplary antibodies that do not have the ability to bind antigens in a pH dependent or calcium ion concentration dependent manner and antigen-antibody complexes of such antibodies are incorporated into cells by non-specific endocytosis and transported to the cell surface by binding to FcRn under the acidic conditions of endosomes. They dissociate from FcRn under neutral conditions on the cell surface and are recycled into plasma. Thus, when an antibody that binds to an antigen in a fully pH dependent manner (which binds under conditions of the neutral pH range and dissociates under conditions of the acidic pH range) or in a completely calcium ion concentration dependent manner (which binds under conditions of high calcium ion concentration and dissociates under conditions of low concentration of calcium ions) binds to the antigen in plasma and dissociates from the antigen in the endosome, the rate of elimination of the antigen is considered equal to the rate of capture of the antibody or antigen-antibody complex into cells through nonspecific endocytosis. When pH or calcium ion dependence of antigen-antibody binding is insufficient, antigens that have not dissociated from antibodies in the endosome are recycled into plasma along with the antibodies. On the other hand, when the pH dependence is high enough, the rate-limiting step of antigen elimination is cellular uptake by non-specific endocytosis. Meanwhile, FcRn transports antibodies from the endosome to the cell surface, and it is expected that part of the FcRn is also distributed on the cell surface.

The inventors of the present invention concluded that IgG immunoglobulins having binding activity or having enhanced binding activity to Fcγ receptors under conditions of the neutral pH range can bind to Fcγ receptors present on the cell surface, and that IgG immunoglobulins are taken up into cells Fcγ receptor dependent manner through binding to Fcγ receptors present on the cell surface. The rate of uptake into cells via Fcγ receptors is higher than the rate of uptake into cells via non-specific endocytosis. Thus, it is believed that the rate of antigen elimination by antigen-binding molecules can be further increased by enhancing the ability to bind to Fcγ receptors under conditions of the neutral pH range. Therefore, antigen-binding molecules that have the ability to bind to Fcγ receptors under conditions of the neutral pH range capture antigens into cells faster than conventional (native human) IgG immunoglobulins and, after binding to FcRn in the endosome and dissociating antigens, they are recycled back into the plasma and they re-bind to antigens and are taken up into cells via Fcγ receptors. Since the turnover of this cycle can be increased by increasing the ability to bind to Fcγ receptors under conditions of the neutral pH range, the rate of antigen elimination from plasma will increase. Moreover, by providing antigen-binding molecules that have reduced antigen-binding activity under acidic pH range conditions compared to antigen-binding activity under neutral pH range conditions, the rate of elimination of antigen from plasma can be further increased. By increasing the number of cycles, as a result of increasing the turnover of this cycle, the number of antigen molecules that can be bound by one antigen-binding molecule can be increased. The antigen binding molecules of the present invention comprise an antigen binding domain and an Fcγ receptor binding domain, and since the Fcγ receptor binding domain does not affect antigen binding and based on the mechanism mentioned above, it is believed that regardless of the type of antigens, uptake of antigen into cells by antigen binding molecules can be enhanced and the rate of antigen elimination can be increased by reducing the binding activity (binding capacity) of the antigen-binding molecule to antigens under ionic concentration conditions, such as acidic pH range or low calcium ion concentration conditions, compared to the binding activity (binding capacity) to antigens under calcium ion concentration conditions, such as a neutral pH range, or high calcium ion concentration conditions and/or increased Fcγ receptor binding activity at plasma pH. Thus, the antigen-binding molecules of the present invention may exhibit better effects than conventional therapeutic antibodies in terms of reducing the side effects caused by antigens, increasing the dose of the antibody, and improving the kinetics of antibodies in vivo.

In FIG. 1 shows an embodiment of a mechanism for the elimination of soluble antigens from plasma by administering antibodies with ion concentration dependent antigen binding having increased binding to Fcγ receptors at neutral pH compared to conventional neutralizing antibodies. Hereinafter, antibodies with hydrogen ion concentration (ie pH) dependent antigen binding are described as an example of antibodies with ion concentration dependent antigen binding, however, the mechanisms are not limited to hydrogen ion concentration. Existing neutralizing antibodies that do not have the ability to pH-dependent antigen binding can be gradually captured, mainly through non-specific interactions with cells after binding to soluble plasma antigens. Complexes of neutralizing antibodies and soluble antigens that have been taken up into cells are translocated to acidic endosomes and recycled to plasma via FcRn. On the other hand, an antibody with pH-dependent antigen binding with increased binding to Fcγ receptors under neutral conditions binds to soluble antigen in plasma, and thereafter it can be rapidly taken up into cells expressing Fcγ receptors on the cell surface through interaction with Fcγ- receptor in addition to non-specific interaction. In this case, soluble antigens bound to the antibody with pH-dependent antigen binding dissociate from the antibody due to the ability to bind pH-dependently in acidic endosomes. Soluble antigens that have dissociated from the antibody then move into lysosomes and are degraded by proteolysis. Meanwhile, antibodies that have released soluble antigens bind to FcRn in the acidic endosome, and then are recycled to the cell membrane by FcRn, and are released back into plasma. Thus, free antibodies that are recycled can be rebound to other soluble antigens. Such pH-dependent antigen-binding antibodies, whose binding to Fcγ receptors under neutral conditions can move large amounts of soluble antigens into lysosomes, reduce total plasma antigen concentration through a repetitive cycle of: uptake into cells via Fcγ receptors; dissociation and degradation of soluble antigens; and recycling of the antibody.

In particular, the present invention also relates to pharmaceutical compositions containing antigen-binding molecules of the present invention, antigen-binding molecules obtained by the modification methods of the present invention, or antigen-binding molecules obtained by the preparation methods of the present invention.

The antigen-binding molecules of the present invention or the antigen-binding molecules obtained by the preparation methods of the present invention are useful as pharmaceutical compositions because they, when administered, have a powerful antigen-lowering effect in plasma compared to typical antigen-binding molecules, and exhibit improved in vivo immune response, pharmacokinetics etc. in animals to which the molecules are administered. Pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers.

Within the scope of the present invention, pharmaceutical compositions generally refer to agents for the treatment or prevention or research and diagnosis of diseases.

Pharmaceutical compositions of the present invention may be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions, including water or other pharmaceutically acceptable liquid. For example, such compositions can be formulated by mixing in the form of a single dose required by generally approved drug manufacturing practice, by combining as appropriate with pharmacologically acceptable carriers or vehicles, in particular, with sterile water, saline, vegetable oil, emulsifier, suspension, a surfactant, a stabilizer, a flavoring agent, an excipient, a carrier, a preservative, a binder, and the like. In such formulations, the amount of the active ingredient is adjusted so as to obtain an appropriate amount within the predetermined range.

Sterile injectable compositions may be formulated using carriers such as distilled water for injection in accordance with standard formulation practice.

Aqueous injections include, for example, physiological saline and isotonic solutions containing dextrose or other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, and sodium chloride). They can also be used in combination with appropriate solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (polysorbate 80(TM), HCO-50 and etc.).

Oils include sesame oil and soybean oils. In combination with solubilizers, benzyl benzoate and/or benzyl alcohol can be used. Buffers (eg, phosphate buffer, sodium acetate buffer), sedatives (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenol), and/or antioxidants can also be combined. The resulting injectables are filled into suitable ampoules.

The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, compositions are administered in dosage form for injection, transnasal, pulmonary or transdermal administration. For example, they can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

Routes of administration can be appropriately selected taking into account the age and symptoms of the patient. The dose of the pharmaceutical composition containing the antigen binding molecule may be, for example, from 0.0001 to 1.000 mg/kg for each administration. Alternatively, the dose may be, for example, from 0.001 to 100,000 mg per patient. However, the present invention is not limited to the numerical values described above. Doses and routes of administration vary depending on the patient's body weight, age, symptoms, and the like. Those skilled in the art can determine suitable doses and routes of administration, taking into account the factors described above.

The amino acids contained in the amino acid sequences of the present invention may be post-translationally modified (eg, modification of the N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the amino acid sequences of the present invention.

Methods Using the Antigen Binding Molecules of the Invention

The present invention also relates to a method comprising contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under conditions of an acidic pH range and comprises an antigen-binding domain and an Fcγ receptor-binding domain in which the antigen-binding activity of the antigen-binding domain varies with ion concentration conditions, and the Fcγ receptor-binding domain has higher Fcγ-receptor binding activity under conditions of the neutral pH range than the native Fcγ-receptor-binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, where the method is any of the following:

(i) a method for increasing the number of antigens to which a single antigen-binding molecule can bind;

(ii) a method for eliminating plasma antigens;

(iii) a method for improving the pharmacokinetics of an antigen-binding molecule;

(iv) a method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen is extracellularly bound to the antigen-binding molecule;

(v) a method for accelerating the release to the outside of the cell of an antigen-binding molecule in a non-antigen bound form; and

(vi) a method of reducing the concentration of free antigen or the total concentration of antigen in plasma.

Moreover, the present invention relates to a method comprising enhancing the binding activity of an Fcγ receptor under conditions of a neutral pH range of an Fcγ receptor binding domain in an antigen binding molecule compared to a native Fcγ receptor binding domain in which the sugar chain linked at position 297 ( EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain and an antigen-binding domain whose antigen-binding activity varies with ion concentration conditions, where the method is any of the following:

(i) a method for modifying an antigen-binding molecule, wherein the intracellular uptake of an antigen to be bound is enhanced;

(ii) a method for increasing the number of antigens to which a single antigen-binding molecule can bind;

(iii) a method for increasing the ability of an antigen-binding molecule to eliminate plasma antigens;

(iv) a method for improving the pharmacokinetics of an antigen-binding molecule;

(v) a method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen is extracellularly bound to the antigen-binding molecule;

(vi) a method for accelerating the release of an antigen-binding molecule to the outside of a cell in a non-antigen-bound form, wherein the antigen-binding molecule has been taken into the cell in an antigen-bound form; and

(vii) a method of altering the antigen-binding molecule, which can reduce the total antigen concentration or the free antigen plasma concentration.

Examples of methods for contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo include (1) the so-called ex vivo method, where plasma containing antigen-binding molecules and antigens that bind to antigen-binding molecules is temporarily removed from a living body; subjected to contact with cells expressing Fcγ receptors; and then, after a certain period of time, the plasma containing the extracellular recycled (or also referred to as resecreted and recirculated) antigen-binding molecules without bound antigens is returned to the living body, and (2) a method of introducing the antigen-binding molecules into the living body. In method (1), it is also possible to use the method of: temporarily extracting from a living body a plasma containing antigens to which antigen-binding molecules bind; contacting cells expressing Fcγ receptors and antigen-binding molecules; and, after a certain period of time, returning the plasma to the living body.

Within the scope of the present invention, Fcγ receptor-expressing cells are not limited to specific cells, and any type of cell can be used, as long as they are cells that express the desired Fcγ receptor(s). Well-known databases such as the Human Protein Atlas (http://www.proteinatlas.org/) can be used to identify cells that express the desired Fcγ receptor(s). Moreover, whether the receptors are expressed in the cells used to contact the antigen-binding molecules of the present invention can be confirmed by a method that confirms the expression of a gene encoding the desired Fcγ receptor(s), or by an immunological method that uses antibodies that bind to the desired Fcγ receptor(s). These methods are also well known. Since contact of Fcγ receptor-expressing cells with antigen-binding molecules and antigens that bind to antigen-binding molecules is also carried out in vivo, contacting Fcγ-receptor-expressing cells with antigen-binding molecules within the scope of the present invention includes introducing antigen-binding molecules into living organisms. The duration of contact is, for example, a suitable duration of one minute to several weeks, 30 minutes to one week, one hour to three days, and two hours to one day. More specifically, the duration required for the capture of antigen-binding molecules or antigens bound to antigen-binding molecules into cells by Fcγ receptor-mediated endocytosis is appropriately used as the duration of contact. For example, various immune cells can be used for Fcγ receptor expressing cells.

A method for enhancing the binding activity of an Fcγ receptor binding domain to an Fcγ receptor under neutral pH conditions relative to a native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain is described. in the section below in relation to the method for producing antigen-binding molecules of the present invention.

Method for modifying an antigen-binding molecule with increased intracellular uptake of antigens that bind to the antigen-binding molecule

The present invention relates to methods of altering an antigen binding molecule with increased intracellular uptake of antigens that bind to the antigen binding molecule, wherein the method comprises enhancing, under neutral pH range conditions, the Fcγ receptor binding activity of the Fcγ receptor binding domain in the antigen binding molecule compared to the native binding molecule. Fcγ receptor domain wherein the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain and an antigen-binding domain , the antigen-binding activity of which varies depending on the conditions of ion concentration.

In the context of the present invention, "intracellular antigen uptake" by an antigen-binding molecule means that antigens are taken into cells by Fcγ receptor-mediated endocytosis and internalization. In the context of the present invention, "increased uptake into cells" means that the rate of uptake into cells of antigen-binding molecules associated with antigens in plasma is increased and/or the amount of captured antigens that have been recycled into plasma is reduced and it is only necessary that the uptake rate into cells be increased. compared to the antigen-binding molecule before the decrease in antigen-binding activity (binding capacity) of the antigen-binding molecule under ionic concentration conditions, such as acidic pH range or low calcium ion concentration compared to antigen-binding activity under ionic concentration conditions, such as neutral pH range or high concentration calcium ions, in addition to enhancing the binding activity of the antigen-binding molecule to the Fcγ receptor in the neutral pH range, and preferably the rate increased compared to native human IgG, in which the sugar chain linked at position 297 ( EU numbering) is a fucose-containing sugar chain, and it is particularly preferred that the rate is increased over any of native human IgG1, IgG2, IgG3 or IgG4. Thus, within the scope of the present invention, whether the uptake of antigens by antigen-binding molecules is enhanced can be determined by observing whether the rate of uptake of antigens into cells is increased. The rate of uptake of antigens into cells can be calculated, for example, by adding an antigen-binding molecule and an antigen to a culture medium containing Fcγ receptor-expressing cells and measuring the decrease in antigen concentration in the culture medium over time, or measuring the amount of antigens taken up by Fcγ receptor-expressing cells with over time. Using the method of increasing the rate of uptake of antigens into cells by an antigen-binding molecule of the present invention, for example, by administering an antigen-binding molecule, the rate of elimination of an antigen from plasma can be increased. Thus, whether uptake of antigen into cells by the antigen-binding molecule is enhanced can also be confirmed, for example, by evaluating whether the rate of elimination of the antigen from plasma increases and whether administration of the antigen-binding molecule reduces the total plasma concentration of the antigen.

In the context of the present invention, "total plasma antigen concentration" means the sum of the concentrations of antigens bound to antigen-binding molecules and non-bound antigens, or "free antigen plasma concentrations", which is the concentration of antigens not bound to antigen-binding molecules. Various methods for measuring "total plasma antigen concentration" or "plasma free antigen concentrations" are well known in the art, as described herein below.

In the context of the present invention, "native human IgG" means unmodified human IgG and is not limited to a particular class of IgG. Moreover, in "native human IgG" it is desirable that the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain. This means that as long as human IgG1, IgG2, IgG3 or IgG4 can bind to human FcRn in the acidic pH range, they can be used as "native human IgG". Preferably, "native human IgG" may be human IgG1.

Method for increasing the number of antigens that can be bound by a single antigen-binding molecule

The present invention relates to a method for increasing the number of antigens to which a single antigen-binding molecule can bind, wherein the method comprises contacting the antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under acidic pH range conditions and contains an antigen-binding domain and an Fcγ receptor-binding domain, in which the antigen-binding activity of the antigen-binding domain changes depending on the conditions of ion concentration, and the Fcγ-receptor binding domain has a higher activity of binding to the Fcγ receptor under conditions of the neutral pH range compared to the native binding An Fcγ receptor domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

Furthermore, the present invention relates to a method for increasing the number of antigens to which a single antigen binding molecule can bind, wherein the method comprises enhancing an Fcγ receptor binding activity under conditions of a neutral pH range of the Fcγ receptor binding domain in the antigen binding molecule compared to a native Fcγ-binding molecule. a receptor domain wherein the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain and an antigen-binding domain, antigen-binding the activity of which varies depending on the conditions of ion concentration.

The expression "number of antigens to which one antigen-binding molecule can bind" in the context of the present invention means the number of antigens to which an antigen-binding molecule can bind until the molecule is degraded or eliminated. The expression "increasing the number of antigens to which one antigen-binding molecule can bind" in the context of the present invention refers to increasing the number of times an antigen molecule bound to an antigen-binding molecule dissociates and binds the antigen-binding molecule back to the antigen molecule. The antigen molecules that bind to the antigen-binding molecule may be the same antigen molecule or different molecules existing in the reaction system where both molecules are present. In other words, it refers to the total number of times an antigen-binding molecule binds to an antigen in a reaction system. In another expression, when one cycle is taken to represent the intracellular uptake of an antigen-binding molecule bound to the antigen, dissociation of the antigen in the endosome, and then the return of the antigen-binding molecule to the outside of the cell, the expression refers to an increase in the number of times this cycle can be repeated until until the antigen-binding molecule is degraded or eliminated. After binding to an Fcγ receptor, antigen-binding molecules of the present invention having an Fcγ receptor binding activity in the neutral pH range are taken up into a cell expressing the Fcγ receptor by endocytosis. The antigen-binding molecule of the present invention, which has dissociated from the Fcγ receptor under ion concentration conditions such as acidic pH range or low calcium ion concentration, is recycled outside the cell by binding to FcRn under acidic pH range conditions. The antigen-binding molecule of the present invention, which is recycled to the outside of the cell after dissociation of the antigen from the antigen-binding molecule under ion concentration conditions such as an acidic pH range or a low calcium ion concentration, can re-bind to the antigen. Thus, whether the number of cycles increases can be assessed by determining whether the aforementioned "increased intracellular uptake" occurs or whether the "improvement in pharmacokinetics" described below occurs.

Method for eliminating plasma antigens or method for increasing the ability of an antigen-binding molecule to eliminate plasma antigens

The present invention relates to a method for eliminating antigens from plasma, which comprises contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn-binding activity under conditions of an acidic pH range and contains an antigen-binding domain and an Fcγ-binding receptor a domain in which the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions and the Fcγ receptor-binding domain has a higher binding activity to the Fcγ receptor under conditions of the neutral pH range compared to the native Fcγ receptor-binding domain in which the chain sugar linked at position 297 (EU numbering) is a fucose-containing sugar chain.

Furthermore, the present invention relates to a method for enhancing the ability of an antigen binding molecule to eliminate plasma antigens, wherein the method comprises enhancing an Fcγ receptor binding activity under conditions of a neutral pH range of an Fcγ receptor binding domain in an antigen binding molecule compared to a native Fcγ receptor binding domain, in wherein the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain, and an antigen-binding domain whose antigen-binding activity is altered depending on the conditions of ion concentration.

In the context of the present invention, the expression "method of increasing the ability to eliminate plasma antigens" has the same meaning as "method of increasing the ability of an antigen-binding molecule to eliminate antigens from plasma".

In the context of the present invention, "the ability to eliminate plasma antigens" refers to the ability to eliminate from plasma antigens present in plasma when antigen-binding molecules are administered in vivo or antigen-binding molecules are secreted in vivo. Thus, within the scope of the present invention, the whole expression "the ability of the antigen-binding molecule to eliminate plasma antigens is increased" means that when the antigen-binding molecule is administered, the rate of elimination of the antigen from plasma increases compared to the rate before the antigen-binding activity of the antigen-binding molecule decreases under conditions of ion concentrations such as as an acidic pH range or low calcium ion concentration, compared to antigen-binding activity in the neutral pH range or high calcium ion concentration, in addition to enhancing the binding activity of the antigen-binding molecule to the Fcγ receptor in the neutral pH range. Whether the ability of antigen-binding molecules to eliminate plasma antigens is increased can be determined, for example, by introducing soluble antigens and antigen-binding molecules into a living body and measuring the plasma concentration of soluble antigens after administration. When the plasma concentration of soluble antigens is reduced following administration of soluble antigens and antigen-binding molecules, by enhancing the binding activity of antigen-binding molecules to Fcγ receptors under conditions of the neutral pH range, or by decreasing the antigen-binding activity of the antigen-binding molecule under conditions of ionic concentration, such as an acidic pH range or low calcium ion concentration, compared with antigen-binding activity under ion concentration conditions such as neutral pH range or high calcium ion concentration, in addition to enhancing Fcγ receptor binding activity, it can be determined that the ability of antigen-binding molecules to eliminate plasma antigens is enhanced. Soluble antigens can be antigens to which antigen-binding molecules actually bind (antigens in the form of an antigen/antigen-binding molecule complex) or antigens to which antigen-binding molecules do not bind, and their concentrations can be defined as "plasma concentration of antigen-binding molecule-bound antigens". and "plasma concentration of non-antigen-binding molecule antigens", respectively (the latter has the same meaning as "plasma free antigen concentration"). Since "total plasma antigen concentration" means the sum of the concentrations of antigen-bound antigens and non-antigen-binding molecule-bound antigens, or "free plasma antigen concentration", which is the concentration of non-antigen-bound antigens, the soluble antigen concentration can be defined as "total plasma antigen concentration". Various methods of measuring "total plasma antigen concentration" or "plasma free antigen concentration" are well known in the art, as described herein below.

Method for improving the pharmacokinetics of antigen-binding molecules

The present invention relates to a method for improving the pharmacokinetics of an antigen-binding molecule, which comprises contacting the antigen-binding molecule with an Fcγ receptor-expressing cell, in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under conditions of an acidic pH range and contains an antigen-binding domain and an Fcγ binding -receptor domain, in which the antigen-binding activity of the antigen-binding domain changes depending on the conditions of ion concentration, and the Fcγ receptor-binding domain has a higher binding activity to the Fcγ receptor under conditions of the neutral pH range compared to the native Fcγ receptor-binding domain, in in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

Moreover, the present invention relates to a method for improving the pharmacokinetics of an antigen binding molecule, wherein the method comprises enhancing an Fcγ receptor binding activity under conditions of a neutral pH range of an Fcγ receptor binding domain in an antigen binding molecule compared to a native Fcγ receptor binding domain in which the sugar chain , linked at position 297 (EU numbering), is a fucose-containing sugar chain, wherein an antigen-binding molecule having human FcRn-binding activity under acidic pH range conditions contains an Fcγ receptor-binding domain and an antigen-binding domain whose antigen-binding activity varies depending on ion concentration conditions.

In the present description, "enhancing pharmacokinetics", "improving pharmacokinetics" and "superior pharmacokinetics" can be reformulated as "enhancing plasma (blood) retention", "improving plasma (blood) retention", "superior plasma (blood) retention", and "prolonged retention in plasma (blood)". These terms are synonyms.

As used herein, "improvement in pharmacokinetics" means not only prolonging the period of time until elimination from plasma (for example, until the antigen-binding molecule is degraded intracellularly, etc. and can return to plasma) after administration of the antigen-binding molecule to a human or non-human animal such as mice, rats, monkeys, rabbits and dogs, but also the prolongation of plasma retention of antigen-binding molecules in a form that permits antigen binding (eg, antigen-free form of the antigen-binding molecule) during the period of administration until elimination due to degradation. Native IgG can bind to FcRn from non-human animals. For example, a mouse can preferably be used for administration in order to confirm the property of an antigen-binding molecule of the invention, since native IgG can bind more strongly to mouse FcRn than to human FcRn (Int Immunol. (2001) 13(12): 1551-1559). As another example, also as an individual for administration in order to confirm the property of the antigen-binding molecule of the invention described later, a mouse deficient in its native FcRn genes and transgene of the human FcRn gene (Methods Mol Biol. 2010; 602: 93- 104). In particular, "improved pharmacokinetics" also includes prolonging the time to elimination due to degradation of the non-antigen-bound antigen-binding molecule (antigen-free form of the antigen-binding molecule). An antigen-binding molecule in plasma cannot bind to a new antigen if the antigen-binding molecule is already bound to the antigen. Thus, the longer the period of time when an antigen-binding molecule is not bound to an antigen, the longer is the period of time when it can bind to a new antigen (higher probability of binding to another antigen). This provides for a reduction in the period of time that the antigen is free from the antigen-binding molecule in vivo and an extension of the period of time that the antigen is bound to the antigen-binding molecule. The plasma concentration of the antigen-free form of the antigen-binding molecule can be increased and the period of time that the antigen is bound to the antigen-binding molecule can be prolonged by accelerating the elimination of the antigen from the plasma by administration of the antigen-binding molecule. Specifically, as used herein, "improving the pharmacokinetics of an antigen-binding molecule" includes improving the pharmacokinetic parameter of the antigen-free form of the antigen-binding molecule (any of prolonging plasma half-life, prolonging mean plasma retention time, and decreasing plasma clearance), prolonging the period of time when the antigen associated with the antigen-binding molecule, after administration of the antigen-binding molecule, and accelerating the antigen-binding molecule-mediated elimination from plasma. Improvement in the pharmacokinetics of an antigen-binding molecule can be assessed by determining any of the parameters: plasma half-life, mean plasma retention time, and plasma clearance, for the antigen-binding molecule or its antigen-free form ("Pharmacokinetics: Enshu-niyoru Rikai (Understanding through practice)" Nanzando). For example, the plasma concentration of an antigen-binding molecule or an antigen-free form thereof is determined after administration of the antigen-binding molecule to mice, rats, monkeys, rabbits, dogs, or humans. Then, each parameter is determined. When the plasma half-life or mean plasma retention time is prolonged, the plasma pharmacokinetics of the antigen-binding molecule can be considered to be increased. The parameters can be determined by methods known to those skilled in the art. Parameters can be assessed as appropriate, for example, by non-compartmental analysis using WinNonlin pharmacokinetic analysis software (Pharsight) according to the instructions for use. The plasma concentration of an antigen-free antigen-binding molecule can be determined by methods known to those skilled in the art, for example using a known assay method (Clin Pharmacol. 2008 Apr; 48(4): 406-417).

Within the scope of the present invention, "improving pharmacokinetics" also includes prolonging the period when an antigen is bound to an antigen-binding molecule after administration of an antigen-binding molecule. Assessing whether the period when the antigen is bound to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule can be made by determining the plasma concentration of free antigen. Prolongation can be inferred from a given plasma concentration of free antigen or the period of time required to increase the ratio of free antigen to total antigen.

The concentration of free antigen not bound to an antigen-binding molecule, or the ratio of free antigen to total antigen, can be determined by methods known to those skilled in the art, for example, the determination method provided in Pharm Res. 2006 Jan; 23(1):95-103. Alternatively, when an antigen exhibits a particular function in vivo, determining whether the antigen is bound to an antigen-binding molecule that neutralizes the function of the antigen (antagonist molecule) can be done by examining whether the function of the antigen is neutralized. Whether antigen function is neutralized can be assessed by in vivo analysis of a marker that reflects antigen function. Whether an antigen is bound to an antigen-binding molecule that activates antigen function (agonist molecule) can be assessed by in vivo analysis of a marker that reflects antigen function.

Determining the plasma concentration of free antigen and the ratio of the amount of free antigen in plasma and the amount of total antigen in plasma, in vivo marker analysis and similar detection methods are not particularly limited; however, the assays are preferably performed after a certain period of time has elapsed after administration of the antigen-binding molecule. Within the scope of the present invention, the period following administration of the antigen-binding molecule is not specifically limited; those skilled in the art can determine the appropriate period depending on properties and the like. administered antigen-binding molecule. Such periods include, for example, one day after administration of the antigen-binding molecule, three days after administration of the antigen-binding molecule, seven days after administration of the antigen-binding molecule, 14 days after administration of the antigen-binding molecule, and 28 days after administration of the antigen-binding molecule. As used herein, "plasma antigen concentration" refers to either "total plasma antigen concentration", which is the sum of the concentration of antigen bound and not bound by an antigen-binding molecule, or to "free plasma antigen concentration", which is the concentration of antigen not bound by an antigen-binding molecule.

By administering the antigen-binding molecule of the present invention, the total plasma antigen concentration can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or even more compared to the introduction of a reference antigen-binding molecule containing the Fc region of native human IgG, in which the sugar chain linked at position 297 (EU numbering) is a sugar chain having fucose as the Fcγ receptor binding domain or compared with when the antigen-binding domain molecule containing the antigen-binding domain of the present invention is not administered.

The molar ratio of antigen/antigen binding molecule can be calculated as shown below;

value A: Molar concentration of antigen at each time point

value B: Molar concentration of the antigen-binding molecule at each time point

C value: Molar concentration of antigen per molar concentration of antigen-binding molecule (molar ratio of antigen/antigen-binding molecule) at each time point

C=A/B.

A lower C value indicates a higher antigen elimination efficiency per antigen binding molecule, while a higher C value indicates a lower antigen elimination efficiency per antigen binding molecule.

The molar ratio of antigen/antigen binding molecule can be calculated as described above.

The molar ratio of antigen/antigen-binding molecule can be reduced by administering the antigen-binding molecule of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or even higher than the introduction of a reference antigen-binding molecule containing the wild-type human IgG Fc region as the human Fcγ receptor-binding domain.

In the context of the present invention, native IgG1, IgG2, IgG3 or IgG4 is preferably used as native human IgG, which is used as a reference native human IgG to be compared with antigen binding molecules for their Fcγ receptor binding activity or in vivo activity. Preferably, a reference antigen-binding molecule containing the same antigen-binding domain as the antigen-binding molecule of interest and a native human IgG Fc region as the Fcγ receptor binding domain can be suitably used. More preferably, native human IgG1 is used as a reference native human IgG to be compared with antigen-binding molecules in terms of their Fcγ receptor binding activity or in vivo activity. Moreover, in the context of the present invention, as a reference antigen-binding molecule to be compared with the antigen-binding molecules of the present invention, an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity does not change with ion concentration, an antigen-binding molecule containing An FcRn-binding domain whose FcRn-binding activity is not enhanced under acidic pH conditions, an antigen-binding molecule containing an Fcγ receptor-binding domain that does not selectively bind to Fcγ receptors, and the like depending on the purpose.

The reduction in total plasma concentration or antigen/antibody molar ratio can be assessed as described in Examples 6, 8 and 13. More specifically, using transgenic mice with human FcRn 32 or 276 (Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104), they can be assessed either using an antigen-antibody co-administration model or a stationary infusion model where the antigen-binding molecule does not cross-react with a mouse antigen analog. When the crosshair antigen binding molecule reacts with a mouse analog, they can be assessed by simply administering the antigen binding molecule to transgenic mouse mice with human FcRn 32 or 276 (Jackson Laboratories). In the mouse co-administration model, a mixture of antigen-binding molecule and antigen is administered. In the stationary infusion model, mice are implanted with an infusion pump containing an antigen solution to achieve a constant plasma concentration of antigen, and then the mice are injected with an antigen-binding molecule. The antigen-binding molecule to be tested is administered at the same dosage. The total plasma antigen concentration, the free antigen plasma concentration, and the plasma concentration of the antigen-binding molecule are measured at the appropriate time point using a method known to those skilled in the art.

To evaluate the effects of an Fcγ receptor-binding domain having selective binding activity for Fcγ receptors when the antigen-binding molecule does not cross-react with the same mouse antigen, the total plasma antigen concentration or reduction in the antigen/antibody molar ratio can be assessed either through the antigen-antigen co-injection model. antibody, or by a steady state antigen injection model using conventionally used C57BL/6J mice (Charles River Japan). When an antigen-binding molecule cross-reacts with a mouse analog, the antigen-binding molecule can simply be injected into conventional C57BL/6J mice (Charles River Japan) for evaluation.

Plasma total or free antigen concentration and antigen/antigen binding molecule molar ratio can be measured 2, 4, 7, 14, 28, 56, or 84 days after administration to assess the long-term effect of the present invention. In other words, long-term plasma antigen concentration is determined by measuring the concentration of total or free antigen in plasma and the molar ratio of antigen/antigen-binding molecule after 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to evaluate the properties of the antigen-binding molecule by the present invention. Whether a reduction in plasma antigen concentration or antigen/antigen binding molecule molar ratio is achieved with an antigen binding molecule of the present invention can be determined by assessing the reduction at any one or more of the time points described above.

Plasma total or free antigen concentration and antigen/antigen-binding molecule molar ratio can be measured 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration to assess the short-term effect of the present invention. In other words, short-term plasma antigen concentration is determined by measuring the concentration of total or free antigen in plasma and the molar ratio of antigen/antigen-binding molecule at 15 min, 1, 2, 4, 8, 12, or 2-4 hours after administration of the antigen-binding molecule to assess the property of the antigen-binding molecule. molecules of the present invention.

The route of administration of the antigen-binding molecule of the present invention can be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral and intramuscular injection.

Within the scope of the present invention, the improvement of pharmacokinetics in humans is preferred. Where human plasma retention is difficult to determine, it can be predicted from plasma retention in mice (eg healthy mice, transgenic mice expressing human antigen, transgenic mice expressing human FcRn) or monkeys (eg cynomolgus monkeys).

A method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, where the antigen is extracellularly bound to the antigen-binding molecule

The present invention relates to a method for accelerating intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen is extracellularly bound to an antigen-binding molecule, wherein the method comprises contacting the antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under range conditions acidic pH values and contains an antigen-binding domain and an Fcγ receptor-binding domain, where the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions and the Fcγ receptor-binding domain has a higher binding activity to the Fcγ receptor under conditions of the neutral pH range compared to a native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

The present invention relates to a method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule, wherein the antigen has intracellularly bound to the antigen-binding molecule, wherein the method comprises enhancing Fcγ receptor binding activity under conditions of a neutral pH range of the Fcγ receptor-binding domain in the antigen-binding molecule compared to native Fcγ binding a receptor domain wherein the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain and an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration.

Within the scope of the present invention, the location where the antigen dissociates from the antigen-binding molecule can be anywhere as long as it is in the cell, but preferably it is in the early endosome. In the context of the present invention, "intracellular dissociation of an antigen from an antigen-binding molecule, where the antigen is extracellularly bound to an antigen-binding molecule," does not mean that all antigens taken up into cells after binding to antigen-binding molecules intracellularly dissociate from antigen-binding molecules, and the ratio of intracellular dissociation of antigens from antigen-binding molecules molecules should only be higher compared to the ratio to reduce the antigen-binding activity of the antigen-binding molecules under ionic concentration conditions, such as an acidic pH range or low calcium ion concentration, than under ionic concentration conditions, such as a neutral pH range or high calcium ion concentration , and there is an increase in Fcγ receptor binding activity in the range of neutral pH values. Moreover, a method for accelerating the intracellular dissociation of an antigen from an antigen-binding molecule can be referred to as a method that enhances the intracellular uptake of antigen-bound antigen-binding molecules and imparts to the antigen-binding molecules the property of facilitating the acceleration of intracellular dissociation of antigens from antigen-binding molecules.

Method for accelerating the extracellular release of an antigen-binding molecule in a non-antigen bound form

The present invention relates to a method for accelerating the extracellular release of an antigen-binding molecule in a non-antigen-bound form, wherein the method comprises contacting the antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under acidic pH range conditions and contains an antigen-binding domain and an Fcγ receptor-binding domain, where the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions and the Fcγ-receptor-binding domain has a higher Fcγ-receptor binding activity under conditions of the neutral pH range compared to the native Fcγ-binding receptor a domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

Moreover, the present invention relates to a method for accelerating the extracellular release of an antigen-binding molecule in a non-antigen bound form, wherein the antigen-binding molecule has been taken up into a cell in an antigen-bound form, wherein the method comprises enhancing Fcγ receptor binding activity under conditions of the neutral pH range of the Fcγ binding α-receptor domain in an antigen-binding molecule compared to a native Fcγ receptor-binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, where the antigen-binding molecule has human FcRn binding activity under acidic range conditions pH and contains a Fcγ receptor-binding domain and an antigen-binding domain, the antigen-binding activity of which varies depending on the ion concentration conditions.

For the purposes of the present invention, the expression "extracellular release of an antigen-binding molecule in non-antigen-bound form, where the antigen-binding molecule has been taken up into the cell in antigen-bound form" should not mean that all antigen-binding molecules that have been taken into the cell in antigen-bound form, are extracellularly released in non-antigen bound form and the ratio of antigen-binding molecules extracellularly released in non-antigen bound form should only be higher compared to the ratio before antigen-binding activity of antigen-binding molecules is reduced under ionic concentration conditions such as an acidic pH range or low calcium ion concentration than under ion concentration conditions such as neutral pH range or high calcium ion concentration, and there is an increase in Fc receptor activity in the neutral pH range. Extracellularly released antigen-binding molecules preferably retain antigen-binding activity. Moreover, a method for promoting the extracellular release of an antigen-binding molecule in a non-antigen-bound form, wherein the antigen-binding molecule has been taken up into a cell in an antigen-bound form, can be referred to as a method that enhances the uptake of antigen-bound antigen-binding molecules into cells and imparts to the antigen-binding molecules the property facilitating the acceleration of the extracellular release of the antigen-binding molecule in a non-antigen bound form.

Method for reducing total antigen concentration or free antigen plasma concentration, or method for altering an antigen-binding molecule that can reduce total antigen concentration or free antigen plasma concentration

The present invention relates to a method for reducing total or free antigen plasma concentration, wherein the method comprises contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo, wherein the antigen-binding molecule has human FcRn binding activity under conditions of an acidic pH range and comprises an antigen-binding domain and an Fcγ receptor-binding domain, in which the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions and the Fcγ receptor-binding domain has a higher binding activity to the Fcγ receptor under conditions of the neutral pH range compared to native Fcγ-binding a receptor domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

Moreover, the present invention relates to a method for modifying an antigen-binding molecule that can reduce total antigen concentration or plasma free antigen concentration, wherein the method comprises enhancing an Fcγ receptor binding activity under conditions of a neutral pH range of an Fcγ receptor-binding domain in an antigen-binding molecule compared to with a native Fcγ receptor-binding domain wherein the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain, wherein the antigen-binding molecule has human FcRn binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain and an antigen-binding domain, the antigen-binding activity of which varies depending on the ion concentration conditions.

A method for evaluating a decrease in total antigen concentration or free antigen concentration in plasma is described in the above-mentioned section on the method for improving the pharmacokinetics of antigen-binding molecules.

Method for elimination of antigens from plasma ex vivo

An example of a non-limiting embodiment of the use of an antigen-binding molecule for the method of eliminating antigens from plasma, which is provided by the present invention, includes the use of an antigen-binding molecule for the so-called ex vivo method for eliminating antigens from plasma, which includes contacting the antigen-binding molecule of the present invention with plasma isolated from individuals to allow the formation of immunocomplexes; and allow the immunocomplexes to contact cells expressing Fcγ receptors and FcRn. The rate of elimination of plasma antigens can be increased by replacing/combining the method of administering antigen-binding molecules in vivo with the so-called ex vivo method, where plasma containing antigen-binding molecules and antigens that bind to antigen-binding molecules is temporarily removed from a living body and then contacted with cells expressing Fcγ receptors and FcRn, and plasma containing extracellularly recycled (or also referred to as resecreted or recirculated) antigen-binding molecules without bound antigen after a certain period of time is returned to the living body.

Moreover, an exemplary non-limiting embodiment of the use of an antigen-binding molecule for a method for eliminating antigens from plasma, which is contemplated within the scope of the present invention, would include the use of an antigen-binding molecule for a so-called ex vivo method for eliminating antigens from plasma, which involves contacting immunocomplexes present in plasma isolated from individuals treated with the antigen-binding molecules of the present invention with cells expressing FcRn and Fcγ receptors.

Confirmation of whether antigens are eliminated from plasma can be carried out, for example, by assessing whether the rate of elimination from plasma of the antigens mentioned above is accelerated when, instead of the antigen-binding molecule of the present invention, an antigen-binding molecule containing an antigen-binding domain is used as a comparison control, in in which the antigen-binding activity does not change with ion concentration, an antigen-binding molecule containing an FcRn-binding domain whose FcRn-binding activity does not increase under acidic pH conditions, or an antigen-binding molecule containing an Fcγ receptor-binding domain that does not have selective binding activity with Fcγ receptors.

Method for producing antigen-binding molecules

The present invention also relates to a method for producing an antigen-binding molecule comprising an antigen-binding domain and an Fcγ receptor-binding domain and having a human FcRn-binding activity under conditions of an acidic pH range, wherein the antigen-binding activity of the antigen-binding domain varies depending on the ion concentration conditions and the Fcγ receptor-binding domain has higher Fcγ receptor binding activity under neutral pH conditions than a native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain.

In particular, the present invention relates to a method for producing an antigen-binding molecule, which includes the following steps (a)-(f):

(a) determining the antigen-binding activity of the antigen-binding domain under high calcium ion conditions;

(b) determining the antigen-binding activity of the antigen-binding domain under low calcium ion conditions;

(c) selecting an antigen-binding domain for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen binding domain selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having human FcRn binding activity in the acidic pH range, which has higher Fcγ receptor binding activity under neutral pH range conditions. pH values than the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced to which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from cell culture according to (e).

The present invention also relates to a method for producing an antigen-binding molecule, which includes the following steps (a)-(f):

(a) determining the antigen-binding activity of the antibody under conditions of high concentration of calcium ions;

(b) determining the antigen-binding activity of the antibody under low calcium ion conditions;

(c) selecting an antibody for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen-binding domain of an antibody selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having a human FcRn binding activity in the acidic pH range and having a higher Fcγ receptor binding activity under the conditions of the range neutral pH values than the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced to which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from cell culture according to (e).

In addition, the present invention relates to a method for producing an antigen-binding molecule, which includes the following steps (a)-(f):

(a) determining the antigen-binding activity of the antigen-binding domain under neutral pH conditions;

(b) determining the antigen-binding activity of the antigen-binding domain under acidic pH conditions;

(c) selecting an antigen-binding domain for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen binding domain selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having human FcRn binding activity under acidic pH range conditions, which has higher Fcγ receptor binding activity under conditions a neutral pH range than the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced to which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from the cell culture according to (e).

In addition, the present invention relates to a method for producing an antigen-binding molecule, which includes the following steps (a)-(f):

(a) determining the antigen-binding activity of the antibody under neutral pH conditions;

(b) determining the antigen-binding activity of the antibody under acidic pH conditions;

(c) selecting an antibody for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b);

(d) linking a polynucleotide encoding an antigen-binding domain of an antibody selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain having a human FcRn binding activity in the acidic pH range and having a higher Fcγ receptor binding activity under the conditions of the range neutral pH values than the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain;

(e) culturing cells into which a vector is introduced to which the polynucleotide obtained according to (d) is operably linked; and

(f) collecting antigen-binding molecules from the cell culture according to (e).

The terms "cell", "cell line", and "cell culture" are used synonymously herein and such designations may include any progeny of a cell or cell line. Thus, for example, the terms "transformants" and "transformed cells" include the primary cells in question and the cultures derived from them, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content due to intentional or accidental mutations. Mutant progeny may also be included that have substantially the same functions or biological activity for which the originally transformed cells were screened. If another designation is intended, this will be apparent from the context of the description.

When referring to the expression of a coding sequence, the term "control sequence" refers to DNA nucleotide sequences that are necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for, for example, prokaryotes include a promoter, an optional operator sequence, a ribosome binding site, and possibly other sequences that are still to be explored. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers to express a coding sequence.

For a nucleic acid, the term "operably linked" means that the nucleic acid has a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a precursor protein that is involved in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. A ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. Typically, the expression "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretory leader, are contiguous and in reading frame. However, enhancers do not need to be contiguous. Linking is carried out by ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with common practice. Moreover, bound nucleic acids can be obtained using the overlapping PCR technology mentioned above.

"Ligation" refers to a method of forming a phosphodiester bond between two nucleic acid fragments. To ligate two fragments, the ends of these fragments must be compatible with each other. In some cases, these ends are compatible immediately after endonuclease cleavage. However, it may be necessary to first convert the sticky ends normally produced by cleavage with an endonuclease to blunt ends in order to be compatible for ligation. For blunt-end conversion, the DNA is treated in an appropriate buffer for at least 15 minutes at 15° C. with approximately 10 Klenow fragment units of DNA polymerase I or T4 DNA polymerase in the presence of four deoxyribonucleotide triphosphates in an appropriate buffer solution. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation, or by silica purification. The DNA fragments to be fused are added in equimolar amounts to the solution. This solution contains ATP and ligase buffer as well as approximately 10 units of ligase such as T4 DNA ligase per 0.5 µg of DNA. If the DNA is to be ligated to a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease. The linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase, thereby preventing the fragment from self-ligating during the ligation step.

In the preparation methods of the present invention, antigen-binding domains or antibodies having higher antigen-binding activity under high calcium ion conditions than under low calcium ion concentration conditions, which are selected by the method described in the "ion concentration conditions" section mentioned above, are isolated. Moreover, antigen-binding domains or antibodies having higher antigen-binding activity under neutral pH range conditions than under acidic pH range conditions, which are selected by the method described in the "ion concentration conditions" section mentioned above, are isolated. For example, when antigen binding domains thus isolated are selected from a library as described in the examples below, polynucleotides encoding antigen binding domains are selected by conventional gene amplification from viruses such as phages. Alternatively, when the antigen-binding domains or antibodies thus isolated are antibodies selected from cell cultures such as hybridomas as described in the antibody section mentioned above, antibody genes, and the like. isolated by conventional gene amplification from these cells.

Further, a polynucleotide encoding an antigen-binding domain isolated as described above is linked in-frame to a polynucleotide encoding an Fcγ receptor-binding domain having a human FcRn binding activity in the acidic pH range and having a higher Fcγ receptor binding activity in the range neutral pH than the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain. A suitable example of an Fcγ receptor binding domain includes the Fc region of an antibody as described in the Fcγ receptor binding domain section mentioned above. Moreover, examples of the Fcγ receptor include FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V) or FcγRIIIa(F).

Suitable examples of the Fc region of an antibody include Fc regions having at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233 , 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265 , 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293 , 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 324 325 326 327 328 , 329, 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 in the region of the Fc region according to EU numbering, which differ from the amino acids in the corresponding regions in the native Fc region. A suitable example of a native Fc region is an Fc region according to any one of IgG1, IgG2, IgG3 and IgG4.

In a non-limiting embodiment, a suitable example of an Fc region of an antibody is an Fc region containing at least one or more amino acids selected from the group consisting of:

either Lys or Tyr at amino acid position 221;

any of Phe, Trp, Glu and Tyr at amino acid position 222;

any of Phe, Trp, Glu and Lys at amino acid position 223;

any of Phe, Trp, Glu and Tyr at amino acid position 224;

any of Glu, Lys and Trp at amino acid position 225;

any of Glu, Gly, Lys and Tyr at amino acid position 227;

any of Glu, Gly, Lys and Tyr at amino acid position 228;

any of Ala, Glu, Gly and Tyr at amino acid position 230;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231;

any of Glu, Gly, Lys and Tyr at amino acid position 232;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 237;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 238;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

any of Ala, Ile, Met and Thr at amino acid position 240;

any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

His at amino acid position 244;

Ala at amino acid position 245;

any of Asp, Glu, His and Tyr at amino acid position 246;

any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

any of Glu, His, Gln and Tyr at amino acid position 249;

either Glu or Gln at amino acid position 250;

Phe at amino acid position 251;

any of Phe, Met and Tyr at amino acid position 254;

any of Glu, Leu and Tyr at amino acid position 255;

any of Ala, Met and Pro at amino acid position 256;

any of Asp, Glu, His, Ser and Tyr at amino acid position 258;

any of Asp, Glu, His and Tyr at amino acid position 260;

any of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

any of Ala, Ile, Met and Thr at amino acid position 263;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264;

any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265;

any of Ala, Ile, Met and Thr at amino acid position 266;

any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271;

any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

either Phe or Ile at amino acid position 273;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

either Leu or Trp at amino acid position 275;

any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

Ala at amino acid position 279;

any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

any of Asp, Lys, Pro and Tyr at amino acid position 281;

any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

any of Glu, Gly, Pro and Tyr at amino acid position 286;

any of Asn, Asp, Glu and Tyr at amino acid position 288;

any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300;

any of Asp, Glu, His and Tyr at amino acid position 301;

Ile at amino acid position 302;

any of Asp, Gly and Tyr at amino acid position 303;

any of Asp, His, Leu, Asn and Thr at amino acid position 304;

any of Glu, Ile, Thr and Tyr at amino acid position 305;

any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

Phe at amino acid position 313;

Leu at amino acid position 315;

either Glu or Gln at amino acid position 317;

any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

Ile at amino acid position 323;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

any of Glu, Lys and Tyr at amino acid position 336;

any of Glu, His and Asn at amino acid position 337;

any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

either Ala or Val at amino acid position 376;

either Gly or Lys at amino acid position 377;

Asp at amino acid position 378;

Asn at amino acid position 379;

any of Ala, Asn and Ser at amino acid position 380;

either Ala or Ile at amino acid position 382;

Glu at amino acid position 385;

Thr at amino acid position 392;

Leu at amino acid position 396;

Lys at amino acid position 421;

Asn at amino acid position 427;

either Phe or Leu at amino acid position 428;

Met at amino acid position 429;

Trp at amino acid position 434;

Ile at amino acid position 436; and

any of Gly, His, Ile, Leu, and Tyr at amino acid position 440;

in the region of the Fc region according to the EU numbering.

Moreover, for the Fc regions of the present invention, Fc regions that have binding activity or enhanced binding activity to FcRn under acidic pH range conditions can be suitably used. Examples of such Fc regions include IgG-type immunoglobulin Fc regions, such as human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof). For variants having alterations to other amino acids, Fc regions with amino acid alterations at any position can be used, provided that there is FcRn binding activity in the acidic pH range or human FcRn binding activity in the acidic pH range can be increased and when the antigen-binding molecule includes the Fc region of human IgG1 as the Fc region, it preferably includes a change that enhances binding to FcRn under acidic pH range conditions compared to the binding activity of the Fc region as the starting material of human IgG1. Suitable examples of amino acids that can be changed include amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447 (EU numbering) as described in WO 2000/042072. Similarly, suitable examples of amino acids that may be altered also include amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436 (EU numbering) as described in WO 2002/060919. Moreover, examples of amino acids that can be changed also include the amino acids at positions 250, 314 and 428 (EU numbering) as described in WO 2004/092219. Other suitable examples of amino acids that can be changed also include the amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434 and/or 436 (EU numbering) as described in WO 2010/045193. Fc regions obtained by enhancing FcRn binding in the acidic pH range of IgG immunoglobulin Fc regions through amino acid changes can be used in the production methods of the present invention.

Moreover, as described below, for the Fc region included in the antigen-binding molecule of the present invention, an Fc region having an FcRn binding activity in the neutral pH range can be suitably used. Such an Fc region can be obtained by any method in accordance with the above-mentioned method for obtaining Fc regions having FcRn binding activity in the acidic pH range. In particular, it is possible to obtain an Fc region containing an FcRn binding domain having a binding activity or having an enhanced binding activity to an FcRn in the neutral pH range due to amino acid changes in the Fc region of a human IgG immunoglobulin used as the starting Fc region. material. Favorable IgG immunoglobulin Fc regions for alteration include human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof). For variants having alterations to other amino acids, Fc regions with amino acid alterations at any position can be used, provided that there is FcRn binding activity in the neutral pH range or human FcRn binding activity in the neutral pH range can be increased. When the antigen binding molecule includes the Fc region of human IgG1 as the Fc region, it preferably includes a change that enhances binding to FcRn in the neutral pH range compared to the binding activity of the Fc region of the starting material of human IgG1. Suitable examples of such altered Fc regions include human Fc regions having at least one or more amino acids selected from the group consisting of amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257 , 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384 , 385, 386, 387, 389, 424, 428, 433, 434 and 436 in the EU numbering portion of the parental Fc region that differ from the corresponding amino acids in the native Fc region.

Suitable examples of such altered Fc regions include Fc regions containing at least one amino acid selected from the group consisting of:

Met at amino acid position 237;

Ala at amino acid position 238;

Lys at amino acid position 239;

Ile at amino acid position 248;

any of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp and Tyr at amino acid position 250;

any of Phe, Trp and Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

any of Asp, Glu and Gln at amino acid position 256;

any of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr and Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Phe at amino acid position 270;

either Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Gly at amino acid position 298;

Ala at amino acid position 303;

Ala at amino acid position 305;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 307;

any of Ala, Phe, Ile, Leu, Met, Pro, Gln and Thr at amino acid position 308;

any of Ala, Asp, Glu, Pro and Arg at amino acid position 309;

any of Ala, His and Ile at amino acid position 311;

either Ala or His at amino acid position 312;

either Lys or Arg at amino acid position 314;

either Ala or His at amino acid position 315;

Ala at amino acid position 317;

Gly at amino acid position 325;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

either Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

either Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 428;

Lys at amino acid position 433;

any of Ala, Phe, His, Ser, Trp and Tyr at amino acid position 434; and

His at amino acid position 436;

in the Fc region according to EU numbering.

For example, by using amino acid changes singly or using more than one of them in combination, FcRn binding of an IgG Fc region in an acidic and/or neutral pH range can be enhanced, and the amino acid changes that are made are not specifically limited, and when provided that plasma retention time is increased, any amino acid change can be made.

The antigen-binding molecule of the present invention is isolated from a culture of cells transformed with a desired expression vector containing an operably linked polynucleotide obtained by linking, as described above, a polynucleotide encoding an antigen-binding domain to a polynucleotide encoding an Fcγ receptor-binding domain having the activity of binding a human FcRn to acidic pH range and having higher binding activity to the Fcγ receptor in the neutral pH range than to the native Fcγ receptor binding domain in which the sugar chain linked at position 297 (EU numbering) is a fucose-containing sugar chain . Antigen-binding molecules of the present invention are obtained using the method in accordance with the method for obtaining antibodies, described in the above-mentioned section on antibodies.

All prior art documents cited in the specification are incorporated herein by reference. Below, the present invention is specifically described by means of examples, however, it should not be considered limited to them.

[Examples]

[Example 1] Preparation of antigen-binding molecules whose mouse FcγR binding activity under neutral pH range conditions is higher than the binding activity of native human IgG Fc region

(1-1) antibodies with pH-dependent binding of the human IL-6 receptor

H54/L28-IgG1, which contains H54-IgG1 (SEQ ID NO: 36) and L28-CK (SEQ ID NO: 37) described in WO 2009/125825, is a humanized anti-IL-6 receptor antibody. Meanwhile, Fv4-IgG1, which contains VH3-IgG1 (SEQ ID NO: 38) and VL3-CK (SEQ ID NO: 39), is a humanized anti-IL-6 receptor antibody that was obtained by reporting H54/L28-IgG1 binding properties to the soluble human IL-6 receptor in a pH dependent manner (which binds at pH 7.4 and dissociates at pH 5.8). An in vivo mouse study described in WO 2009/125825 demonstrated that, in a group administered with a mixture of Fv4-IgG1 and soluble human IL-6 receptor as antigen, the elimination of soluble human IL-6 receptor from plasma was significantly accelerated. compared to the group administered with a mixture of H54/L28-IgG1 and soluble human IL-6 receptor as antigen.

Soluble human IL-6 receptor coupled to H54/L28-IgG1, which is an antibody that binds to soluble human IL-6 receptor, is recycled to plasma via FcRn along with another antibody. Meanwhile, Fv4-IgG1, which is an antibody that binds to the soluble human IL-6 receptor in a pH dependent manner, dissociates from the soluble human IL-6 receptor under acidic conditions in the endosome. The dissociated soluble human IL-6 receptor is degraded in lysosomes, and thus it provides a significant acceleration in the elimination of the soluble human IL-6 receptor. Moreover, after binding to FcRn in the endosome, Fv4-IgG1, which is an antibody that binds to the soluble human IL-6 receptor in a pH-dependent manner, is recycled into plasma. Because the recycled antibody can re-bind to the soluble human IL-6 receptor, the antibody repeatedly binds to the antigen (soluble human IL-6 receptor) and is recycled via FcRn to plasma. It is believed that, as a result, a single antibody molecule can re-bind several times to the soluble human IL-6 receptor (WO 2009/125825).

(1-2) Production of anti-human IL-6 receptor antibody with enhanced mouse FcγR binding and anti-IL-6 receptor antibody without mouse FcγR binding

VH3-IgG1-F1022 (SEQ ID NO: 40), an antigen-binding molecule with enhanced mouse FcγR binding, was generated by replacing Lys with Asp at position 326 (EU numbering) and Leu with Tyr at position 328 (EU numbering) in VH3-IgG1. Fv4-IgG1-F1022 containing VH3-IgG1-F1022 as a heavy chain and VL3-CK as a light chain was obtained using the method described in Reference Example 2.

Meanwhile, VH3-IgG1-F760 (SEQ ID NO: 41), an antigen-binding molecule without mouse FcγR binding, was obtained by replacing Leu with Arg at position 235 and Ser with Lys at position 239 (EU numbering) in VH3-IgG1. Fv4-IgG1-F760 containing VH3-IgG1-F760 as a heavy chain and VL3-CK as a light chain was obtained using the method described in Reference Example 2.

(1-3) Evaluation of mouse FcγR binding

VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F1022 and VH3/L(WT)-IgG1-F760, which contained VH3-IgG1, VH3-IgG1-F1022 and VH3-IgG1-F760 as heavy chain and L(WT)-CK (SEQ ID NO: 42) as light chain were prepared using the method described in Reference Example 2. These antibodies were subjected to kinetic analysis for their binding to mouse FcγR as described below.

(1-4) Kinetic analysis of mouse FcγR binding

Antibody binding kinetic analysis was performed with mouse FcγRI, FcγRIIb, FcγRIII and FcγRIV (hereinafter referred to as mouse FcγR) (prepared according to Example 26) using Biacore T100 and T200 (GE Healthcare). An appropriate amount of L protein (ACTIGEN) was immobilized onto a CM4 sensor chip (GE Healthcare) by the amine addition method and antibodies of interest were captured thereon. Then, diluted solutions of mouse FcγR and running buffer were injected as a blank, and mouse FcγR was allowed to interact with antibodies captured on the sensor chip. The running buffer used was 20 mmol/L ACES, 150 mmol/L NaCl, 0.05% (w/v) Tween20, pH 7.4. This buffer was also used to dilute mouse FcγR. The sensor chip was regenerated using 10 mmol/l glycine-HCl, pH 1.5. All measurements were carried out at 25°C. The binding rate constant ka (1/M s) and the dissociation rate constant kd (1/s), which are kinetic parameters, were calculated from the sensorgrams obtained by measurement. Based on these values, the K _D (M) of each antibody against human FcγR was calculated. Each parameter was calculated using Biacore T100 or T200 Evaluation Software (GE Healthcare).

By measurement, the result shown in Table 7 was obtained. It was demonstrated that VH3/L(WT)-IgG1-F1022 has increased binding activity to mFcγRI, mFcγRIIb and mFcγRIII compared to VH3/L(WT)-IgG1. As for VH3/L(WT)-IgG1-F760, binding to various mouse FcγRs was not detectable, demonstrating that VH3/L(WT)-IgG1-F760 lacks binding activity to various mouse FcγRs.

(1-5) Production of low fucose antibodies

Known methods for increasing antibody FcγR binding activity include methods for converting antibody-bound sugar chains to low-fucose sugar chains (J. Biol. Chem. (2003) 278, 3466-3473), in addition to methods for making amino acid changes to Fc -area of the antibody. Low-fucose Fv4-IgG1 (hereinafter abbreviated as Fv4-IgG1-Fuc) was acquired by Fv4-IgG1 expression using fucose transporter gene-deficient CHO cells (WO 2006067913) as host cells according to the method described in Reference Example 4. Among mFcγRs (mouse Fcγ receptors), low-fucose antibodies have been reported to have selectively increased FcγRIV binding activity (Science, 2005, 310 (5753) 1510-1512).

[Example 2] Effect of eliminating antigens from plasma by antigen-binding molecules whose FcγR binding activity is higher than the binding activity of native human IgG Fc region

(2-1) Effect of H54/L28-IgG1 and Fv4-IgG1 on elimination of antigens from plasma

H54/L28-IgG1, which is an antibody against human IL-6 receptor, and Fv4-IgG1, which has the property of binding to the human IL-6 receptor in a pH-dependent manner, were obtained by the method described in Reference Example 1. In vivo infusion studies were performed using obtained H54/L28-IgG1 and Fv4-IgG1 in the manner described below.

(2-1-1) In vivo infusion studies using transgenic mice with human FcRn

An animal model in which the concentration of soluble human IL-6 receptor is maintained constant in plasma was created by implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet) containing soluble human IL-6 receptor under the dorsal skin of transgenic mice with human FcRn ( B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol Biol (2010) 602, 93-104). In an animal model, in vivo dynamics were evaluated after administration of an antibody against the human IL-6 receptor. To suppress the production of neutralizing antibodies against the soluble human IL-6 receptor, an anti-mouse CD4 monoclonal antibody (prepared in a known manner) was injected once at 20 mg/kg into the tail vein. An infusion pump containing 92.8 μg/ml of soluble human IL-6 receptor was then implanted into the dorsal area of the mice. Three days after implantation of the infusion pump, an antibody against the human IL-6 receptor was injected once a day at a dose of 1 mg/kg into the tail vein. The mice were bled 15 minutes, seven hours, one day, two days, four days and seven days after administration of the anti-human IL-6 receptor antibody. Immediately thereafter, the collected blood was centrifuged at 15,000 rpm. and 4°C for 15 minutes to obtain plasma. The isolated plasma was stored in a freezer set at -20° C. or lower until use.

(2-1-2) Determination of human IL-6 receptor (hsIL-6R) concentration in plasma by electrochemiluminescence method

Concentrations of human IL-6 receptor in mouse plasma were determined by the electroluminescence method. Samples for the hsIL-6R standard curve prepared at 2000, 1000, 500, 250, 125, 62.5 and 31.25 pg/mL and mouse plasma samples diluted 50-fold or more were mixed with anti- Human IL-6R (R&D), biotinylated anti-human IL-6R antibody (R&D), tocilizumab, ruthenated with SULFO-TAG NHS Ester (Meso Scale Discovery). The mixtures were incubated at 37°C overnight. Tocilizumab was prepared at a final concentration of 333 μg/ml. The reaction mixtures were then aliquoted in a Streptavidin MA400 PR plate (Meso Scale Discovery). The solution, reacted at room temperature for one hour, was washed off and then aliquoted with Reading Buffer T (x4) (Meso Scale Discovery). Immediately after this, the measurement was carried out using a SECTOR PR 400 Reader (Meso Scale Discovery). Human IL-6 receptor concentration was determined from the standard curve response using SOFTmax PRO analytical software (Molecular Devices).

Monitoring of human IL-6 receptor concentration over time is shown in FIG. 2. Compared to H54/L28-IgG1, Fv4-IgG1, which binds to the human IL-6 receptor in a pH dependent manner, could reduce the concentration of human IL-6 receptor but could not lower it below baseline without antibody administration. Therefore, an administered antibody that binds to an antigen in a pH-dependent manner could not reduce the concentration of the antigen below the level prior to administration of the antibody.

(2-2) Effect of Antigen Elimination from Plasma by an Antibody with Increased or Decreased FcγR Binding Activity

Whether the concentration of human IL-6 receptor is affected over time by an increase or decrease in the activity of Fv4-IgG1, which is an antibody with pH-dependent binding of the human IL-6 receptor, in relation to FcγR binding, was evaluated by the method described below. Using Fv4-IgG1, Fv4-IgG1-F760, Fv4-IgG1-F1022 and Fv4-IgG1-Fuc prepared as described in Example 1, in vivo infusion tests were carried out by the method described below.

(2-2-1) In vivo infusion tests using transgenic mice with human FcRn

An animal model in which the concentration of soluble human IL-6 receptor is maintained constant in plasma was created by implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet) containing soluble human IL-6 receptor under the dorsal skin of transgenic mice with human FcRn ( B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol Biol (2010) 602, 93-104). In an animal model, an anti-human IL-6 receptor antibody was administered simultaneously with Sanglopor (CSL Behring), which is an immunoglobulin preparation, to assess the in vivo dynamics of the soluble human IL-6 receptor following antibody administration. To suppress the production of neutralizing antibodies against the soluble human IL-6 receptor, an anti-mouse CD4 monoclonal antibody (prepared in a known manner) was injected once at 20 mg/kg into the tail vein. An infusion pump containing 92.8 μg/ml of soluble human IL-6 receptor was then implanted into the dorsal region of the mice. Three days after implantation of the infusion pump, anti-human IL-6 receptor antibody and Sanglopor were administered once at a dose of 1 mg/kg and 1000 mg/kg, respectively, into the tail vein. The mice were bled 15 minutes, seven hours, one day, two days, four days and seven days after administration of the anti-human IL-6 receptor antibody. The mice were bled 15 minutes, seven hours, one day, two days, three days and seven days after administration of the anti-human IL-6 receptor antibody. Immediately thereafter, the collected blood was centrifuged at 15,000 rpm. and 4°C for 15 minutes to obtain plasma. The isolated plasma was stored in a freezer set at -20° C. or lower until use.

(2-2-2) Plasma concentration determination of soluble human IL-6 receptor (hsIL-6R) by electrochemiluminescence method

Concentrations of human hsIL-6R in mouse plasma were determined by the electroluminescence method as described in (2-1-2).

The result is shown in Fig. 3. The plasma concentration of human IL-6 receptor over time in Fv4-IgG1-F760-treated mice, in which Fv4-IgG1 binding to mouse FcγR is eliminated, has been shown to be comparable to that in Fv4-IgG1-treated mice. . Cytotoxic activity against membrane antigen depends on FcγR binding and thus cytotoxic activity is lost when FcγR binding is eliminated. On the other hand, even when an antibody in which mouse FcγR binding is eliminated against human IL-6 receptor, which is a soluble antigen, was administered, there was no effect on the plasma concentration of human IL-6 receptor over time in administered mice. Thus, it could be assumed that the binding of the antibody against the soluble antigen to the FcγR did not contribute to the concentration of the antigen over time in the plasma of mice injected with the antibody.

Surprisingly, however, the plasma concentration of human IL-6 receptor in Fv4-IgG1-F1022-treated mice with enhanced mouse FcγR binding was significantly reduced compared to the plasma concentration of human IL-6 receptor in Fv4-IgG1-treated mice. With regard to the degree of reduction, it was confirmed that the concentration decreased below the initial concentration of human IL-6 receptor without administration of the antibody. In particular, the plasma concentration of human IL-6 receptor in Fv4-IgG1-F1022-treated mice was reduced to approximately 1/100 three days post-administration compared to Fv4-IgG1 administration. These data demonstrate that by administering to mice an antibody that binds to the human IL-6 receptor in a pH dependent manner and the binding to the FcγR of which is enhanced, the plasma concentration of the human IL-6 receptor in mice can be reduced and, in terms of the degree of reduction, the concentration Plasma antigen levels may be reduced below levels prior to administration of the antibody.

Moreover, it has been demonstrated that, compared to mice injected with Fv4-IgG1, the plasma concentration of human IL-6 receptor was reduced in mice with Fv4-IgG1-Fuc, which has a low fucose sugar chain and has increased activity. mouse FcγR IV binding. In particular, the plasma concentration of human IL-6 receptor in Fv4-IgG1-Fuc-treated mice was reduced to about 1/2 after seven days of administration compared with the case of Fv4-IgG1 administration. The above data demonstrate that by administering to mice a pH-dependent antigen-binding molecule that binds to the human IL-6 receptor in a pH-dependent manner and whose FcγR binding is enhanced, the concentration of soluble antigen in the plasma of mice can be reduced. In this case, methods for enhancing FcγR binding are not specifically limited to making amino acid changes. It has been demonstrated that such enhancement can be achieved, for example, using a human IgG Fc region to which a low-fucose sugar chain is linked at position 297 (EU numbering); however, the effect of Fv4-IgG1-Fuc in reducing antigen concentration was less than that of Fv4-F1022. Based on this result, it was believed that among several FcγRs (FcγRI, II, III and IV for mouse), mFcγIV, to which Fv4-IgG1-Fuc binding is enhanced, does not contribute much to the reduction of antigen concentration as FcγR.

Thus, it has been found that by administering to an individual an antibody that binds to a soluble antigen in a pH dependent manner and whose binding to FcγR is enhanced, the plasma concentration of the soluble antigen in the individual's plasma can be significantly reduced.

Without wishing to be bound by a particular theory, the unexpected decrease in plasma concentration of soluble antigen that was observed upon administration of an antigen-binding molecule that contains an antigen-binding domain whose binding to FcγR is enhanced and whose antigen-binding activity varies depending on ion concentration conditions such as pH, and FcRn The α-binding domain, which has FcRn binding activity under acidic pH range conditions, can be explained as follows.

IgG antibodies that are non-specifically incorporated into cells return to the cell surface by binding to FcRn under acidic conditions in the endosome and then dissociated from FcRn under neutral conditions in plasma. In such a case, when an antibody that neutralizes the function of the soluble antigen by binding to the antigen is administered to mice in which the plasma concentration of the soluble antigen is kept constant, the plasma soluble antigen forms a complex with the administered antibody. It is believed that the soluble antigen incorporated into cells, remaining as a complex, is recycled, in the antibody-bound state, into plasma along with the antibody, since the Fc region of the antibody binds to the FcRn under acidic conditions in the endosome.

Meanwhile, when an antibody against a soluble antigen is an antibody that binds to an antigen in a pH-dependent manner (i.e., an antibody that dissociates from a soluble antigen under acidic conditions in the endosome), the soluble antigen that is non-specifically incorporated into cells while remaining in as a complex with an antibody, dissociates from the antibody in the endosome and degrades in the lysosome; thus, the soluble antigen is not recycled into plasma. Therefore, it is believed that Fv4-IgG1 incorporated as a complex with a soluble antigen into cells can dissociate from the soluble antigen in the endosome and thus accelerate the elimination of the soluble antigen.

As described above, antigen-binding molecules such as Fv4-IgG1, which contain an antigen-binding domain whose antigen-binding activity varies with ion concentration, are believed to be able to bind to antigens multiple times. It is believed that the effect of accelerating the elimination of soluble antigens from plasma by dissociating them in the endosome depends on the rate of incorporation of the antigen/antigen-binding molecule complex into the endosome. An antigen-binding molecule that contains an antigen-binding domain whose binding activity to various FcγRs is increased and whose antigen-binding activity changes depending on ion concentration conditions, is actively incorporated into cells by binding to various FcγRs expressed on the cell membrane, and can migrate back into plasma by recycling through binding between FcRn and an FcRn binding domain contained in a molecule that has FcRn binding activity under acidic pH range conditions. Therefore, it is believed that since the above antigen-binding molecule, which forms a complex with soluble antigen in plasma, is actively incorporated into cells via FcγR expressed on the cell membrane, its effect of accelerating the elimination of soluble antigen from plasma is more pronounced than that of antigen-binding molecules, the activity the binding of which to various FcγRs was not increased.

In a living organism, various FcγRs are expressed on the cell membrane of immune cells and perform different functions. Any FcγR can be used to incorporate antibodies into cells. In particular, inhibitory FcγRIIb and activating FcγRs are known to be present in humans, including FcγRI, FcγRIIa, and FcγRIIIa, and antibodies can be incorporated through any of them. Antibodies can be incorporated through all or any of the FcγRs. Alternatively, antibodies can be turned on indirectly by various activating FcγRs only or by inhibitory FcγRIIb only.

On the other hand, to achieve the above goals, it is possible to use any methods to increase the FcγR binding activity at the FcγR-binding domain of antigen-binding molecules. For example, as shown in Example 1, amino acid mutations can be introduced into the FcγR-binding domain of antigen-binding molecules to increase FcγR binding activity, or low-fucose antibodies can be used. Meanwhile, the effect of increasing FcγR binding activity that is provided by such methods may be an effect of enhancing binding to any FcγR. In particular, it is possible to increase binding activity to any one, some or all of the FcγRs. Moreover, one can only increase the activity of binding to various activating FcγR or inhibitory FcγRIIb.

The FcγR binding activity of an antibody that binds to a membrane antigen plays an important role in the cytotoxic activity of the antibody. Thus, when an antibody used as a pharmaceutical agent is required to have cytotoxic activity, a human IgG1 isotype with high FcγR binding activity is used. In addition, methods for enhancing the cytotoxic activity of such antibodies by increasing the antibody's FcγR binding activity are widely used in the art.

Meanwhile, the role of the FcγR binding activity of antibodies that bind to soluble antigens and that are used as pharmaceuticals has not been known in the art. It has not been sufficiently appreciated what difference in effect on the living body to which the antibodies are administered is provided by the difference in FcγR binding activity between human IgG1 with high FcγR binding activity and human IgG2 and human IgG4 with low FcγR binding activity. In fact, in the present example, it was demonstrated that there was no effect on plasma soluble antigen concentration over time in individuals administered an antibody that lacks FcγR binding activity. Meanwhile, within the framework of the present invention, it was found that the concentration of soluble antigen was significantly reduced in the plasma of individuals who were administered an antigen-binding molecule, the FcγR binding activity of which was increased and which contains an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration. In particular, it can be said that the present inventors have for the first time recognized the benefit of enhancing FcγR binding by combining an FcRn-binding domain that has FcRn-binding activity under acidic pH range conditions with an antigen-binding domain whose binding to soluble antigen varies depending on ion concentration conditions. contained in an antigen-binding molecule that targets a soluble antigen.

[Example 3] The effect of eliminating antigens from plasma by antigen-binding molecules whose FcγR binding activity exceeds that of native human IgG Fc region and whose human FcRn binding activity is increased under conditions of acidic pH range

(3-1) Production of antigen-binding molecules whose FcγR binding activity exceeds the binding activity of native human IgG Fc region and whose binding activity to human FcRn is increased under conditions of acidic pH range

A disclosed method for increasing plasma retention of an IgG antibody is to increase FcRn binding under acidic pH conditions. It is believed that when FcRn binding under conditions of the acidic pH range is increased by introducing an amino acid substitution into the Fc region of an IgG antibody, this increases the efficiency of endosome-to-plasma recycling, resulting in increased plasma retention time of the IgG antibody.

There are many reports of amino acid changes to increase plasma retention time by increasing human FcRn binding activity under conditions of the acidic pH range. Such changes include, for example:

a method for replacing Met with Leu at position 428 and Asn with Ser at position 434 (EU numbering) in an IgG antibody (Nat. Biotechnol, (2010) 28, 157-159); a method for replacing Asn with Ala at position 434 (Drug. Metab. Dispos. (2010) 38 (4), 600-605); a method for replacing Met with Tyr at position 252, Ser with Thr at position 254, and Thr with Glu at position 256 (J. Biol. Chem. (2006) 281, 23514-23524); a method for replacing Thr with Gln at position 250 and Met with Leu at position 428 (J. Immunol. (2006) 176 (1) 346-356); a method for replacing Asn with His at position 434 (Clin. Pharm. & Ther. (2011) 89 (2) 283-290.); and WO 2010/106180; W02010/045193; W02009/086320; W02009/058492; W02008/022152; WO 2006/050166, WO 2006/053301, WO 2006/031370; WO2005/123780; W02005/047327; W02005/037867; WO2004/035752; and WO 2002/060919.

VH3-IgG1-F1093 (SEQ ID NO: 43) with Met replaced by Leu at position 428 and Asn by Ser at position 434 (EU numbering) in VH3-IgG1-F1022 was generated to improve the pharmacodynamics of Fv4-IgG1-F1022, which was demonstrated that when administered, it causes the effect of significantly reducing the concentration of soluble antigen in plasma, as described in example 2. Fv4-IgG1-F1093 containing VH3-IgG1-F1093 as a heavy chain and VL3-CK as a light chain was constructed using the method described in Reference Example 2.

(3-2) The effect of eliminating antigens from plasma by antigen-binding molecules whose binding activity to FcγR is higher than that of native human IgG Fc region and whose binding activity to human FcRn is increased under conditions of acidic pH range

An in vivo infusion test was performed for Fv4-IgG1-F1093 in the same manner as described in Example (2-1-1) using human FcRn transgenic mice in which the concentration of soluble human IL-6 receptor is kept constant in plasma. Plasma concentrations of soluble human IL-6 receptor in mice were determined by the method described in Example (2-1-2). The result is shown in Fig. four.

(3-2-1) Determination of anti-human IL-6 receptor antibody concentration in plasma by ELISA method

Mouse plasma concentrations of anti-human IL-6 receptor antibody were determined by ELISA. First, the idiotypic Fv4 antibody F(ab') MABTECH was aliquoted into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International). The plate was allowed to stand overnight at 4° C. to give plates immobilized with the idiotypic anti-Fv4 antibody. An idiotypic antibody was obtained by immunizing a rabbit with Fv4-M73 (WO 2009/125825). After serum purification using an ion exchange resin, the antibody was subjected to affinity purification using a column on which Fv4-M73 was immobilized, followed by adsorption using a column with immobilized human antigen. Curve standard samples containing anti-human IL-6 receptor antibody (plasma concentration: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 and 0.0125 μg/mL) and samples mouse plasma for analysis, diluted 100-fold or more. 100 μl of each standard curve sample and assay sample was combined with 200 μl of soluble human IL-6 receptor at a concentration of 20 ng/ml. The resulting mixtures were allowed to stand at room temperature for one hour and aliquoted into each well of the anti-Fv4 idiotypic antibody immobilized plate. The tablet was allowed to stand at room temperature for another hour. Then, the biotinylated anti-human IL-6R antibody (R&D) was reacted with it at room temperature for one hour. Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was then reacted with it at room temperature for one hour. The chromatographic reaction of the reaction solution was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After completion of the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance at 450 nm of the reaction solution in each well was measured using a microplate reader. Mouse plasma antibody concentrations were determined from an absorbance standard curve using SOFTmax PRO analysis software (Molecular Devices).

The result is shown in Fig. 5.

(3-3) Improved pharmacodynamics by increasing FcRn binding activity under acidic pH conditions

As shown in FIG. 5, in the group in which Fv4-IgG1-F1022 was administered, obtained by enhancing the binding activity of Fv4-IgG1 to FcγR under the conditions of the neutral pH range, it was demonstrated that the plasma retention of the administered antibody is reduced compared to the group in which Fv4 was administered. -IgG1. Meanwhile, in the group in which Fv4-IgG1-F1093 was administered due to enhanced binding activity of Fv4-IgG1-F1022 to human FcRn under acidic pH range conditions, it was demonstrated that the plasma retention of the administered antibody was significantly increased compared to the group in which Fv4-IgG1-F1022 was administered.

Moreover, as shown in FIG. 4, the concentration of soluble human IL-6 receptor in plasma over time in the Fv4-IgG1-F1022 administration group was equivalent to that in the Fv4-IgG1-F1093 administration group, up to three days after antibody administration. On the third day after administration, compared to the Fv4-IgG1 administration group, plasma concentrations of soluble human IL-6 receptor decreased by up to approximately 100-fold in both the Fv4-IgG1-F1022 and Fv4-IgG1-F1093 administration groups. However, on the seventh day after administration of the antibody, it was observed that the plasma concentration of soluble human IL-6 receptor was increased in the Fv4-IgG1-F1022 administration group compared to the third day after administration. On the other hand, in the Fv4-IgG1-F1093 administration group, no increase in the plasma concentration of soluble human IL-6 receptor was observed, which demonstrates that the effect of reducing the concentration of human soluble IL-6 receptor was long-lasting in this administration group.

In particular, Fv4-IgG1-F1093, when administered, reduced the concentration of soluble human IL-6 receptor in plasma in an individual after administration by up to approximately 1/100 compared to Fv4-IgG1, and, in addition, it maintained this state for a long time. period of time. Thus, Fv4-IgG1-F1093 has been demonstrated to be an excellent antigen-binding molecule. Without being bound by a particular theory, the phenomenon observed in this case can be explained as follows. It is believed that Fv4-IgG1-F1022, in which Fv4-IgG1 binding activity to FcγR is increased under neutral pH range conditions, is incorporated in a large amount mainly in immune cells expressing FcγR on the cell membrane. The incorporated antibody is transferred to the endosome and, by binding to the FcRn in the endosome, the antibody is recycled to the plasma. It is believed that when the binding activity of the antibody to FcRn is not high enough under acidic pH conditions in the endosome, the antibody incorporated into the endosome is not able to be sufficiently recycled. In particular, a possible reason for the reduced plasma retention of Fv4-IgG1-F1022 relative to Fv4-IgG1 is that FcRn binding activity under conditions of the acidic pH range is insufficient to sufficiently recycle an endosome-embedded antibody into plasma via FcRn binding, and an antibody that not recycled, degraded in the lysosome.

On the other hand, as in the case of Fv4-IgG1-F1022, Fv4-IgG1-F1093 obtained by enhancing the binding activity of Fv4-IgG1-F1022 to human FcRn under conditions of the acidic pH range is believed to be included in a large amount mainly in immune cells expressing FcγR on the cell membrane. The antibody incorporated and transferred into the endosome is recycled to plasma by binding to the FcRn in the endosome. Because its human FcRn binding activity is enhanced under acidic pH conditions, Fv4-IgG1-F1093 is believed to have sufficient FcRn binding activity in the endosome. Thus, after incorporation into cells, most of the Fv4-IgG1-F1093 is recycled to plasma. Thus, it could be assumed that the plasma retention of Fv4-IgG1-F1093 was increased in individuals who received its introduction, compared with Fv4-IgG1-F1022.

On the other hand, plasma retention of conventional antibodies is known to be improved when their FcRn binding activity is increased under acidic pH range conditions. However, it is believed that when the plasma retention time of an antibody is increased, the plasma retention time of antibody-bound antigens is also increased, and this results in an increase in plasma antigen concentration. In fact, as described in WO 2010/088444, antibody 18E, which introduced a YTE change in antibody 18, which is a human IgG1 antibody against IL-6, to increase FcRn binding activity under conditions of the acidic pH range, showed an increased retention time in plasma of cynomolgus monkeys and, at the same time, the concentration of IL-6 antigen was also increased in plasma.

Surprisingly, however, when Fv4-IgG1-F1093 is introduced, which is modified similar to YTE to increase FcRn binding activity under acidic pH range conditions in Fv4-F1022, which binds to antigen in a pH-dependent manner and has increased FcγR binding activity , the plasma retention time of the antibody was significantly increased in the individuals to whom it was administered, without increasing the concentration of the soluble human IL-6 receptor, which is an antigen. Instead, on the seventh day after antibody administration, the concentration of soluble human IL-6 receptor remained low in Fv4-IgG1-F1093-treated individuals compared to Fv4-F1022-treated individuals.

Without being bound by a particular theory, the phenomenon observed in this case can be explained as follows. When introduced into a living organism, an antibody without pH-dependent antigen binding is non-specifically incorporated into cells. Antigens that remain bound to the antibody are recycled into plasma to the same extent as the antibody. Meanwhile, for an antibody with increased FcRn binding activity under acidic pH range conditions, the recycling rate to plasma in the living body to which the antibody is administered is higher than the recycling rate of an antibody without increased FcRn binding activity, and this results in an increased recycling rate. antibodies bound to an antigen in the plasma of a living organism. Thus, it is believed that due to the increased plasma retention of an antibody introduced into a living body, the plasma concentration of the antigen to which the antibody binds also increases in the living body.

Meanwhile, when administered to a living body, an antibody which binds to an antigen in a pH-dependent manner and which has an increased FcγR binding activity is mainly included in immune cells expressing FcγR on the cell membrane, and this reduces plasma retention. Moreover, after incorporation into cells, in the antibody-bound state, the antigen dissociates from the antibody in the endosome and then is degraded in the lysosome, which leads to a decrease in the plasma concentration of the antigen in a living organism. When FcRn binding activity is increased under acidic pH range conditions, plasma retention time of the antibody, even if reduced due to increased FcγR binding activity, is increased by increasing the recycling rate by FcRn. In this case, since the antigen bound to the antibody, which binds to the antigen in a pH-dependent manner, is believed to dissociate from the antibody in the endosome and be directly degraded in the lysosome, the plasma concentration of the antigen increases. Moreover, it is believed that the increased plasma retention of an antibody introduced into a living organism provides the effect of prolonged elimination of the antibody and maintenance of the antigen concentration at a low level for a longer period of time.

The data described above demonstrate that the plasma retention time of an administered antibody is increased in a living organism to which the antibody is administered, where the human FcRn binding activity under acidic pH range conditions is enhanced in an antigen-binding molecule whose FcγR binding activity is higher than that of native Fc- human IgG regions. Moreover, it was found that in this case, the retention time of the antibody in plasma increases without worsening the effect of antigen elimination.

[Example 4] Further Evaluation of the Effect of Antigen Binding Molecules in Eliminating Plasma Antigens whose FcγR Binding Activity is Higher Than Native Human IgG Fc Region and whose Binding Activity to Human FcRn is Increased Under Acid pH Range Conditions

(4-1) Effect of antigen elimination in a living body injected with an antibody whose FcγR binding activity is higher than that of the native human IgG Fc region and which has a human FcRn binding activity increased under conditions of acidic pH range

As described in Example 2, the plasma antigen concentration was significantly reduced in the group administered with Fv4-IgG1-F1022 with enhanced mouse FcγR binding. Meanwhile, as shown in Example 3, the reduced plasma retention time observed in the Fv4-IgG1-F1022 management group was significantly increased by increasing the binding activity of Fv4-IgG1-F1022 to human FcRn under acidic pH range conditions. Further, as described below, the effect of eliminating soluble antigens from plasma by enhancing the binding of mouse FcγR and the effect of increasing the plasma retention time of the antibody in a living body to which it was administered by enhancing the binding activity of human FcRn under acidic pH conditions were further evaluated.

(4-2) Production of anti-human IL-6 receptor antibody with enhanced mouse FcγR binding

VH3-IgG1-F1087 (SEQ ID NO: 123) obtained by replacing Lys with Asp at position 326 (EU numbering) in VH3-IgG1, and VH3-IgG1-F1182 (SEQ ID NO: 124) obtained by replacing Ser with Asp at position 239 and Ile at Glu at position 332 (EU numbering) in VH3-IgG1 were generated as antigen-binding molecules with increased mouse FcγR binding. Fv4-IgG1-F1087 which contains VH3-IgG1-F1087 as heavy chain and VL3-CK as light chain, and Fv4-IgG1-F1182 which contains VH3-IgG1-F1182 as heavy chain and VL3-CK as light chain was obtained using the method described in Reference Example 2.

(4-3) Evaluation of mouse FcγR binding activity

VH3/L (WT)-IgG1-F1087 and VH3/L (WT)-IgG1-F1182, which contain VH3-IgG1-F1087 and VH3-IgG1-F1182 as heavy chain, respectively, and L (WT)-CK ( SEQ ID NO: 42) as a light chain was obtained by the method described in Reference Example 2. These antibodies and VH3/L (WT)-IgG1-F1022 were evaluated for their mouse FcγR binding activity by the method described in Reference Example 2. Result is presented in Table 8. In addition, the ratio of increase in FcγR binding activity of each variant relative to IgG1 before the change is presented in Table 9.

As shown in Table 9, it was demonstrated that F1087 and F1022 had increased binding activity to mouse FcγRI, mouse FcγRIIb and mouse FcγRIII compared to IgG1, while their binding activity to mouse FcγRIV was not increased. With regard to the binding activity of F1087 to mouse FcγRI, mouse FcγRIIb, mouse FcγRIII and mouse FcγRIV, it was found that the increase was less than that of F1022. Meanwhile, it was shown that the binding activity of F1182 to mouse FcγRI and mouse FcγRIV was significantly increased, while the degree of increase in its binding activity to FcγRIIb and FcγRIII was less than that of F1022 and F1087. As mentioned above, these three types of variants showed enhanced binding to several mouse FcγRs; however, FcγR binding activity has been shown to selectively increase and the degree of increase varies depending on the variant.

(4-4) The effect of elimination of antigens from plasma in an individual administered with Fv4-IgG1-F1087 and Fv4-IgG1-F1182

Using the same method as described in Example 2, in vivo infusion tests using transgenic mice with human FcRn were performed to determine plasma concentrations of soluble IL-6 receptor in mice. The result is shown in Fig. 6.

In both groups administered in vivo with Fv4-IgG1-F1087 and Fv4-IgG1-F1182, which have increased mouse FcγR binding activity compared to Fv4-IgG1, the in vivo plasma concentration of soluble human IL-6 receptor was reduced compared to group in which Fv4-IgG1 was administered. The effect of reducing the above plasma concentration of soluble human IL-6 receptor was high, especially in the group administered with Fv4-IgG1-F1087, which has increased binding to mouse FcγRII and mouse FcγRIII. Meanwhile, the effect of F1182 administration on reducing the plasma concentration of human soluble IL-6 receptor was small in the in vivo administration group of F1182, which has a significantly increased binding activity to mouse FcγRI and mouse FcγRIV (as well as several-fold increased binding to mouse FcγRII and mouse FcγRIII). Based on these results, it was suggested that the mouse FcγRs that contribute significantly more to the effect of effectively lowering plasma antigen concentration in mice administered with the pH-dependent antigen-binding antibody are mouse FcγRII and/or mouse FcγRIII. In particular, it is believed that the plasma concentration of antigen can be more effectively reduced in vivo by introducing into a living organism an antibody with pH-dependent antigen binding with enhanced binding to mouse FcγRII and/or mouse FcγRIII.

(4-5) Production of antigen-binding molecules whose FcγR binding activity is higher than the binding activity of native human IgG Fc region and which has increased human FcRn binding activity under acidic pH range conditions

As described in Example 3, compared to human FcRn transgenic mice administered Fv4-IgG1-F1022, the plasma retention of the administered antibody was significantly increased in human FcRn transgenic mice administered Fv4-IgG1-F1093, obtained by an increase in binding activity to Human FcRn under acidic pH range conditions in Fv4-IgG1-F1022, whose binding activity to mouse FcγR is increased. Whether this effect is also seen in human FcRn transgenic mice injected with Fv4-IgG1-F1087 and Fv4-IgG1-F1182, and whether the same effect is seen in mice injected with variants whose binding activity to human FcRn is increased under acidic pH range conditions, by adding a change different from the change evaluated in Example 3, it was evaluated as follows.

VH3-IgG1-F1180 (SEQ ID NO: 125) and VH3-IgG1-F1181 (SEQ ID NO: 126) were generated by replacing Met with Leu at position 428 and Asn with Ser at position 434 (EU numbering) in VH3- heavy chains. IgG1-F1087 and VH3-IgG1-F1182, to increase the binding activity of Fv4-IgG1-F1087 and Fv4-IgG1-F1182 to human FcRn under acidic pH conditions. Moreover, VH3-IgG1-F1412 (SEQ ID NO: 127) was obtained by replacing Asn with Ala at position 434 (EU numbering) in the VH3-IgG1-F1087 heavy chain, to increase the binding activity of Fv4-IgG1-F1087 to human FcRn in acidic pH range conditions. Fv4-IgG1-F1180, Fv4-IgG1-F1181 and Fv4-IgG1-F1412, which contain the heavy chains described above and VL3-CK as a light chain, were prepared using the method described in Reference Example 2.

(4-6) Improved pharmacodynamics of antibodies with enhanced human FcRn binding activity under acidic pH conditions

In vivo infusion tests were performed by administering Fv4-IgG1-F1180, Fv4-IgG1-F1181 and Fv4-IgG1-F1412 to human FcRn transgenic mice according to the same method as described in Example 2 to determine the concentration of soluble IL receptor -6 in mouse plasma. The results for plasma antibody concentrations in groups of mice administered with Fv4-IgG1-F1087, Fv4-IgG1-F1180, Fv4-IgG1-F1412 and Fv4-IgG1 are shown in FIG. 7. The results for plasma antibody concentrations in groups of mice administered with Fv4-IgG1-F1182, Fv4-IgG1-F1181 and Fv4-IgG1 are shown in FIG. 8. Meanwhile, plasma antibody concentrations in the mouse groups were measured by the method described in Example 3. Results for plasma concentrations of soluble IL-6 receptor in the Fv4-IgG1-F1087, Fv4-IgG1-F1180, Fv4-IgG1- F1412 and Fv4-IgG1 are shown in FIG. 9; and the results for plasma concentrations of soluble IL-6 receptor in the Fv4-IgG1-F1182, Fv4-IgG1-F1181 and Fv4-IgG1 groups are shown in FIG. ten.

It was confirmed that, compared with the group of mice injected with Fv4-IgG1-F1182, the plasma retention time of the injected antibodies was increased in the group of mice injected with Fv4-IgG1-F1181, resulting in an increase in Fv4-IgG1-F1182 binding activity. with human FcRn in the acidic pH range. Meanwhile, the plasma concentration of soluble IL-6 receptor in Fv4-IgG1-F1181-treated mice was comparable to that of Fv4-IgG1-F1182-treated mice. Compared to Fv4-IgG1-treated mice, the plasma concentration of soluble IL-6 receptor was reduced in both groups.

On the other hand, compared with the group of mice administered with Fv4-IgG1-F1087, the plasma retention time of the administered antibodies was increased in both groups of mice administered with Fv4-IgG1-F1180 and Fv4-IgG1-F1412 obtained by increasing the activity binding of Fv4-IgG1-F1087 to human FcRn in the acidic pH range, and unexpectedly, plasma retention time increased to a level comparable to that in mouse groups administered with Fv4-IgG1. Moreover, the duration of the effect of reducing the concentration of soluble IL-6 receptor in plasma was increased by increasing the retention time of the antibody in plasma in the groups of injected mice. In particular, in groups of administered mice, plasma concentrations of soluble IL-6 receptor 14 days and 21 days after administration of Fv4-IgG1-F1180 and Fv4-IgG1-F1412 were significantly reduced compared with concentrations 14 days and 21 days after administration of Fv4 -IgG1-F1087.

In view of the above, with respect to groups of mice administered with the four exemplary antibodies Fv4-IgG1-F1093, Fv4-IgG1-F1181, Fv4-IgG1-F1180, and Fv4-IgG1-F1412, it has been demonstrated that plasma retention time can be increased in vivo. an organism into which the antibody has been administered, wherein the binding activity to a human FcRn under acidic pH conditions is enhanced in an antigen-binding molecule whose binding activity to an FcγR is higher than the binding activity of a native human IgG Fc region. It has also been demonstrated that in a living body into which an antigen-binding molecule has been introduced, the plasma retention time is increased without impairing the effect of eliminating antigens from the living body, and rather, the antigen-eliminating effect can be long lasting.

(4-7) Generation of antigen-binding molecules with increased binding activity to human FcRn under acidic pH conditions and reduced binding to rheumatoid factor

In recent years, it has been reported that an antibody molecule obtained by replacing Asn with His at position 434 (EU numbering) in a humanized anti-CD4 antibody to increase plasma retention time by increasing its binding activity to human FcRn under acidic pH range conditions binds to rheumatoid factor (RF) (Clin. Pharmacol. Ther. (2011) 89 (2), 283-290). This antibody has a human IgG1 Fc region and an Asn to His substitution at position 434 (EU numbering) in the FcRn binding site. It has been demonstrated that rheumatoid factor recognizes and binds to the area with the replacement.

As described in Example (4-6), compared with the case where Fv4-IgG1-F1087 was administered to transgenic mice with human FcRn, Fv4-IgG1-F1180 obtained by increasing the binding activity of Fv4-IgG1-F1087 under acidic pH conditions with human FcRn with increased mouse FcγR binding activity showed increased plasma retention time. Various alterations have been reported to increase human FcRn binding activity under acidic pH range conditions. Among these modifications, the heavy chain variant replacing Met with Leu at position 428 and Asn with Ser at position 434 (EU numbering) in the heavy chain has been reported to show increased binding to rheumatoid factors.

However, a variant that has a Tyr to Thr substitution at position 436 (EU numbering) in addition to the above described substitutions at positions 428 and 434 (EU numbering) shows significantly reduced binding to rheumatoid factors while maintaining increased binding activity to human FcRn under acidic range conditions. pH values.

Therefore, antigen-binding molecules that have increased human FcRn binding activity under conditions of the acidic pH range, but do not have binding to rheumatoid factor, can be obtained by making a change in the Fc region that reduces rheumatoid factor binding activity alone without reducing FcRn binding activity. in the acidic pH range.

Such changes used to reduce rheumatoid factor binding activity include changes at positions 248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439 and 441-444 (numbering EU), preferably at positions 387, 422, 424, 426, 433, 436, 438 and 440 (EU numbering) and particularly preferably a change that changes Val to Glu or Ser at position 422, a change that changes Ser to Arg at position 424, a change that changes His to Asp at position 433, a change that changes Tyr to Thr at position 436, a change that changes Gln to Arg or Lys at position 438, and a change that changes Ser to Glu or Asp at position 440 ( EU numbering). These changes can be used alone or in combination.

Alternatively, it is possible to introduce N-type glycosylation sequences to reduce rheumatoid factor binding activity. In particular, known N-type glycosylation sequences include Asn-Xxx-Ser/Thr (Xxx is an arbitrary amino acid other than Pro). This sequence can be inserted into the Fc region to add an N-type sugar chain and binding to RF can be inhibited due to steric hindrance by the N-type sugar chain. Changes used to add an N-type sugar chain preferably include a change that changes Lys to Asn at position 248, a change that changes Ser to Asn at position 424, a change that changes Tyr to Asn at position 436, and Gln to Thr at position 436. position 438, and a change that replaces Qln with Asn at position 438, according to EU numbering, a change that replaces Ser with Asn at position 424 (EU numbering) is particularly preferred.

(4-8) Evaluation of the effect of improving the pharmacodynamics of antigen-binding molecules with increased human FcRn binding activity under conditions of acidic pH range and reduced binding to rheumatoid factor

To assess the effect of antibodies with a change that reduces the rheumatoid factor binding activity mentioned above, Fv4-IgG1-F1782 with Met replaced by Leu at position 428, Asn by Ser at position 434 and Tyr by Thr at position 436 (EU numbering) in the heavy chain Fv4-IgG1-F1087 was obtained using the method described in Reference Example 2. As described in Example (4-7), Fv4-IgG1-F1782 is an antibody whose binding activity to human FcRn under acidic pH conditions and binding activity which is increased with mouse FcγR, but its rheumatoid factor binding activity is not increased compared to native human IgG1. To investigate whether the retention time of the Fv4-IgG1-F1782 antibody is increased compared to Fv4-IgG1-F1087, antibody pharmacodynamics in the plasma of human FcRn transgenic mice injected with the antibodies were assessed using the same method as described in Example 2. Plasma concentration of soluble human IL-6 receptor was determined by the method described in Example (2-1-2), while plasma antibody concentrations were determined by the method described in Example (3-2-1).

Plasma antibody concentration over time is shown in FIG. 11 and the plasma concentration of soluble human IL-6 receptor over time is shown in FIG. 12. Fv4-IgG1-F1782 showed an increased retention time of the antibody in plasma compared to Fv4-IgG1-F1087. Meanwhile, the plasma concentration of soluble human IL-6 receptor was significantly reduced in the groups in which the above-mentioned antibodies were administered compared with the Fv4-IgG1 administration group.

Without being bound by a particular theory, the results described above can be interpreted as follows. The results described in Examples 3 and 4 demonstrate that plasma retention can be extended in a living organism administered with an antibody obtained by increasing human FcRn binding activity under acidic pH range conditions of an antigen-binding molecule whose FcγR binding activity is higher than the activity of binding to the Fc region in natural human IgG. It has also been demonstrated that in a living body to which such an antigen-binding molecule has been introduced, the plasma retention is prolonged without impairing the effect of eliminating antigens from the living body, and rather, the antigen-eliminating effect can be long lasting.

However, there is concern that antigen-binding molecules that have been modified to increase human FcRn binding activity under acidic pH range conditions may be at risk of increased rheumatoid factor binding activity. Thus, plasma retention time can be increased without increasing rheumatoid factor binding activity by introducing a mutation in the binding molecules that reduces rheumatoid factor binding activity while maintaining human FcRn binding activity under acidic pH range conditions.

In other words, it has been found that, when introduced into a living body, antigen-binding molecules that have the property of binding to antigens in a pH-dependent manner and have an increased binding activity of human FcRn under acidic pH range conditions and whose binding activity to FcγR is higher than in the Fc region of native human IgG, and which have reduced rheumatoid factor binding activity, have the excellent property that antigen-binding molecules exhibit increased plasma retention time without increasing their rheumatoid factor binding activity, and effectively reduce the concentration of soluble antigen in the living body.

[Example 5] The effect of eliminating antigens from the plasma of a living body into which an antigen-binding molecule has been introduced whose FcγR binding activity is higher than the binding activity of native mouse IgG Fc region

(5-1) Effect of eliminating antigen from the plasma of a living body injected with mouse antibody with enhanced FcγR binding activity

As described in Examples 1-4, elimination of soluble human IL-6 receptor from mouse plasma was demonstrated to be accelerated in groups of human FcRn transgenic mice administered antigen-binding molecules obtained by increasing mouse FcγR binding activity in antigen-binding molecules that possess Fc- a human antibody region; and a human IL-6 receptor binding property in a pH-dependent manner. Whether this effect was achieved in normal mice having mouse FcRn injected with antigen-binding molecules that have the Fc region of mouse antibodies and the property of binding to the human IL-6 receptor in a pH dependent manner was assessed in normal mice having mouse FcRn as follows. way.

(5-2) Generation of mouse antibodies with enhanced FcγR binding activity

For a mouse IgG1 antibody having the property of binding to the human IL-6 receptor in a pH dependent manner, a VH3-mIgG1 heavy chain (SEQ ID NO: 128) and a VL3-mk1 light chain (SEQ ID NO: 129) were constructed using the method described in Reference Example 2. Meanwhile, in order to increase the binding activity of VH3-mIgG1 to mouse FcγR, VH3-mIgG1-mF44 (SEQ ID NO: 130) was obtained by replacing Ala with Asp at position 327 (EU numbering). Similarly, VH3-mIgG1-mF46 (SEQ ID NO: 131) was generated by replacing Ser with Asp at position 239 and Ala with Asp at position 327, according to EU numbering, in VH3-mIgG1. Fv4-mIgG1, Fv4-mIgG1-mF44, and Fv4-mIgG1-mF46, which contain VH3-mIgG1, VH3-mIgG1-mF44, and VH3-mIgG1-mF46, respectively, as the heavy chain and VL3-mk1 as the light chain, were obtained using the method described in Reference Example 2.

(5-3) Evaluation of mouse FcγR binding activity of mouse antibody with enhanced FcγR binding activity

VH3/L (WT)-mIgG1, VH3/L (WT)-mIgG1-mF44, and VH3/L (WT)-mIgG1-mF46, which contain VH3-mIgG1, VH3-mIgG1-mF44, and VH3-mIgG1-mF46, respectively , as a heavy chain and L (WT)-CK (SEQ ID NO: 42) as a light chain, were obtained by the method described in Reference Example 2. These antibodies were evaluated for their mouse FcγR binding activity by the method described in Reference Example 25 The result is shown in Table 10. In addition, the ratio of increase in mouse FcγR binding activity of each variant relative to mIgG1 before the change is shown in Table 11.

The result of evaluating Example 4 demonstrating that VH3/L (WT)-mIgG1 having the Fc region of native mouse IgG1 antibody only binds to mouse FcγRIIb and mouse FcγRIII but not to mouse FcγRI and mouse FcγRIV indicates that Mouse FcγRs important for reducing antigen concentration are mouse FcγRII and/or mouse FcγRIII. It has been demonstrated that VH3/L (WT)-mIgG-mF44 and VH3/L (WT)-mIgG1-mF46, in which a change is introduced that presumably increases the binding activity of VH3/L (WT)-mIgG1 to FcγR, has an increased activity binding to both mouse FcγRIIb and mouse FcγRIII.

(5-4) Evaluation of the effect on reducing the plasma concentration of soluble IL-6 receptor in normal mice

The elimination effect of soluble IL-6 receptor from the plasma of normal mice injected with anti-human IL-6 receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44 or Fv4-mIgG1mF46 was evaluated as follows.

An animal model in which the concentration of soluble human IL-6 receptor is maintained constant in plasma was created by implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet) containing soluble human IL-6 receptor under the back skin of normal mice (C57BL/ mouse). 6J, Charles River Japan). In an animal model, in vivo dynamics were evaluated after administration of an antibody against the human IL-6 receptor. To suppress the production of neutralizing antibodies against the soluble human IL-6 receptor, anti-mouse CD4 monoclonal antibody was injected once at 20 mg/kg into the tail vein. An infusion pump containing 92.8 μg/ml of soluble human IL-6 receptor was then implanted into the dorsal area of the mice. Three days after implantation of the infusion pump, an antibody against the human IL-6 receptor was injected once a day at 1 mg/kg into the tail vein. The mice were bled 15 minutes, seven hours, one day, two days, four days and seven days after administration of the anti-human IL-6 receptor antibody. Immediately thereafter, the collected blood was centrifuged at 15,000 rpm. and 4°C for 15 minutes to obtain plasma. The isolated plasma was stored in a freezer set at -20° C. or lower until use.

Soluble human IL-6 receptor plasma concentrations were determined by the method described in (2-1-2). The result is shown in Fig. 13.

Surprisingly, it was demonstrated that in mice administered with mF44 and mF46, modified to increase the binding activity of mIgG1 (mice native IgG1) to mouse FcγRIIb and mouse FcγRIII, the plasma concentration of IL-6 receptor was significantly reduced compared to mice which were injected with mIgG1. In particular, even at 21 days after mF44 administration, the plasma concentration of IL-6 receptor in the mF44 administration group was reduced by approximately 6-fold compared with the plasma IL-6 receptor concentration in the group without antibody administration and approximately 10-fold compared with with the mIgG1 administration group. On the other hand, on the seventh day after mF46 administration, the plasma concentration of IL-6 receptor in the mF46 administration group was significantly reduced by approximately 30-fold compared with the plasma concentration of IL-6 receptor in the group without antibody administration and approximately 50-fold compared with the mIgG1 injection group.

The above data demonstrate that elimination of the soluble IL-6 receptor from plasma was also accelerated in antibody-treated mice, where the mouse FcγR binding activity of an antigen-binding molecule having mouse IgG1 Fc regions is increased, as is the case for antibodies, wherein the mouse FcγR binding activity of an antigen-binding molecule having the Fc region of a human IgG1 antibody is increased. Without being bound by a particular theory, the observed phenomenon described above can be explained as follows.

When administered to mice, antibodies that bind to soluble antigen in a pH-dependent manner and have increased FcγR binding activity are actively incorporated primarily into cells expressing FcγR at the cell membrane. Incorporated antibodies dissociate from soluble antigen under acidic pH conditions in the endosome and are then recycled to plasma via FcRn. Thus, the factor that provides the plasma elimination effect of soluble antigen from such an antibody is the level of FcγR binding activity of the antibody. In particular, since the FcγR binding activity is higher, incorporation into FcγR expressing cells is more active and this makes the elimination of soluble antigens from plasma faster. Moreover, provided that the FcγR binding activity is increased, the effect can be assessed in a similar manner whether the Fc region contained in the antibody is from human or mouse IgG1. In particular, the evaluation can be performed on the Fc region of any animal species, such as any of human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, rat IgG, monkey IgG, and rat IgG. provided that the FcγR binding activity in the animal species to be administered is increased.

[Example 6] Antigen Elimination Effect of Antibodies with Binding Activity Enhanced in a FcγRIIb Selective Way

(6-1) Antigen elimination effect of antibodies in which FcγRIIb binding activity is selectively increased

FcγRIII deficient mice (B6 mouse, 129P2-FcgrFcγR3tm1Sjv/J, Jackson Laboratories) express mouse FcγRI, mouse FcγRIIb, and mouse FcγRIV, but not mouse FcγRIII. Meanwhile, Fc receptor γ-chain deficient mice (Fcer1g mouse, Taconic, Cell (1994) 76, 519-529) only express mouse FcγRIIb but not mouse FcγRI, mouse FcγRIII and mouse FcγRIV.

As described in Example 5, mF44 and mF46 with increased native IgG1 binding activity to mouse FcγR were shown to selectively enhance binding to mouse FcγRIIb and mouse FcγRIII. It has been hypothesized that, using selectively enhanced antibody binding activity, the conditions under which an antibody with selectively enhanced mouse FcγRIIb binding can be administered can be mimicked by administering mF44 and mF46 to FcγRIII deficient mice or Fc receptor γ chain deficient mice that do not express mouse FcγRIII.

(6-2) Evaluation of the effect of antigen elimination in a living body injected with mouse FcγRIIb binding selectively enhanced antibody using FcγRIII deficient mice

The elimination effect of soluble IL-6 receptor from plasma in FcγRIII-deficient mice treated with anti-human IL-6 receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1-mF46 was evaluated in the same manner as described in Example 5 Plasma concentrations of soluble human IL-6 receptor in mice were determined by the method described in Example (2-1-2). The result is shown in Fig. fourteen.

Surprisingly, it was demonstrated that plasma concentrations of IL-6 receptor in FcγRIII-deficient mice treated with mF44 and mF46, which mimic conditions in which mIgG1 (native mouse IgG1) binding activity to mouse FcγRIIb is selectively increased, were significantly reduced compared to with the plasma concentration of IL-6 receptor in mice injected with mIgG1. In particular, the plasma concentration of IL-6 receptor in the mF44 administration group was reduced by approximately three times compared with the mIgG1 administration group, and the increase in the concentration of accumulated antigen due to the administration of the antibody was suppressed. Meanwhile, on the third day after administration, the plasma concentration of IL-6 in the mF46 administration group was significantly reduced by approximately six times compared with the plasma concentration of IL-6 receptor in the group without antibody administration and approximately 25 times higher than the concentration of IL-6 receptor. 6 in plasma in the mIgG1 administration group. This result shows that since the mouse FcγRIIb binding activity of the anti-human IL-6 receptor antibody, which binds to the antigen in a pH-dependent manner, is higher, the IL-6 receptor concentration can be more reduced in mice administered with the antibody. .

(6-3) Evaluation of the effect of antigen elimination in the plasma of a living body injected with an antibody with selective enhancement of mouse FcγRIIb binding using Fc receptor γ-chain deficient mice

The elimination effect of soluble IL-6 receptor from plasma in mice deficient in the Fc receptor γ-chain, which were injected with an antibody against human IL-6 receptor Fv4-mIgG1, Fv4-mIgG1-mF44 or Fv4-mIgG1mF46, was evaluated in the same way as described in Example 5. Soluble human IL-6 receptor plasma concentrations in mice were determined by the method described in Example (2-1-2). The result is shown in Fig. fifteen.

As in the case where mF44 and mF46 were administered to FcγRIII deficient mice, it was demonstrated that the plasma concentration of IL-6 receptor in Fc receptor γ chain deficient mice administered mF44 and mF46, which mimic conditions for selectively increasing binding activity mIgG1 with mouse FcγRIIb (native mouse IgG1) was significantly reduced compared to IL-6 receptor concentration in Fc receptor γ-chain deficient mice injected with mIgG1. In particular, the plasma concentration of IL-6 receptor in the mF44 administration group was reduced by approximately three times compared with the mIgG1 administration group, and the increase in the concentration of accumulated antigen due to the administration of the antibody was suppressed. Meanwhile, on the third day after administration, the plasma concentration of IL-6 receptor in the mF46 administration group was significantly reduced by about five times compared with the group without antibody administration and approximately 15 times compared with the mIgG1 administration group.

The results described in Examples (6-2) and (6-3) demonstrate that the plasma concentration of soluble antigen is significantly reduced in the group administered with an antibody that binds to the soluble antigen in a pH-dependent manner and has a selectively increased binding activity. Mouse FcγRIIb.

[Example 7] Effect of Antibodies on Antigen Elimination with Selective Enhancement of FcγRIII Binding

(7-1) Effect of antigen elimination in the plasma of a living body injected with antibodies with selectively enhanced binding of FcγRIII

FcγRIIb deficient mice (FcgrFcγR2b mouse (FcγRII), Taconic) (Nature (1996) 379 (6563), 346-349) express mouse FcγRI, mouse FcγRIII and mouse FcγRIV, but not mouse FcγRIIb. As described in Example 5, it was demonstrated that mF44 and mF46 obtained by increasing the binding activity of native mouse IgG1 to FcγR show selectively enhanced binding to mouse FcγRIIb and mouse FcγRIII. It has been suggested that, based on the use of antibodies with selectively enhanced binding activity, the conditions for administering an antibody with selectively enhanced binding to mouse FcγRIII can be mimicked by administering mF44 or mF46 to FcγRIIb deficient mice that do not express mouse FcγRIIb.

As described in Example 6, the plasma concentration of soluble antigen was reduced in FcγRIII deficient mice, which mimic the administration state of an antibody with selectively increased mouse FcγRIIb binding activity. Meanwhile, whether the plasma concentration of soluble antigen decreases in FcγRIIb-deficient mice that mimic the administration conditions of an antibody with selectively increased mouse FcγRIII binding activity was evaluated by the assay described below.

(7-2) Evaluation of antigen elimination effect by selectively enhancing mouse FcγRIII binding using FcγRIIb deficient mice

The elimination effect of soluble IL-6 receptor from plasma FcγRIIb-deficient mice treated with anti-human IL-6 receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1mF46 was evaluated in the same manner as described in Example 5. Concentrations soluble human IL-6 receptor in plasma was determined by the method described in Example (2-1-2). The result is shown in Fig. 16.

Surprisingly, in groups administered with mF44 and mF46, which mimic a selective increase in mIgG1 (native mouse IgG1) binding activity to mouse FcγRIII, the plasma concentration of IL-6 receptor was reduced, however, the reduction was not confirmed compared to that shown in example 6.

Without being bound by a particular theory, based on the results described in examples 5, 6 and 7, the following discussion is possible. Elimination of the soluble IL-6 receptor from plasma was found to be significantly accelerated in normal mice expressing both mouse FcγRIIb and mouse FcγRIII injected with mF44 and mF46 with selectively increased binding activity of mIgG1 (native mouse IgG1) to mouse FcγRIIb and mouse FcγRIII . Moreover, when mF44 and mF46 were administered to mice that express mouse FcγRIIb but not mouse FcγRIII (i.e., FcγRIII deficient mice and Fc receptor γ chain deficient mice), elimination of the soluble IL receptor -6 from plasma in mice was also significantly accelerated. Meanwhile, when mF44 and mF46 were administered to mice that express mouse FcγRIII but not mouse FcγRIIb (i.e., FcγRII deficient mice), elimination of the soluble IL-6 receptor from plasma was not accelerated in mice to the same extent as when they were administered FcγRIII deficient mice or Fc receptor γ chain deficient mice.

From the above data, mF44 and mF46 antibodies, in which the binding activity of mIgG1 (native mouse IgG1) to mouse FcγRIIb and mouse FcγRIII is increased, are thought to be incorporated into FcγR-expressing cells primarily via mouse FcγRIIb and thus a soluble plasma antigen. that binds to antibodies is eliminated. Meanwhile, FcγRIII-mediated incorporation of antibody/antigen complexes into FcγR-expressing cells is not believed to significantly contribute to the elimination of soluble antigen from plasma.

Moreover, as shown in Example 4, the plasma concentration of soluble IL-6 receptor was significantly reduced in mice treated with Fv4-IgG1-F1087, which has increased binding activity to, in particular, mouse FcγRIIb and mouse FcγRIII. Meanwhile, the elimination effect of the soluble IL-6 receptor from the plasma of mice injected with Fv4-IgG1-F1182 with increased binding activity to mouse FcγRI and mouse FcγRIV in particular was less than that of Fv4-IgG1-F1087.

Moreover, as shown in Example 2, in mice administered with Fv4-IgG1-Fuc, the binding activity of which to mouse FcγRIV is significantly increased due to the presence of a low-fucose sugar chain (Science (2005) 310 (5753) 1510-1512), Plasma concentrations of soluble IL-6 receptor were reduced compared to those of soluble IL-6 receptor in Fv4-IgG1-treated mice; however, the reduction effect was about half that. Thus, it is believed that mouse FcγRIV-mediated incorporation of antibodies into FcγR-expressing cells does not significantly contribute to the elimination of soluble antigens from plasma.

In view of the above, it has been demonstrated that of several mouse FcγR, mouse FcγRIIb and mouse FcγIII, in particular mouse FcγRIIb, it has been shown to play a major role in antibody incorporation into FcγR expressing cells in mice. Thus, mutations introduced into the mouse FcγR binding domain would preferably include, but are not limited to, mutations that enhance binding to mouse FcγRIIb and mouse FcγIII, in particular binding to mouse FcγRIIb.

The data described above demonstrate that in mice, the binding activity of the antibody to be administered to mouse FcγRIIb and mouse FcγIII is more preferable, in particular, the binding activity to FcγRIIb is increased to accelerate the elimination of soluble antigens from living body plasma by introducing antigen-binding molecules that bind to soluble antigens in a pH dependent manner and have increased FcγR binding activity. In particular, when administered to a living body, antigen-binding molecules that bind to soluble antigens in a pH-dependent manner and have increased activity with mouse FcγRIIb and mouse FcγIII, in particular, increased FcγRIIb binding activity, can accelerate the elimination of soluble antigens from plasma and effectively reduce the concentration of soluble antigens in plasma and thus it was found that antigen-binding molecules show a very effective action.

[Example 8] Evaluation of the aggregation ability of antibodies containing an Fc region that has a known change that enhances FcγRIIb binding

(8-1) Production of antibodies containing an Fc region that has been modified to enhance FcγRIIb binding

As described in Reference Example 7, antigens can be effectively eliminated from the plasma of a living body by introducing antibodies with a selectively increased FcγRIIb binding activity into a living body. Moreover, administration of antibodies containing an Fc region with a selectively increased FcγRIIb binding activity is believed to be preferable from the point of view of safety and side effects in a living organism into which such antibodies are administered.

However, both mouse FcγRIIb binding and mouse FcγRIII binding are enhanced in mF44 and mF46, and thus binding enhancement is not selective for mouse FcγRIIb. Because the homology between mouse FcγRIIb and mouse FcγRIII is high, it would be difficult to find a change that enhances selective binding to FcγRIIb that distinguishes them. Moreover, there are no previous reports of Fc regions with selectively enhanced binding of mouse FcγRIIb. It is also known that the homology between human FcγRIIb and human FcγRIIa (two allotypes, 131Arg and 131His) is high. Moreover, there are no reports of Fc regions that contain a change that enhances selective binding to human FcγRIIb distinguishing them (Seung et al., (Mol. Immunol. (2008) 45, 3926-3933); Greenwood et al., (Eur. J. Immunol. (1993) 23 (5), 1098-1104)). Moreover, FcγRIIa binding has been reported to play an important role in the platelet aggregation activity of the antibody (Meyer et al., (J. Thromb. Haemost. (2009), 7 (1), 171-181); Robles-Carrillo et al. ., (J. Immunol. (2010), 185 (3), 1577-1583)). In view of these reports, it is possible that an antibody with enhanced FcγRIIa binding may increase the risk of thrombosis in the living organism to which it is administered. Thus, whether antibodies with enhanced FcγRIIa binding have increased platelet aggregation activity was evaluated as follows.

(8-2) Evaluation of human FcγR binding activity in antibodies containing an Fc region that has a known alteration that enhances FcγRIIb binding

Antibodies containing an Fc region tampered with with a known alteration that enhances human FcγRIIb binding were analyzed for their affinity for human FcγRIa, R-type and H-type FcγRIIa, FcγRIIb, and FcγRIIIa by the following procedure. The H chain was designed to have as the variable region of the human H chain the variable region of the anti-human interleukin 6 receptor antibody IL6R-H (SEQ ID NO: 132) as described in WO 2009/125825 and, as the constant region H β-chain of an antibody, IL6R-G1d (SEQ ID NO: 133), which has G1d obtained by removing the C-terminal Gly and Lys from human IgG1. Then, IL6R-G1d-v3 (SEQ ID NO: 138) was constructed by changing the Fc region of IL6R-G1d by replacing Ser with Glu at position 267 (EU numbering) and Leu with Phe at position 328 (EU numbering), as described in Seung et al., (Mol. Immunol. (2008) 45, 3926-3933). IL6R-L (SEQ ID NO: 135), which is the L chain of an antibody against human interleukin 6 receptor, was used as the L chain of a conventional antibody and expressed in combination with the corresponding H chains according to the method described in Reference Example 1 and the resulting antibodies were purified. Further, antibodies containing IL6R-G1d and IL6R-G1d-v3 as a heavy chain are referred to as IgG1 and IgG1-v3, respectively.

The interaction between FcγR and the antibodies described above was then kinetically analyzed using Biacore T100 (GE Healthcare). Interaction assays were performed at 25° C. using HBS-EP+ (GE Healthcare) as running buffer. The chip used was a CM5 Series S sensor chip (GE Healthcare) onto which protein A was immobilized by amine addition. Each FcγR diluted with running buffer was allowed to interact with antibodies of interest captured on the chip to measure antibody binding to each FcγR. After measurement, 10 mM glycine-HCl (pH 1.5) was reacted with the chip to wash off the captured antibodies to reuse the regenerated chip. The sensorgram obtained from the measurement was analyzed using a 1:1 Langmuir binding model using the Biacore Evaluation Software, and the binding rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) were calculated, and from these values, the dissociation constant KD (mol/L) was calculated. The KD values of IgG1 and IgG1-v3 for each FcγR are shown in Table 12 (KD values of each antibody against each FcγR), while the relative KD values of IgG1-v3, which are obtained by dividing the KD of IgG1 for each FcγR by the KD of IgG1 -v3 for each FcγR are presented in Table 13.

(8-3) Onenka ability to aggregate platelets

Next, an assessment of whether increased/decreased affinity for FcγRIIa of an antibody containing an Fc region with Ser replaced by Glu at position 267 and Leu by Phe at position 328 (EU numbering) in the IgG1 Fc region alters platelet aggregation ability was performed with using platelets obtained from donors with FcγRIIa type H or type R. Antibody containing as a light chain omalizumab_VL-CK (SEQ ID NO: 137) and omalizumab_VH-G1d (SEQ ID NO: 136), which contain the variable region of the heavy chain of the antibody hIgG1 (human IgG1 constant region) that binds to IgE and heavy chain constant region G1d were constructed using the method described in Reference Example 2. Moreover, omalizumab_VH-G1d-v3 (SEQ ID NO: 138) was constructed by replacing Ser to Glu at position 267 and Leu to Phe at position 328 (EU numbering) in omalizumab_VH-G1d. Omalizumab-G1d-v3, which contains omalizumab_VH-G1d-v3 as a heavy chain and omalizumab_VL-CK as a light chain, was prepared using the method described in Reference Example 2. This antibody was evaluated for platelet aggregation ability.

Platelet aggregation was analyzed using a HEMA TRACER 712 platelet aggregometer (LMS Co.). First, approximately 50 ml of whole blood was collected in a fixed amount in 4.5 ml evacuated blood collection tubes containing 0.5 ml of 3.8% sodium citrate and centrifuged at 200 g for 15 minutes. The resulting supernatant was collected and used as platelet-rich plasma (PRP). After PRP was washed with buffer A (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.42 mM NaH ₂ PO ₄ , 2 mM MgCl ₂ , 5 mM HEPES, 5.55 mM dextrose, 1, 5 U/ml apyrase, 0.35% BSA), buffer was replaced with buffer B (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.42 mM NaH ₂ PO ₄ , 2 mM MgCl ₂ , 5 mM HEPES, 5.55 mM dextrose, 2 mM CaCl ₂ , 0.35% BSA). This provided washed platelets at a density of approximately 300,000/µl. 156 μl of washed platelets were aliquoted into assay cuvettes containing stirrer in a platelet aggregometer. Platelets were stirred at 1000 rpm. stirrer in cuvettes maintained at 37.0°C in a platelet aggregometer. 44 μl of the immune complex omalizumab-G1d-v3 and IgE in a 1:1 molar ratio prepared at final concentrations of 600 μg/ml and 686 μg/ml, respectively, was added to the cuvette. Platelets were subjected to reaction with the immune complex for five minutes. Then, at a concentration that does not allow secondary platelet aggregation, adenosine diphosphate (ADP, SIGMA) was added to the reaction mixture to examine whether aggregation increased.

The result of platelet aggregation for each donor with the FcγRIIa (R/H or H/H) polymorphic form obtained in the analysis above is shown in FIG. 17 and 18, respectively. From the result in FIG. 17 Platelet aggregation is observed when the immune complex is added to donor platelets with the FcγRIIa (R/H) polymorph. Meanwhile, as shown in FIG. 18, no platelet aggregation was observed when the immune complex was added to donor platelets with the FcγRIIa (H/H) polymorph.

Further, platelet activation was assessed using activation markers. Platelet activation can be measured based on increased expression of an activation marker such as CD62p (p-selectin) or active integrin on the platelet membrane surface. 2.3 μl of the immune complex was added to 7.7 μl of washed platelets prepared as described above. After reacting for five minutes at room temperature, activation was induced by adding ADP at a final concentration of 30 μM and it was assessed whether the immune complex enhances ADP-dependent activation. A sample to which buffer (pH 7.4) (Gibco) was added in place of the immune complex was used as a negative control. Staining was performed by post-reaction addition of PE-labeled anti-CD62 antibody (BECTON DICKINSON), PerCP-labeled anti-CD61 antibody, and FITC-labeled PAC-1 antibody (BD bioscience) to each sample post-reaction. The fluorescence intensity of each dye was measured using a flow cytometer (FACS CantoII, BD bioscience).

The result of CD62p expression obtained by the above method is shown in FIG. 19. The result of activated expression of integrins is shown in FIG. 20. The washed platelets used were from a healthy individual with the FcγRIIa R/H polymorph. The expression level of both CD62p and active integrin expressed on the platelet membrane surface, which are induced by ADP stimulation, increased in the presence of the immune complex.

The above results demonstrate that an antibody having an Fc region that has a known change that enhances binding of human FcγRIIb, which is the replacement of Ser with Glu at position 267 and Leu with Phe at position 328 (EU numbering) in the Fc region IgG1 accelerates the aggregation of platelets expressing FcγRIIa with the FcγRIIa allotype, in which the amino acid at position 131 is R, compared to platelets expressing FcγRIIa with the FcγRIIa polymorph, in which the amino acid at position 131 is H. Thus, it was proposed that that the risk of developing thrombosis due to platelet aggregation can be increased when an antibody containing an Fc region in which a known alteration is made that enhances binding to a known human FcγRIIb is administered to a human having an R-type FcγRIIa in at least one allele. It has been shown that antigen-binding molecules containing the Fc region of the present invention, which enhances FcγRIIb binding more selectively, not only increase antigen plasma retention time, but also possibly solve the above problems. Thus, the applicability of the antigen-binding molecules of the present invention is clear.

(8-4) Comparison of platelet aggregation ability between an antibody (BP230) containing an Fc with selectively enhanced FcγRIIb binding and an antibody containing a known Fc with selectively enhanced FcγRIIb binding

(8-4-1) Production of antibodies with selectively enhanced FcγRIIb binding

As described in Example (8-3), FcγRIIa-enhanced binding antibodies have been shown to have enhanced platelet aggregation ability and increased platelet activation ability, and may increase the risk of thrombosis when administered to a human. Meanwhile, given that among the FcγRs, only FcγRIIa is expressed on platelets, it is believed that antibodies with selectively enhanced FcγRIIb binding do not show increased platelet aggregation or activation ability or increase the risk of thrombosis when administered to a human. To confirm this, antibodies with selectively enhanced FcγRIIb binding were actually investigated for their ability to aggregate platelets and their ability to activate platelets.

In particular, omalizumab_VH-G1d (SEQ ID NO: 136) and omalizumab_VL-CK (SEQ ID NO: 137), as the heavy chain and light chain of hIgG1 antibody (human IgG1 constant region) that binds to IgE, respectively, were obtained omalizumab_VH-BP230 (SEQ ID NO: 140) was then obtained by replacing Glu with Asp at position 233, Gly with Asp at position 237, Pro with Asp at position 238, His at Asp at position 268, Pro at Gly at position 271, Tyr at Asp at position 296, Ala at Arg at position 330, and Lys at Glu at position 439 (EU numbering). Similarly, to increase the binding activity of human FcγRIIb and FcγRIIa R in omalizumab_VH-G1d, omalizumab_VH-G1d-v3 (SEQ ID NO: 138) was generated by replacing Ser with Glu at position 267 and Leu with Phe at position 328 (EU numbering).

Omalizumab-BP230, which contains omalizumab_VH-BP230 (SEQ ID NO: 140) as a heavy chain and omalizumab_VH-CK (SEQ ID NO: 137) as a light chain, and omalizumab-G1d-v3, which contains omalizumab_VH-G1d-v3 (SEQ ID NO: 138) as a heavy chain and omalizumab_VL-CK (SEQ ID NO: 137) as a light chain were obtained using the method described in Reference Example 2. These antibodies were evaluated for their ability to aggregate and activate platelets.

(8-4-2) Evaluation of omalizumab-BP230 and omalizumab-G1d-v3 for their human FcγR binding activity

The results of the affinity analysis between each human FcγR and the Fc region of omalizumab-G1d-v3 by enhancing the binding of human FcγRIIb to omalizumab, which is a known antibody, are shown in Example (8-2). The affinity of the Fc region of omalizumab-BP230 for each human FcγR was also analyzed in a similar manner and the results are shown in Table 22.

(8-4-3) Assessing the ability to aggregate platelets

Then, omalizumab-BP230 and omalizumab-G1d-v3 prepared as described in Example (8-4-1) were analyzed for their binding using platelets from a donor with the FcγRIIa (R/H) polymorph. The result of the analysis was used to assess whether the ability to aggregate platelets, depending on the level of affinity for FcγRIIa.

Platelet aggregation analysis was performed using a HEMA TRACER 712 platelet aggregometer (LMS Co.). First, approximately 50 ml of whole blood was collected in a fixed amount in 4.5 ml evacuated blood collection tubes containing 0.5 ml of 3.8% sodium citrate and centrifuged at 200 g for 15 minutes. The resulting supernatant was collected and used as platelet-rich plasma (PRP). After PRP was washed with buffer A (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.42 mM NaH ₂ PO ₄ , 2 mM MgCl ₂ , 5 mM HEPES, 5.55 mM dextrose, 1, 5 U/ml apyrase, 0.35% BSA), buffer was replaced with buffer B (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.42 mM NaH ₂ PO ₄ , 2 mM MgCl ₂ , 5 mM HEPES , 5.55 mM dextrose, 2 mM CaCl ₂ , 0.35% BSA). This provided washed platelets at a density of approximately 300,000/µl. 150.2 μl of washed platelets were aliquoted into assay cuvettes containing stirrer in a platelet aggregometer. Platelets were stirred at 1000 rpm. stirrer in cuvettes maintained at 37.0°C in a platelet aggregometer. 24.3 μl of omalizumab-G1d-v3 or omalizumab-BP230 prepared at a final concentration of 600 μg/ml was added to the cuvettes. After reacting for one minute, 25.4 μl of IgE was added, which was prepared so that its molar ratio to antibody was 1:1. The mixtures were incubated for five minutes. Adenosine diphosphate (ADP, SIGMA) was then added to the reaction mixture at a concentration that did not induce secondary platelet aggregation to investigate whether aggregation was enhanced. The washed platelets used were from one healthy individual with the FcγRIIa R/H polymorph.

The result obtained by the method described above is shown in Fig. 21. The result shown in FIG. 21 showed that platelets aggregated strongly when the immune complex containing omalizumab-G1d-v3 was added. On the other hand, there was no difference in platelet aggregation between when PBS was added and when the immune complex containing omalizumab-BP230 was added.

Further, platelet activation was evaluated by omalizumab-G1d-v3 and omalizumab-BP230. Platelet activation can be measured based on increased expression of an activation marker such as CD62p (p-selectin) or active integrin on the platelet membrane surface. 1.22 µl of omalizumab-G1d-v3 or omalizumab-BP230 prepared at a final concentration of 600 µg/ml was added to 7.51 µl of washed platelets prepared as described above. After reacting for five minutes at room temperature, 1.27 μl of IgE was added, which was obtained so that its molar ratio to antibody was 1:1. Then, after reacting for five minutes at room temperature, it was evaluated whether platelet activation was induced. The negative control used was a sample in which phosphate buffer (pH 7.4, Gibco) was added in place of the immune complexes. After the reaction, each sample was subjected to fluorescent staining with PE-labeled anti-CD62 antibody (BECTON DICKINSON), PerCP-labeled anti-CD61 antibody, and FITC-labeled PAC-1 antibody (BD bioscience) and determined the fluorescence intensity using a flow cytometer (FACS CantoII, BD Bioscience). The washed platelets used were from one healthy individual with the FcγRIIa R/H polymorph.

The results obtained using the analysis method described above are shown in FIG. 22 and 23. Both CD62p and active integrin expression on the platelet membrane surface was increased by the addition of an immune complex containing omalizumab-G1d-v3. Meanwhile, the expression of molecules on the platelet membrane surface when the immune complex omalizumab-BP230 was added was comparable to the expression on the platelet membrane surface when phosphate buffer was added.

The above results demonstrate that an antibody that contains a known modified Fc region with Ser to Glu at position 267 and Leu to Phe at position 328 (EU numbering) in the IgG1 Fc region and which has enhanced binding to both human FcγRIIb and and with human FcγRIIa R, accelerates platelet aggregation and activation when the amino acid at position 131 is Arg in at least any of the alleles of the FcγRIIa polymorphic gene compared to an antibody that contains an Fc region with Glu to Asp substitutions at position 233, Gly to Asp at position 237, Pro to Asp at position 238, His to Asp at position 268, Pro to Gly at position 271, Tyr to Asp at position 296, Ala to Arg at position 330, and Lys to Glu at position 439 (numbering EU) and which has selectively enhanced binding to human FcγRIIb. In particular, it has been suggested that an antibody that contains a variant of the known Fc region with enhanced binding of human FcγRIIb has the problem that it may increase the risk of thrombosis due to platelet aggregation in a human with the R allele of FcγRIIa; on the other hand, a variant Fc region with selectively enhanced FcγRIIb binding has been shown to overcome this problem.

[Example 9] Detailed analysis of FcγRIIb binding for variants that have a change in the hinge region in addition to the P238D change

In Fc obtained by replacing Pro at position 238 (EU numbering) with Asp in naturally occurring human IgG1, the expected combinatorial effect could not be achieved even by combining it with another change predicted to further increase FcγRIIb binding, based on from analysis of naturally occurring antibodies. Thus, in order to find variants that further enhance FcγRIIb binding, extensive changes were made to the altered Fc obtained by replacing Pro at position 238 (EU numbering) with Asp. IL6R-F11 (SEQ ID NO: 141) was made by changing Met at position 252 (EU numbering) to Tyr and changing Asn at position 434 (EU numbering) to Tyr in IL6R-G1d (SEQ ID NO: 133), which was used as the H chain of the antibody. Moreover, IL6R-F652 (SEQ ID NO: 142) was obtained by making a change by replacing Pro at position 238 (EU numbering) with Asp in IL6R-F11. Expression plasmids containing the antibody H chain sequence were generated for each of the antibody H chain sequences obtained by replacing the region near the residue at position 238 (EU numbering) (positions 234 to 237, and 239 (EU numbering)) in IL6R- F652, in each case 18 amino acids, excluding the original amino acids and cysteine. For all antibodies, IL6R-L (SEQ ID NO: 135) was used as the antibody L chain. These variants were expressed and purified by the method of Reference Example 2. These Fc variants are referred to as PD variants. The interactions of each PD variant with FcγRIIa type R and FcγRIIb were evaluated in detail by the method of Reference Example 25.

A graph showing the results of the analysis of interaction with the corresponding FcγR was built in accordance with the following method. The value obtained by dividing the value of the binding value of each PD variant to each FcγR by the value of the binding value of the FcγR of the previously altered antibody that was used as a control (IL6R-F652/IL6R-L, which has the Pro substitution change at position 238 (EU numbering) by Asp and then multiplying the result by 100 is shown as the relative binding activity of each PD variant to each FcγR The horizontal axis shows the relative binding activity of FcγRIIb for each PD variant and the vertical axis shows the relative binding activity of R type FcγRIIa for each variant PD (Fig. 24).

As a result, it was found that the binding of FcγRIIb in the case of eleven types of change was increased compared to the antibody before the change, and they have the effects of maintaining or enhancing the binding of FcγRIIa type R. The activity of these eleven variants in relation to the binding of FcγRIIb and FcγRIIa type R is summarized in table 14. In the table, the sequence ID numbers refer to the H chain numbers of the variants and the change refers to the change made in IL6R-F11 (SEQ ID NO: 141).

In FIG. 25 shows the relative values for the FcγRIIb binding activity obtained by further making the above eleven changes to a variant having the P238D change and the relative values for the FcγRIIb binding activity of the variant obtained by making these changes to an Fc that does not contain P238D. These eleven changes enhanced FcγRIIb binding compared to before they were added to the P238D variant. On the contrary, the effect of reducing FcγRIIb binding was observed for eight of these changes, with the exception of G237F, G237W and S239D, when they were introduced into a variant that does not contain P238D (data not shown).

These results showed that, based on the effects of making changes to naturally occurring IgG1, it is difficult to predict the effects of combining and making the same changes to a variant containing the P238D change. In other words, it is not possible to identify the eight changes identified this time without this study, in which these changes are combined and made into a variant containing the P238D change.

The results of measuring the KD values of the variants listed in Table 14 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIaV by the method of Reference Example 25 are summarized in Table 15. In the table, the change refers to the change made to IL6R-F11 (SEQ ID NO: 141) . The template used to obtain IL6R-F11, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD(IIaR)/KD(IIb) and KD(IIaH)/KD(IIb) in the table, respectively, show the value obtained by dividing the KD value of each FcγRIIaR option by the KD value of each FcγRIIb option, and the value obtained by dividing the KD value of each FcγRIIaH variant by the KD value of each FcγRIIb variant. The parent polypeptide/KD(IIb) variant KD(IIb) refers to the value obtained by dividing the parent polypeptide's FcγRIIb KD value by the FcγRIIb KD value of each FcγRIIb variant.

In addition, Table 15 shows KD values for stronger FcγRIIaR and FcγRIIaH binding activity of each variant/KD values for stronger FcγRIIaR and FcγRIIaH binding activity of the parent polypeptide. In this case, the parent polypeptide refers to the variant that has IL6R-F11 (SEQ ID NO: 141) as the H chain. It was determined that, due to the weak binding of FcγR to IgG, it could not be accurately analyzed using kinetic analysis, and thus the gray cells in Table 15 show the values calculated using Equation 2 of Reference Example 25.

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C.

Table 15 shows that for all variants, the binding affinity for FcγRIIb increased compared to IL6R-F11, and the improvement ranged from 1.9-fold to 5.0-fold. The ratio of FcγRIIaR KD value of each variant/FcγRIIb KD value of each variant, and the FcγRIIaH KD value of each variant/FcγRIIb KD value of each variant correspond to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant for FcγRIIb, and a higher value indicates a higher binding selectivity for FcγRIIb. For the parent IL6R-F11/IL6R-L polypeptide, the ratio of FcγRIIaR KD value/FcγRIIb KD value and the FcγRIIaH KD value/FcγRIIb KD value ratio is 0.7 in both cases, and, accordingly, all variants presented in the table 15 demonstrated an increase in binding selectivity for FcγRIIb over the parent polypeptide. When the ratio of KD value for the stronger of the FcγRIIaR and FcγRIIaH variant binding activities/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide is 1 or more, it means that the stronger of the FcγRIIaR and FcγRIIaH binding activities of the variant has an equivalent or reduced binding versus binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 0.7 to 5.0 for the variants obtained this time, it can be said that the binding at the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variants obtained this time was substantially the same or reduced compared to original polypeptide. These results showed that, compared to the parent polypeptide, the variants obtained in this case have retained or reduced binding activity to FcγRIIa type R and type H, and enhanced binding activity to FcγRIIb and thus have increased selectivity for FcγRIIb. . Moreover, compared to IL6R-F11, all variants had lower affinity for FcγRIa and FcγRIIIaV.

[Example 10] X-ray diffraction analysis of the complex formed between Fc containing P238D and the extracellular region of FcγRIIb

As shown earlier in Example 9, even though a change is made that is predicted from analysis of naturally occurring IgG1 antibodies to increase FcγRIIb binding activity or FcγRIIb selectivity, in Fc containing P238D, it was found that the activity FcγRIIb binding is reduced, and the reason for this may be that the structure on the interaction surface between Fc and FcγRIIb is changed due to the introduction of P238D. Thus, in order to establish the cause of this phenomenon, the three-dimensional structure of the complex formed between the IgG1 Fc containing the P238D mutation (hereinafter referred to as Fc(P238D)) and the extracellular region of FcγRIIb was determined by X-ray diffraction analysis, and it was compared with the three-dimensional structure of the complex formed by between the Fc of naturally occurring IgG1 (hereinafter referred to as Fc(WT)) and the extracellular region of FcγRIIb, and the binding methods were compared. Many reports have been made about the three-dimensional structure of the complex formed between Fc and the extracellular region of FcγR; and analyzed the three-dimensional structures of the Fc(WT) complex/FcγRIIIb extracellular region (Nature, 2000, 400: 267-273; J. Biol. Chem. 2011, 276: 16469-16477), the Fc(WT) complex/FcγRIIIa extracellular region ( Proc. Natl. Acad. Sci. USA, 2011, 108: 12669-126674), and the Fc(WT)/extracellular region FcγRIIa complex (J. Immunol. 2011, 187: 3208-3217). Although the three-dimensional structure of the Fc(WT) complex/FcγRIIb extracellular region has not been analyzed, the three-dimensional structure of the complex formed with Fc(WT) is known for FcγRIIa and FcγRIIb, and their extracellular regions share 93% amino acid sequence and have very high homology. Thus, the three-dimensional structure of Fc(WT)/FcγRIIb extracellular region was predicted by modeling using the crystal structure of the Fc(WT)/FcγRIIa extracellular region.

Fc(WT) и соответствующей внеклеточной областью FcγRI На, FcγRIIIb или FcγRIIa, проанализированных до настоящего времени.The three-dimensional structure of the Fc(P238D)/FcγRIIb extracellular region was determined by X-ray analysis at a resolution of 2.6 Å. The structure resulting from this analysis is shown in FIG. 26. The extracellular region of FcγRIIb binds between the two CH2 domains of Fc, and this was similar to the three-dimensional structures of the complexes formed between

Fc(WT) and the corresponding extracellular region of FcγRI Ha, FcγRIIIb or FcγRIIa analyzed to date.

Further, for detailed comparison, the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the structure of the Fc(WT) complex/FcγRIIb extracellular region were superimposed on each other using a least squares approximation based on the distances between pairs of Cα atoms in relation to the extracellular region of FcγRIIb and CH2 -domain A Fc (Fig. 27). In this case, the degree of overlap between the CH2 domains of B Fc was not satisfactory, and conformational differences were found in this part. Moreover, using the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, pairs of atoms were selected that have a distance of 3.7 Å or less between the selected FcγRIIb extracellular region and CH2- domain B of Fc and compared to compare the interatomic interaction between FcγRIIb and CH2 domain B in Fc(WT) with the interatomic interaction between FcγRIIb and Fc(P238D). As shown in Table 16, the interatomic interactions between the CH2 domain of Fc and FcγRIIb in Fc(P238D) and Fc(WT) did not match.

Moreover, the detailed structures in the P238D region were compared by overlaying the X-ray crystal structure of the Fc (P238D) complex/FcγRIIb extracellular region with the model structure of the Fc (WT) complex/FcγRIIb extracellular region using the least squares method based on the Ca atomic distance between the CH2 domains of A and B Fc separately. Since the position of the amino acid residue at position 238 (EU numbering), i.e. the mutagenesis position of Fc (P238D) is changed relative to Fc (WT), it was found that the structure of the loop in the region of the amino acid residue at position 238 after the hinge region differs between Fc (P238D) and Fc (WT) (Fig. 28). Pro at position 238 (EU numbering) is initially located within Fc (WT), forming a hydrophobic core with residues in the region of position 238. However, if Pro at position 238 (EU numbering) is changed to a highly hydrophilic and charged Asp, the presence of an altered Asp residue in the hydrophobic core is energetically advantageous in terms of desolvation. Thus, in Fc(P238D), to eliminate this energetically disadvantageous situation, the amino acid residue at position 238 (EU numbering) changes its orientation towards the solvent, and this can cause this change in the structure of the loop near the amino acid residue at position 238. Moreover, since this loop is located close to the S-S-linked hinge region, its structural change will not be limited to local change, and will affect the relative positioning of the A CH2 domain and the Fc B domain. As a result, the interatomic interactions between FcγRIIb and the CH2 domain of Fc are changed. Thus, the predicted effects could not be observed when changes that increase the selectivity and binding activity for FcγRIIb in naturally occurring IgG were combined with an Fc containing the P238D change.

Moreover, as a result of structural changes due to the introduction of P238D into the Fc A CH2 domain, a hydrogen bond was detected between the Gly backbone at position 237 (EU numbering), which is adjacent to the mutant P238D and Tyr at position 160 in FcγRIIb (Fig. 29 ). The residue in FcγRIIa that corresponds to this Tyr160 is Phe; and when binding to FcγRIIa occurs, this hydrogen bond is not formed. Considering that the amino acid at position 160 represents one of a small number of differences between FcγRIIa and FcγRIIb at the interface with Fc, and the presence of this hydrogen bond, which is specific to FcγRIIb, presumably resulted in an increase in FcγRIIb binding activity and a decrease in FcγRIIa binding activity in Fc (P238D) and increase its selectivity. Moreover, in the CH2 domain of B Fc, an electrostatic interaction was observed between Asp at position 270 (EU numbering) and Arg at position 131 in FcγRIIb (FIG. 30). In FcγRIIa type H, which is one of the allotypes of FcγRIIa, the residue corresponding to Arg at position 131 of FcγRIIb is His, and thus cannot form this electrostatic interaction. This may explain why Fc(P238D) binding activity is reduced in type H FcγRIIa compared to type R FcγRIIa. domains causes the formation of new interactions that do not occur with the binding of naturally occurring IgG and FcγR, and this could lead to a selective binding profile of P238D variants in relation to FcγRIIb.

[Expression and purification of Fc(P238D)]

Fc containing the change P238D was obtained as follows. First, the Cys at position 220 (EU numbering) of hIL6R-IgG1-v1 (SEQ ID NO: 143) was changed to Ser. Then, the Fc(P238D) genetic sequence from Glu at position 236 (EU numbering) to its C-terminus was cloned by PCR. Using this cloned genetic sequence, expression vectors were produced and Fc(P238D) was expressed and purified according to the method of Reference Examples 1 and 2. The Cys at position 220 (EU numbering) forms a disulfide bond with the L chain Cys in general IgG1. The L chain is not co-expressed when Fc is prepared alone, and thus the Cys residue was changed to Ser to avoid the formation of unnecessary disulfide bonds.

[Expression and purification of the extracellular region of FcγRIIb]

The extracellular region of FcγRIIb was obtained according to the method of Reference Example 25.

[Sedum complex Fc(P238D)/extracellular region FcγRIIb]

To 2 mg of the FcγRIIb extracellular region sample prepared for use in crystallization was added 0.29 mg of Endo F1 (Protein Science (1996) 5: 2617-2622), expressed and purified from Escherichia coli, as glutathione-S-transferase fusion protein . The mixture was allowed to stand at room temperature for three days in 0.1 M Bis-Tris buffer at 6.5 and the N-linked oligosaccharide was cleaved off except for the N-acetylglucosamine directly bound to the Asn of the extracellular region of FcγRIIb. Next, a sample of the extracellular region of FcγRIIb subjected to carbohydrate cleavage treatment, which was concentrated by ultrafiltration with 5000 MWCO, was purified by gel filtration (Superdex200 10/300) using a column equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl. Moreover, Fc(P238D) was added to the fraction of the extracellular region of FcγRIIb obtained after carbohydrate cleavage, so that the extracellular region of FcγRIIb was present in a molar ratio in a slight excess. The mixture, concentrated by ultrafiltration with 10,000 MWCO, was subjected to purification by gel filtration (Superdex200 10/300) using a column equilibrated in 20 mm HEPS at pH 7.5 containing 0.05 M NaCl. Thus, a sample of the Fc(P238D)/FcγRIIb extracellular region complex was obtained.

[Crystallization of the Fc(P238D) complex/extracellular region of FcγRIIb]

Using a sample of the Fc(P238D)/FcγRIIb extracellular region complex, which was concentrated to approximately 10 mg/ml by ultrafiltration with 10,000 MWCO, the complex was crystallized by the sessile drop vapor diffusion method. Hydra II Plus One (MATRIX) was used for crystallization; and as a reservoir solution containing 100 mM Bis-Tris pH 6.5, 17% PEG3350, 0.2 M ammonium acetate, and 2.7% (w/v) D-galactose, a crystallization drop was obtained by mixing the ratio reservoir solution : crystallization sample = 0.2 µl: 0.2 µl. The container was closed and the crystal was allowed to stand at 20°C, and thus flat crystals were obtained.

[Measurement of X-ray diffraction data in the crystal of the Fc(P238D) complex/extracellular region of FcγRIIb]

One of the obtained single crystals of the Fc(P238D)/FcγRIIb extracellular region was immersed in a solution of 100 mM Bis-Tris pH 6.5, 20% PEG3350, ammonium acetate, 2.7% (w/v) D-galactose, 22, 5% (v/v) ethylene glycol. The single crystal was fished out of solution using a pin with a thin nylon loop attached and frozen in liquid nitrogen. Next, crystal X-ray diffraction data was measured with a Photon Factory BL-1A bremsstrahlung equipment at the High Energy Accelerator Research Organization. During the measurement, the crystal was constantly placed in a flow of nitrogen at -178°C to maintain it frozen, and a total of 225 X-rays were obtained using a Quantum 270 CCD (ADSC) detector connected to the beam line, with a crystal rotation of 0.8° per one once. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data from the obtained X-ray patterns were performed using the Xia2 program (ССР4 Software Suite), XDS Package (Walfgang Kabsch) and Scala (CCP4 Software Suite); and finally, crystal diffraction intensity data were obtained at resolutions up to 2.46 Å. The crystal belongs to the space group P2 ₁ and has the following cell parameters; a=48.85 Å, d=76.01 Å, c=115.09 Å, α=90°, β=100.70°, γ=90°.

[X-ray diffraction analysis of the Fc(P238D) complex/extracellular region of FcγRIIb]

The crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region was determined by the molecular displacement method using the Phaser program (CCP4 Software Suite). From the resulting crystal lattice size and the molecular weight of the Fc(P238D)/FcγRIIb extracellular region, it was predicted that the number of complexes in the asymmetric element was one. From the structural coordinates of the PDB code: 3SGJ, which is the crystal structure of the Fc(WT) complex/FcγRIIIa extracellular region, the portions of the amino acid residues of the A chain at positions 239-340 and the B chain at positions 239-340 were taken as separate coordinates, and they were adopted, respectively, as models for the search for Fc CH2 domains. The amino acid residue regions of the A chain at positions 341-444 and the B chain at positions 341-443 were taken as a single set of coordinates from the same structural coordinates of the PDB code: 3SGJ; and they were adopted as a model for searching for Fc CH3 domains. Finally, from the structural coordinates of the PDB code: 2FCB, which corresponds to the crystal structure of the extracellular region of FcγRIIb, the regions of A chain amino acid residues at positions 6-178 were chosen and adopted as a model for searching for the extracellular region of FcγRIIb. The orientation and position of each retrieval pattern in the crystal lattice was determined in the order of Fc CH3 domain, FcγRIIb extracellular region, and Fc CH2 domain, based on rotation function and translation function, to obtain an initial crystal structure model of the Fc(P238D)/FcγRIIb extracellular region. When the obtained initial model was refined into a rigid body that moves two Fc CH2 domains, two Fc CH3 domains, and an FcγRIIb extracellular region, the crystallographic confidence factor, R-value, became 40.4%, and the free R-value became equal to 41.9% for diffraction intensity data from 25 Å to 3.0 Å at this point in time. Moreover, structure refinement using Refmac5 (CCP4 Software Suite), and revising the model to identify electron density maps whose coefficient has 2Fo-Fc or Fo-Fc, which are calculated from the experimentally determined structure factor Fo, the calculated structure factor Fc and calculated phase using the model were performed using the Coot program (Paul Emsley). The model was refined by repeating these steps. Finally, by incorporating water molecules into the electron density map model using 2Fo-Fc or Fo-Fc as a coefficient and then refining, the crystallographic confidence factor, R-value, and free R-value of the model containing 4846 non-hydrogen atoms became 23.7% and 27.6% for 24291 diffraction intensity data at a resolution of 25 Å to 2.6 Å, respectively.

[Obtaining a model structure of the Fc(WT) complex/extracellular region of FcγRIIb]

Based on the structural coordinates of the PDB code: 3RY6, which corresponds to the crystal structure of the Fc(WT)/FcγRIIa extracellular region complex, the Build Mutants function of Discovery Studio 3.1 (Accelrys) was used to introduce mutations to match the FcγRIIb amino acid sequence in FcγRIIa in these structural coordinates. In this case, the optimization level was set to high, the cutoff radius was set to 4.5, five models were obtained, and the model with the best energy estimate among them was set as the model structure for the Fc(WT)/FcγRIIb extracellular region complex.

[Example 11] FcγR binding analysis of Fc variants in which sites of change were determined based on crystal structures.

Based on the results of X-ray diffraction analysis of the complex formed between Fc(P238D) and the extracellular region of FcγRIIb obtained in example 10, variants were constructed by making complex changes in the regions of the altered Fc, having the replacement of Pro at position 238 (EU numbering) with Asp, which, as predicted to affect interaction with FcγRIIb (residues 233, 240, 241, 263, 265, 266, 267, 268, 271, 273, 295, 296, 298, 300, 323, 325, 326, 327, 328, 330 , 332 and 334 (EU numbering)), and investigated whether combinations of changes could be obtained that further enhance FcγRIIb binding, in addition to the P238D change.

IL6R-B3 (SEQ ID NO: 144) was obtained by introducing into IL6R-G1d (SEQ ID NO: 134) the change obtained by replacing Lys at position 439 (EU numbering) with Glu. Further, IL6R-BF648 was obtained by making a substitution in IL6R-B3 obtained by replacing Pro at position 238 (EU numbering) with Asp. IL6R-L (SEQ ID NO: 135) was used as the total L chain of the antibody. These expressed antibody variants were purified according to the method of Reference Example 2. The binding of each of these antibody variants to each of the FcγRs (FcγRIa, FcγRIIa type H, FcγRIIa type R, FcγRIIb and FcγRIIIa type V) was assessed in detail by the method of Reference Example 25.

In accordance with the following method, a graph was built to show the results of the analysis of interactions with the corresponding FcγR. The binding value of each variant to each FcγR was divided by the binding value of the pre-modified control antibody (IL6R-BF648/IL6R-L, modified by replacing Pro at position 238 (EU numbering) with Asp) to each FcγR, and then the resulting value was multiplied by 100 , and is shown as a measure of the relative binding activity of each variant to each FcγR. The horizontal axis shows the relative binding activity of each variant to FcγRIIb, and the vertical axis shows the relative binding activity of each variant to type R FcγRIIa (FIG. 31).

As shown in FIG. 31, the results show that of all the changes found, 24 types of changes retain or enhance FcγRIIb binding compared to the pre-modified antibody. The association of these variants with each of the FcγRs is shown in Table 17. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 144). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*).

The results of measuring the KD values of the variants shown in Table 17 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb and FcγRIIIa type V using the method of Reference Example 25 are summarized in Table 18. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 144). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD (IIaR) /KD (IIb) and KD (IIaH) / KD (IIb) in the table, respectively, correspond to the value obtained by dividing the KD value of each FcγRIIaR option by the KD value of each FcγRIIb option, and the value obtained by dividing the KD value of each FcγRIIaH variant by the KD value of each FcγRIIb variant. The KD(IIb) of a parent polypeptide/KD(IIb) of an altered polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of each FcγRIIb variant. In addition, the KD value for the stronger of the FcγRIIaR and FcγRIIaFI binding activities of each variant/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide are shown in Table 18. Here, the parent polypeptide refers to the variant that has IL6R-B3 (SEQ ID NO: 144) as the H chain. It was determined that, due to the weak binding of FcγR to IgG, it could not be accurately analyzed by kinetic analysis, and thus the gray cells in Table 18 show values calculated using Equation 2 of Reference Example 25.

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C

Table 18 shows that, compared to IL6R-B3, all variants showed an improvement in affinity for FcγRIIb and the improvement ranged from 2.1-fold to 9.7-fold. The ratio of KD value of each FcγRIIaR variant/KD value of each FcγRIIb variant, and the ratio of KD value of each FcγRIIaH variant/KD value of each FcγRIIb variant corresponds to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant to FcγRIIb, and a higher value indicates higher binding selectivity to FcγRIIb. Since the ratio of FcγRIIaR KD value/FcγRIIb KD value, and the ratio of FcγRIIaH KD value/FcγRIIb KD value in the original IL6R-B3/IL6R-L polypeptide were 0.3 and 0.2, respectively, all variants in Table 18 showed improved binding selectivity for FcγRIIb over the parent polypeptide. When the ratio of the KD value for the stronger of the FcγRIIaR and FcγRIIaH variant binding activities/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide is 1 or more, this means that the stronger of the FcγRIIaR and FcγRIIaH binding activities of the variant has an equivalent or reduced binding versus binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 4.6 to 34.0 for the variants obtained this time, it can be said that compared to the original polypeptide, the variants obtained this time had reduced binding with the stronger of the binding activities of FcγRIIaR and FcγRIIaH. These results indicated that, compared to the parent polypeptide, the variants generated this time have retained or reduced type R and type H FcγRIIa binding activity, enhanced FcγRIIb binding activity, and increased FcγRIIb selectivity. Moreover, compared to IL6R-B3, all variants had lower affinity for FcγRIa and FcγRIIIaV.

With regard to the promising options among the obtained combined options, the factors providing their effects, investigated using the crystal structure. In FIG. 32 shows the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex. In this figure, the H chain on the left is the A chain of Fc and the H chain on the right is the B chain of Fc. Here it can be seen that the site at position 233 (EU numbering) in the A chain of Fc, is located near Lys at position 113 of FcγRIIb. However, in this crystal structure, the E233 side chain is in a highly mobile state, and its electron density is not clearly visible. Thus, the change obtained by replacing Glu at position 233 (EU numbering) with Asp results in a decrease in the degree of freedom of the side chain, since the side chain becomes one carbon atom shorter. As a result, the loss of entropy when forming an interaction with Lys at position 113 of FcγRIIb can be reduced, and therefore, presumably, this leads to an increase in the binding free energy.

Similarly, in FIG. 33 shows the surroundings near the region at position 330 (EU numbering) in the structure of the Fc(P238D)/FcγRIIb extracellular region complex. This figure shows that the environment of the region at position 330 (EU numbering) of the Fc A chain in Fc (P238D) is a hydrophilic environment consisting of Ser at position 85, Glu at position 86, Lys at position 163, and the like. FcγRIIb. Thus, the change obtained by replacing Ala at position 330 (EU numbering) with either Lys or Arg is expected to result in a stronger interaction with Ser at position 85 or Glu at position 86 in FcγRIIb.

In FIG. 34 shows the structures of Pro at position 271 (EU numbering) of the Fc B chain after superposition of the crystal structure of the Fc(P238D)/FcγRIIb extracellular region and Fc(WT)/FcγRIIIa extracellular region by least squares approximation based on distances between pairs of Cα atoms with respect to the Fc B-chain. The two structures match well, but have different 3D Pro structures at position 271 (EU numbering). When the low electron density in this region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region is also taken into account, it is assumed that there is a possibility that Pro at position 271 (EU numbering) in Fc(P238D)/FcγRIIb causes a large strain in the structure , thus upsetting the balance of the loop structure to achieve the optimal structure. Thus, the change obtained by replacing Pro at position 271 (EU numbering) with Gly mobilizes this loop structure and presumably leads to increased binding by lowering the energy barrier when the optimal structure is allowed to form during interaction with FcγRIIb.

[Example 12] Examining the combinatorial effect of changes that enhance FcγRIIb binding when combined with P238D.

Of the changes obtained according to Examples 9 and 11, changes that enhanced FcγRIIb binding or retained FcγRIIb binding and exhibited effects of suppressing binding to other FcγRs were combined with each other and their effect was examined.

Particularly advantageous changes selected from Tables 14 and 18 were introduced into the H chain of the IL6R-BF648 antibody similarly to the method of Example 22. IL6R-L was used as the L chain, the expressed antibodies were purified according to the method of Reference Example 1. Binding to each of FcγR (FcγRIa, H-type FcγRIIa, R-type FcγRIIa, FcγRIIb, and V-type FcγRIIIa) were evaluated in detail by the method of Reference Example 25.

In accordance with the following method, the relative binding activity was calculated from the results of the analysis of interactions with the corresponding FcγR. The binding value of each variant to each FcγR was divided by the binding value of the pre-modified control antibody (IL6R-BF648/IL6R-L with Pro substitutions at position 238 (EU numbering) by Asp to each FcγR, and multiplied by 100; and then this value were presented as relative binding activity of each variant to each FcγR (Table 19).

In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 144). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*).

The results of measuring the KD values of the variants shown in Table 19 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb and FcγRIIIa type V by the method of Reference Example 25 are summarized in Tables 20-1 and 20-2. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 144). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD(IIaR)/KD(IIb) and KD(IIaH)/KD(IIb) in the table, respectively, correspond to the value obtained by dividing the KD value of each FcγRIIaR variant by the KD value of the FcγRIIb variant and the value obtained by dividing the KD value of the FcγRIIaH variant by the KD value of each FcγRIIb variant. The KD(IIb) of a parent polypeptide/KD(IIb) of an altered polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of the FcγRIIb variant. In addition, Tables 20-1 and 20-2 show the ratio of KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of each variant/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Here, the original polypeptide refers to a variant that has IL6R-B3 (SEQ ID NO: 144) as the H chain. It was determined that due to the weak binding of FcγR to IgG, it could not be accurately analyzed by analyzing the kinetics, and thus the values in the shaded boxes in Tables 20-1 and 20-2 show the values calculated using equation 2 of the reference example 25.

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C

Tables 20-1 and 20-2 show that, compared to IL6R-B3, all variants showed an increase in affinity for FcγRIIb and the improvement ranged from 3.0-fold to 99.0-fold. The ratio of FcγRIIaR KD value of each variant/FcγRIIb KD value of each variant, and the FcγRIIaH KD value of each variant/FcγRIIb KD value of each variant correspond to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant to FcγRIIb, and a higher value indicates higher binding selectivity to FcγRIIb. Since the ratio of FcγRIIaR KD value/FcγRIIb KD value and the FcγRIIaH KD value/FcγRIIb KD value ratio of the parent IL6R-B3/IL6R-L polypeptide were 0.3 and 0.2, respectively, all variants in tables 20-1 and 20-2 showed an increase in the selectivity of binding to FcγRIIb compared to the original polypeptide. When the ratio of KD value for the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variant/KD value of the stronger of the binding activities of the parent polypeptide is 1 or more, it means that the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variant has equivalent or reduced binding compared to with binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 0.7 to 29.9 for the variants obtained this time, it can be said that the binding at the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variants obtained this time is practically equivalent or reduced compared to the original polypeptide . These results indicated that, compared to the parent polypeptide, the variants generated this time have retained or reduced type R and type H FcγRIIa binding activity, enhanced FcγRIIb binding activity, and increased FcγRIIb selectivity. Moreover, compared to IL6R-B3, all variants had lower affinity for FcγRIa and FcγRIIIaV.

[Example 13] Preparation of FcγRIIb Enhanced Binding Variants

As shown in Example 8, when FcγRIIb binding is increased, it is preferred that FcγRIIb binding is increased while suppressing binding to other activating FcγRs as much as possible. Thus, the present inventors have further obtained variants with enhanced FcγRIIb binding or increased FcγRIIb selectivity by combining changes that enhance FcγRIIb binding or increased FcγRIIb selectivity. In particular, the changes described in Examples 9, 11 and 12, which were found to be effective when combined with the P238D change, were combined with each other, based on the P238D change, which showed an excellent effect of enhancing FcγRIIb binding and increasing selectivity to FcγRIIb.

Variants were generated by combining the Fc regions of IL6R-G1d (SEQ ID NO: 133) and IL6R-B3 (SEQ ID NO: 144) with changes E233D, L234Y, G237D, S267Q, H268D, P271G, Y296D, K326D, K326A, A330R and A330K described in examples 9, 11 and 12, which were found to be effective when combined with the change P238D. Using IL6R-L (SEQ ID NO: 135) as the antibody L chain, antibodies containing the above-described variants in the heavy chain were expressed and purified according to the method described in Reference Examples 1 and 2. The resulting variants, respectively, was evaluated for binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) by the method described in Reference Example 25.

The KD of each variant for each FcγR is shown in Table 21. "Change" refers to a change made to IL6R-B3 (SEQ ID NO: 144). IL6R-B3/IL6R-L, which is used as a template for obtaining each option, indicated by an asterisk (*). "KD(IIaR)/KD(IIb)" in the table shows the value obtained by dividing the KD of each FcγRIIaR variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb. "KD(IIb) of the original polypeptide/KD(IIb) of the modified polypeptide" shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant in relation to FcγRIIb. Meanwhile, the KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIaR by the KD value of each variant in relation to FcγRIIaR. In Table 21, the value in gray cells indicates that it was concluded that the binding of FcγR to IgG is too weak to be analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

Taking the binding of IL6R-B3/IL6R-L obtained by modifying the K439E in the H chain of IL6R-G1d/IL6R-L containing the sequence of native human IgG1 to each FcγR as 1, the binding of IL6R-G1d/IL6R-L to FcγRIa was 1 ,3 times; IL6R-G1d/IL6R-L binding to FcγRIIaR was 1.1-fold; the binding of IL6R-G1d/IL6R-L to FcγRIIaH was 1.1 times, the binding of IL6R-G1d/IL6R-L to FcγRIIb was 1.2 times, and the binding of IL6R-G1d/IL6R-L to FcγRIIIaV was 0.9 times. Thus, for any given FcγR type, IL6R-B3/IL6R-L binding to FcγR was comparable to IL6R-G1d/IL6R-L binding to FcγR. Thus, comparing the binding of each variant to each FcγR to IL6R-B3/IL6R-L before making the change is presumably equivalent to comparing the binding of each variant to each FcγR to the binding of IL6R-G1d/IL6R-L containing the sequence of native human IgG1 to each of each FcγR . For this reason, in the examples below, the binding activity of each variant to each FcγR will be compared with the binding activity of IL6R-B3/IL6R-L to each FcγR before modification. Table 21 shows that all variants have increased FcγRIIb binding activity compared to IL6R-B3 before the change. The binding activity of IL6R-BF648/IL6R-L, which was the lowest, was increased by 2.6 times, while the binding activity of IL6R-BP230/IL6R-L, which was the highest, was increased by 147.6 times. As for the KD(IIaR)/KD(IIb) value, which corresponds to the selectivity, the value for IL6R-BP234/IL6R-L, which was the lowest, was 10.0, while the value for IL6R-BP231/IL6R-L , which was the highest, was 32.2. Compared to 0.3 for IL6R-B3/IL6R-L before the change, these values imply that all variants have increased selectivity. All variants showed lower binding activity to FcγRIa, FcγRIIaH and FcγRIIIaV than IL6R-B3/IL6R-L prior to modification.

[Example 14] X-ray diffraction analysis of complexes of FcγRIIb extracellular region or FcγRIIaR extracellular region and FcγRIIb enhanced binding Fc region

As shown in Example 13, binding to FcγRIIb of the IL6R-BP230/IL6R-L variant whose FcγRIIb binding activity was most enhanced was approximately 150-fold enhanced compared to pre-change IL6R-B3/IL6R-L, in while its binding to FcγRIIaR was suppressed approximately 1.9-fold. Thus, IL6R-BP230/IL6R-L is excellent in terms of both FcγRIIb binding and selectivity. However, the present inventors were looking to create more preferred variants with even more enhanced FcγRIIb binding while suppressing FcγRIIaR binding as much as possible.

As shown in FIG. 30 described in Example 10, in the Fc region with the P238D change, Asp at position 270 (EU numbering) in its CH2 domain B forms a strong electrostatic interaction with Arg at position 131 in FcγRIIb. This amino acid residue at position 131 is His in FcγRIIIa and FcγRIIaH while it is Arg in FcγRIIaR as in FcγRIIb. Thus, there is no difference between FcγRIIaR and FcγRIIb in terms of the interaction of the amino acid residue at position 131 with Asp at position 270 (EU numbering) in the B CH2 domain. .

On the other hand, the extracellular regions of FcγRIIa and FcγRIIb are 93% identical in amino acid sequence and thus have very high homology. Based on the crystal structure analysis of the complex of the Fc region of native IgG1 (hereinafter abbreviated as Fc (WT)) and the extracellular region of FcγRIIaR (J. Imunol. (2011) 187, 3208-3217), a difference in the contact surface between FcγRIIaR and FcγRIIb their interaction, which is only three amino acids (Gln127, Leu132, Phe160). Thus, the authors of the present invention suggested that it is extremely difficult to increase the selectivity of the Fc region between FcγRIIb binding and FcγRIIaR binding.

In this context, the present inventors suggested that in order to further enhance the binding activity of the Fc region to FcγRIIb and to increase the selectivity of the Fc regions for binding to FcγRIIb and FcγRIIaR, it was important to establish subtle differences between the Fc-region-FcγRIIb interaction and the Fc- region-FcγRIIaR by analyzing not only the 3D structure of the complex of the Fc region with enhanced FcγRIIb binding and the extracellular region of FcγRIIb, but also the 3D structure of the complex of the Fc region with enhanced FcγRIIb binding and the extracellular region of FcγRIIaR. First, the present inventors analyzed the X-ray crystal structure of the complex of the extracellular region of FcγRIIb or FcγRIIaR and Fc (P208) obtained by removing the K439E change from the Fc region of IL6R-BP208/IL6R-L obtained as described in Example 12, which was the variant used in as a basis for obtaining IL6R-BP230/IL6R-L.

(14-1) X-ray diffraction analysis of the Fc complex (P208) and the extracellular region of FcγRIIb

[Expression and purification of Fc (P208)]

Fc (P208) was obtained as described below. First, IL6R-P208 was generated by replacing Glu with Lys at position 439 (EU numbering) in IL6R-BP208, as with the native human IgG1 sequence. Then, the sequence of the Fc gene (P208), which was cloned by PCR, was cloned from Glu at position 216 (EU numbering) to the C-terminus, using as template DNA encoding the Cys to Ser variant at position 220 (EU numbering). Expression vector construction, expression and purification were carried out according to the method described in Reference Examples 1 and 2. Meanwhile, Cys at position 220 (EU numbering) in normal IgG1 forms a disulfide bond with Cys in the L chain. When the Fc region is obtained alone, the L chain is not expressed in combination. Thus Cys at position 220 was replaced with Ser to avoid unnecessary disulfide bond formation.

[Expression and purification of the extracellular region of FcγRIIb]

The extracellular region of FcγRIIb was obtained according to the method described in Reference Example 25.

[Purification of the Fc complex (P208)/extracellular region of FcγRIIb]

To 0.15 mg of the purified Endo F1 product (Protein Science (1996) 5, 2617-2622) expressed in E. coli as a glutathione S-transferase fusion protein, 1.5 samples were added to crystallize the extracellular region of FcγRIIb. This image in 0.1 M Bis-Tris buffer (pH 6.5) was allowed to stand at room temperature for three days to cleave off the N-type sugar chain, except for the N-acetylglucosamine directly bound to Asn in the FcγRIIb extracellular region sample. Then, a sample of the extracellular region of FcγRIIb treated to cleave the sugar chain was concentrated with a 5000MWCO ultrafiltration filter and purified by chromatography with a gel filtration column (Superdex200 10/300) equilibrated with 20 mM HEPES (pH 7.5)/0 .1 M NaCl. Next, Fc (P208) was added so that the extracellular region of FcγRIIb was present in a slightly excess molar ratio. After concentration with a 10000MWCO ultrafiltration filter, the purified fraction of the extracellular region of FcγRIIb subjected to sugar chain cleavage was purified by chromatography with a gel filtration column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH 7.5)/0.1 M NaCl. The purified fraction obtained as described above was used as a sample of the Fc(P208)/FcγRIIb extracellular region complex in the subsequent evaluation.

[Crystallization complex Fc (P208)/extracellular region FcγRIIb]

A sample of the Fc (P208)/FcγRIIb extracellular region concentrated to approximately 10 mg/mL using a 10000MWCO ultrafiltration filter was crystallized using the hanging drop method by vapor diffusion in combination with the seeding method. A VDXm plate (Hampton Research) was used for crystallization. Using a reservoir solution of 0.1 M Bis-Tris (pH 6.5)/19% (w/v) PEG3350/0.2 M dibasic potassium phosphate, crystallization drops were obtained in a mixing ratio of reservoir solution : crystallization sample = 0.85 µl: 0.85 µl. Crystals of the complex obtained under the same conditions were crushed with a Seed Bead (Hampton Research) to obtain a solution with seed crystals. To crystallization drops was added 0.15 μl of the diluted solution obtained from the seed solution, and allowed to stand at 20°C in closed reservoir wells. This provided flat crystals.

[Measurement of X-ray diffraction data for a crystal of the Fc (P208)/FcγRIIb complex]

. Кристалл принадлежит к пространственной группе C222₁ с константой решетки а=156,69

, b=260,17

, с=56,85

, α=90°, β=90° и γ=90°.One crystal of the Fc (P208)/FcγRIIb extracellular region prepared as described above was immersed in a solution of 0.1 M Bis-Tris (pH 6.5)/24% (w/v) PEG3350/0.2 M dibasic potassium phosphate/20% (v/v) ethylene glycol. Then one crystal was fished out of the solution using a pin with a nylon loop attached to it and frozen in liquid nitrogen. Single crystal X-ray diffraction data were obtained using a Spring-8 BL32XU. During the measurement, the crystal was constantly placed in a stream of nitrogen at -178°C to keep it frozen. A total of 300 x-ray diffraction images of a single crystal were obtained using an MX-225HE (RAYONIX) CCD detector connected to the beamline with the single crystal rotated 0.6° at a time. Based on the obtained diffraction images, determination of the lattice constant, indexing of diffraction spots, diffraction data, processing was carried out using the Xia2 program (J. Appl. Cryst. (2010) 43, 186-190), XDS Package (Acta Cryst. (2010) D66 , 125-132) and Scala (Acta Cryst. (2006) D62, 72-82). Finally, single crystal intensity data were obtained at resolutions up to 2.81

. The crystal belongs to the space group C222 ₁ with a lattice constant a=156.69

, b=260.17

, s=56.85

, α=90°, β=90° and γ=90°.

[X-ray diffraction analysis of the Fc complex (P208)/extracellular region of FcγRIIb]

составляла 42,6% и величина свободного R составляла 43,7% после детализации жесткого каркаса, где двум СН2-доменам и двум СН3-доменам Fc-области и внеклеточной области FcγRIIb позволяли отклоняться от полученной исходной структурной модели. Затем уточнения структурной модели достигали посредством повторения детализации структуры с использованием программы REFMAC5 (Acta Cryst. (2011) D67, 355-367) с последующей проверкой структурной модели, проведенной с использованием программы Coot (Acta Cryst. (2010) D66, 486-501) с помощью карт электронной плотности, где вычисляли коэффициенты 2Fo-Fc и Fo-Fc с использованием экспериментально определенного структурного фактора Fo, структурного фактора Fc, вычисленного в соответствии со структурной моделью, и фаз, вычисленных в соответствии со структурной моделью. Затем дальнейшую детализацию проводили на основе карт электронной плотности с коэффициентами 2Fo-Fc и Fo-Fc посредством включения молекул воды в структурную модель. С помощью 27259 данных интенсивности дифракции при разрешении от 25 до 2,81

, конечная величина кристаллографического фактора достоверности R составляла 24,5% и свободная величина R составляла 28,2% для структурной модели, содержащей 4786 атомов, не являющихся атомами водорода.The structure of the Fc (P208)/FcγRIIb extracellular region was determined by the molecular displacement method using the Phaser program (J. Appl. Cryst. (2007) 40, 658-674). The number of complexes in the asymmetric element was estimated based on the size of the resulting crystal lattice and the molecular weight of the complex Fc (P208)/extracellular region of FcγRIIb. Segments spanning amino acid residues at positions 239-340 of the A chain and at positions 239-340 of the B chain, which were established as an independent coordinate from the structural coordinate of the PDB code: 3SGJ for the crystal structure of the Fc (WT) complex/FcγRIIIa extracellular region, used as a model to search for the CH2 domain of the Fc region. Similarly, the segments spanning amino acid residues at positions 341-444 of the A chain and at positions 341-443 of the B chain, which were established as a coordinate from the structural coordinate of the PDB code: 3SGJ, were used as a model to search for the CH3 domain of Fc- areas. Finally, the segment spanning amino acid residues at positions 6-178 of the A chain, which was derived from the structural coordinate of the PDB code: 2FCB for the crystal structure of the extracellular region of FcγRIIb, was used as a model for searching for Fc (P208). The present inventors attempted to determine the orientations and positions of the respective patterns for searching for the Fc region CH3 domain, the FcγRIIb extracellular region, and the Fc region CH2 domain in crystal lattices based on the rotation function and the shear function, but could not determine the position of one of the CH2- domains. Then, referring to the crystal structure of the Fc(WT) complex/FcγRIIIa extracellular region, the position of the last CH2 domain of A was determined from the electron density map, which was calculated from the phase determined for the remaining three parts. Thus, the authors of the present invention have received the original model of the crystal structure of the complex Fc (P208)/extracellular region of FcγRIIb. The value of the crystallographic confidence factor R of the structural model for diffraction intensity data at 25 to 3.0

was 42.6% and the free R value was 43.7% after rigid framework detailing, where two CH2 domains and two CH3 domains of the Fc region and the extracellular region of FcγRIIb were allowed to deviate from the resulting original structural model. Structural model refinements were then achieved by repeating the structure refinement using the REFMAC5 program (Acta Cryst. (2011) D67, 355-367) followed by a structural model check performed using the Coot program (Acta Cryst. (2010) D66, 486-501) using electron density maps, where the coefficients 2Fo-Fc and Fo-Fc were calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated in accordance with the structural model, and the phases calculated in accordance with the structural model. Then, further detailing was carried out on the basis of electron density maps with coefficients 2Fo-Fc and Fo-Fc by including water molecules in the structural model. Using 27259 diffraction intensity data at resolutions from 25 to 2.81

, the final value of the crystallographic confidence factor R was 24.5% and the free value R was 28.2% for a structural model containing 4786 non-hydrogen atoms.

посредством анализа структуры. Структура, полученная с помощью этого анализа, представлена на фиг. 35. Было выявлено, что внеклеточная область FcγRIIb связана и расположена между двумя СН2-доменами Fc-области, что напоминает трехмерные структуры ранее анализированных комплексов Fc (WT), которые представляют собой Fc нативного IgG и каждую из внеклеточных областей FcγRIIIa (Proc. Natl. Acad. Sci. USA (2011) 108, 12 669-12 6674), FcγRIIIb (Nature (2000) 400, 267-273; J. Biol. Chem. (2011) 276, 16469-16477) и FcγRIIa (J. Immunol. (2011) 187 (6), 3208-3217).The three-dimensional structure of the Fc (P208) complex/FcγRIIb extracellular region was determined at a resolution of 2.81

through structure analysis. The structure obtained from this analysis is shown in FIG. 35. The extracellular region of FcγRIIb was found to be associated and located between the two CH2 domains of the Fc region, resembling the three-dimensional structures of the previously analyzed Fc (WT) complexes, which are native IgG Fc and each of the extracellular regions of FcγRIIIa (Proc. Natl. Acad. Sci. USA (2011) 108, 12 669-12 6674), FcγRIIIb (Nature (2000) 400, 267-273; J. Biol. Chem. (2011) 276, 16469-16477) and FcγRIIa (J. Immunol (2011) 187(6), 3208-3217).

Careful observation of the Fc (P208)/FcγRIIb extracellular region revealed a change in the loop structure at positions 233-239 (EU numbering) after the hinge region in the CH2 domain A of the Fc region, due to the influence of the introduced G237D and P238D changes compared to the Fc complex ( WT)/extracellular region FcγRIIaR (Fig. 36). This caused the Asp backbone at position 237 (EU numbering) in Fc (P208) to form a strong hydrogen bond with the Tyr sidechain at position 160 in FcγRIIb (FIG. 37). In both FcγRIIaH and FcγRIIaR, the amino acid residue at position 160 is Phe, which is incapable of forming such a hydrogen bond. This indicates that the hydrogen bond described above makes an important contribution to enhancing FcγRIIb binding and acquiring Fc (P208) binding selectivity to FcγRIIa, i.e. an increase in Fc (P208) binding activity to FcγRIIb and a decrease in Fc (P208) binding activity to FcγRIIa.

On the other hand, the Asp side chain at position 237 (EU numbering) in Fc (P208) does not form either a particularly significant FcγRIIb binding interaction or interaction with other residues in the Fc region. Ile at position 332, Glu at position 333 and Lys at position 334 (EU numbering) in the Fc region are located near Asp at position 237 (EU numbering) (FIG. 38). When the amino acid residues at these positions are replaced by hydrophilic residues to form an interaction with the Asp side chain at position 237 (EU numbering) in Fc (P208) and the loop structure can be stabilized through the interaction, this can lead to a reduction in entropy energy loss due to the formation of hydrogen bonds between Fc-region and Tyr at position 160 in FcγRIIb and thus increase the binding free energy, i.e. an increase in binding activity.

When comparing the X-ray crystal structure of the Fc complex (P238D) with the P238D change and the extracellular region of FcγRIIb described in Example 10 with the X-ray crystal structure of the Fc complex (P208) and the extracellular region of FcγRIIb, deviations are observed in five regions of Fc (P208) compared to Fc (P238D) and most of the changes are observed only at the level of the side chain. Meanwhile, a positional deviation at the backbone level due to a change from Pro to Gly at position 271 (EU numbering) is also observed in the CH2 domain of the B Fc region, and in addition there is a structural change in the loop at positions 266-270 (EU numbering) (Fig. 39). As described in Example 11, it is hypothesized that when Asp at position 270 (EU numbering) in Fc (P238D) forms a strong electrostatic interaction with Arg at position 131 in FcγRIIb, the interaction can induce a stereochemical strain at Pro at position 271 (EU numbering) . The experiment described herein indicates that the structural change observed with the change to Gly for the amino acid at position 271 (EU numbering) is presumably the result of elimination of the structural distortion centered at Pro prior to the change and elimination results in an increase in free energy for binding FcγRIIb, i.e. an increase in binding activity.

Moreover, it has been demonstrated that, due to a change in the structure of the loop at positions 266-271 (EU numbering), Arg at position 292 (EU numbering) undergoes a structural change, being in two states. In this case, the electrostatic interaction (FIG. 39) formed between Arg at position 292 (EU numbering) and Asp at position 268 (EU numbering), which is a modified residue in Fc (P208), can lead to stabilization of the loop structure. Since the electrostatic interaction between Asp at position 270 (EU numbering) in the loop and Arg at position 131 in FcγRIIb largely contributes to the activity of Fc (P208) binding to FcγRIIb, it is likely that stabilization of the loop structure in the binding conformation will reduce the entropy energy loss at binding. Thus, the change is expected to lead to an increase in the binding free energy, i.e. an increase in binding activity.

Moreover, based on the result of the structural analysis, the possibility of changing to increase the activity was investigated. The Ser at position 239 (EU numbering) was found to be a candidate for the patch site. As shown in FIG. 40, Ser at position 239 (EU numbering) in the CH2 domain of B is present at the position most naturally faced in the structure by Lys at position 117 in FcγRIIb. However, since electron density was not observed for Lys at position 117 in FcγRIIb using the analysis described above, Lys does not have a refined structure. In this situation, Lys117 probably only has a limited effect on interaction with Fc (P208). When Ser at position 239 (EU numbering) in the CH2 domain of B is replaced by a negatively charged Asp or Glu, such a change is expected to cause an electrostatic interaction with a positively charged Lys at position 117 in FcγRIIb, thereby leading to an increase in FcγRIIb binding activity. .

On the other hand, the observation of the Ser structure at position 239 (EU numbering) in the CH2 domain of A showed that, due to the formation of a hydrogen bond with the Gly backbone at position 236 (EU numbering), the side chain of this Ser stabilized the loop structure at positions 233- 239, including Asp at position 237 (EU numbering), which forms a hydrogen bond with the Tyr side chain at position 160 in FcγRIIb after the hinge region (FIG. 37). Stabilization of the loop structure in the binding conformation can reduce the loss of entropy energy during binding and lead to an increase in the free energy of binding, i.e. an increase in binding activity. Meanwhile, when Ser at position 239 (EU numbering) in CH2 domain A is replaced by Asp or Glu, the loop structure may become unstable due to loss of hydrogen bonding to the Gly backbone at position 236 (EU numbering). In addition, the change may lead to electrostatic repulsion from Asp at position 265 (EU numbering) adjacent, leading to further destabilization of the loop structure. The energy of destabilization corresponds to the loss of free energy for binding FcγRIIb, which can lead to a decrease in binding activity.

(14-2) X-ray diffraction analysis of the Fc complex (P208) and the extracellular region of FcγRIIaR

[Expression and purification of the extracellular region of FcγRIIaR]

The extracellular region of FcγRIIaR was obtained according to the method described in Reference Example 2.

[Purification of the Fc complex (P208)/extracellular region FcγRIIaR]

To 1.5 mg of purified FcγRIIaR extracellular region sample was added 0.15 mg of purified Endo F1 product (Protein Science (1996) 5, 2617-2622) expressed in E. coli as S-transferase fusion protein, 20 µl 5 U /ml Endo F2 (QA-bio) and 20 µl 5 U/ml Endo F3 (QA-bio). After incubation for 9 days at room temperature in 0.1 M Na-acetate buffer (pH 4.5), 0.07 mg of Endo F1 described above, 7.5 μl of Endo F2 described above and 7.5 μl of Endo F3 described above, and incubated for three days to cleave the N-type sugar chain, except for N-acetylglucosamine directly bound to Asn in the FcγRIIaR extracellular region sample. Then, a sample of the extracellular region of FcγRIIaR, concentrated with a 10,000MWCO ultrafiltration filter and subjected to the sugar chain cleavage treatment described above, was purified by chromatography with a gel filtration column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH 7)/0.1 M NaCl. Next, Fc (P208) was added so that the extracellular region of FcγRIIaR was present in a slightly excess molar ratio. After concentration with a 10000MWCO ultrafiltration filter, the purified extracellular region fraction of FcγRIIaR subjected to the above sugar chain cleavage treatment was purified by chromatography with a gel filtration column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH 7)/0.1 M NaCl. The purified fraction obtained as described above was used as a sample of the Fc(P208)/FcγRIIaR extracellular region in the subsequent evaluation.

[Crystallization of the Fc complex (P208)/extracellular region FcγRIIaR]

A sample of Fc (P208)/FcγRIIaR extracellular region concentrated to approximately 10 mg/mL using a 10000MWCO ultrafiltration filter was crystallized using the hanging drop method by vapor diffusion. Using a reservoir solution of 0.1 M Bis-Tris (pH 7.5)/26% (w/v) PEG3350/0.2 M ammonium sulfate, crystallization drops were obtained in a mixing ratio of reservoir solution : crystallization sample = 0 .8 µl:1.0 µl. The drops were tightly closed and allowed to stand at 20°C. This provided flat crystals.

[Measurement of X-ray diffraction data for a crystal of the Fc complex (P208)/extracellular region of FcγRIIaR]

. Кристалл принадлежит к пространственной группе C222₁ с константой решетки а=154,31

, b=257,61

, с=56,19

, α=90°, β=90° и γ=90°.One crystal of the Fc (P208)/FcγRIIaR extracellular region prepared as described above was immersed in a solution of 0.1 M Bis-Tris (pH 7.5)/27.5% (w/v) PEG3350/0, 2 M dibasic potassium phosphate/20% (v/v) glycerin. Then one crystal was fished out of the solution using a pin with a nylon loop attached to it and frozen in liquid nitrogen. Single crystal X-ray diffraction data were obtained at the Photon Factory BL-17A of the Synchrotron Radiation Institute at the High Energy Accelerator Research Organization. During the measurement, the crystal was constantly placed in a stream of nitrogen at -178°C to keep it frozen. A total of 225 x-ray diffraction images of a single crystal were obtained using a Quantum 315r CCD detector (ADSC) equipped with a beam line rotating the single crystal 0.6° at a time. Based on the obtained diffraction images, determination of the lattice constant, indexing of diffraction spots, diffraction data, processing was carried out using the Xia2 program (J. Appl. Cryst. (2010) 43, 186-190), XDS Package (Acta Cryst. (2010) D66 , 125-132) and Scala (Acta Cryst. (2006) D62, 72-82). Finally, single crystal intensity data were obtained at resolutions up to 2.87

. The crystal belongs to the space group C222 ₁ with a lattice constant a=154.31

, b=257.61

, s=56.19

, α=90°, β=90° and γ=90°.

[X-ray diffraction analysis of the Fc complex (P208)/extracellular region of FcγRIIaR]

составляла 38,4% и величина свободного R составляла 30,0% после детализации жесткого каркаса, где двум СН2-доменам и двум СН3-доменам Fc-области и внеклеточной области FcγRIIaR позволяли отклоняться от полученной исходной структурной модели. Затем уточнения структурной модели достигали посредством повторения детализации структуры с использованием программы REFMAC5 (Acta Cryst. (2011) D67, 355-367) с последующей проверкой структурной модели, проведенной с использованием программы Coot (Acta Cryst. (2010) D66, 486-501) с помощью карт электронной плотности, где вычисляли коэффициенты 2Fo-Fc и Fo-Fc с использованием экспериментально определенного структурного фактора Fo, структурного фактора Fc, вычисленного в соответствии со структурной моделью, и фаз, вычисленных в соответствии со структурной моделью. Наконец, дальнейшую детализацию проводили на основе карт электронной плотности с коэффициентами 2Fo-Fc и Fo-Fc посредством включения молекул воды в структурную модель. С помощью 24838 данных интенсивности дифракции при разрешении от 25 до 2,87

, конечная величина кристаллографического фактора достоверности R составляла 26,3% и свободная величина R составляла 38,0% для структурной модели, содержащей 4758 атомов, не являющихся атомами водорода.The structure of the Fc(P208)/FcγRIIaR extracellular region was determined by the molecular displacement method using the Phaser program (J. Appl. Cryst. (2007) 40, 658-674). The number of complexes in the asymmetric element was estimated based on the size of the resulting crystal lattice and the molecular weight of the complex Fc (P208)/extracellular region FcγRIIaR. Using, as a search model, the crystallographic structure of the Fc(P208)/FcγRIIb extracellular region obtained as described in Example (14-1), the orientation and position of the Fc(P208)/FcγRIIaR extracellular region in the crystal lattice was determined by basis of the rotation function and the shift function. The value of the crystallographic confidence factor R of the structural model for diffraction intensity data at 25 to 3.0

was 38.4% and the free R value was 30.0% after rigid scaffold detailing, where two CH2 domains and two CH3 domains of the Fc region and extracellular region of FcγRIIaR were allowed to deviate from the resulting original structural model. Structural model refinements were then achieved by repeating the structure refinement using the REFMAC5 program (Acta Cryst. (2011) D67, 355-367) followed by a structural model check performed using the Coot program (Acta Cryst. (2010) D66, 486-501) using electron density maps, where the coefficients 2Fo-Fc and Fo-Fc were calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated in accordance with the structural model, and the phases calculated in accordance with the structural model. Finally, further refinement was carried out on the basis of electron density maps with coefficients 2Fo-Fc and Fo-Fc by including water molecules in the structural model. With 24838 diffraction intensity data at resolutions from 25 to 2.87

, the final value of the crystallographic confidence factor R was 26.3% and the free value R was 38.0% for a structural model containing 4758 non-hydrogen atoms.

посредством анализа структуры. Сравнение кристаллической структуры между комплексом Fc (Р208)/внеклеточная область FcγRIIaR и комплексом Fc (Р208)/внеклеточная область FcγRIIb, описанным в примере (14-1), практически не выявило отличий на уровне общей структуры (фиг. 41), что отражает очень высокую идентичность аминокислот между двумя Fcy-рецепторами.The three-dimensional structure of the Fc (P208) complex/FcγRIIaR extracellular region was determined at a resolution of 2.87

through structure analysis. Comparison of the crystal structure between the Fc (P208)/FcγRIIaR extracellular region and the Fc (P208)/FcγRIIb extracellular region described in Example (14-1) showed little or no difference at the overall structure level (FIG. 41), reflecting the very high amino acid identity between the two Fcy receptors.

However, precise observation of structures at the electron density level revealed some differences that may lead to increased selectivity between Fc region binding to FcγRIIb and to FcγRIIaR. The amino acid residue at position 160 in FcγRIIaR is not Tyr, but Phe. As shown in FIG. 42, a hydrogen bond between the amino acid residue at position 237 of the main chain (EU numbering) in the CH2 domain A of the Fc region and Tyr at position 160 in FcγRIIb is expected, although it was formed by binding between FcγRIIb and the Fc region with the P238D change, will not form when binding between FcγRIIb and the Fc region with the P238D change. The absence of hydrogen bonding may be the main factor in increasing the selectivity between the binding of the Fc region modified P238D with FcγRIIb and with FcγRIIaR. Further comparison at the electron density level showed that in the Fc-region/FcγRIIb complex, electron density was clearly observed for the Leu side chains at positions 235 (EU numbering) and 234 (EU numbering), while the electron density of the side chains was unclear at Fc-region/FcγRIIaR complex. This indicates that the loop near position 237 (EU numbering) fluctuates due to reduced FcγRIIaR interaction at this position. Meanwhile, a structural comparison of the CH2 domain B of the Fc region (Fig. 43) in the same region revealed that in the complex of the Fc region and FcγRIIb, an electron density was observed up to Asp at position 237 (EU numbering), while in the structure of the Fc-region and FcγRIIaR complex, electron density was observed up to three residues before Asp at position 237 (EU numbering), i.e. up to approximately Leu at position 234 (EU numbering), indicating that FcγRIIaR binding forms an interaction over a larger area compared to FcγRIIb binding. The binding described above indicates the likelihood that in the CH2 domain A of the Fc region, the region at positions 234 to 238 (EU numbering) contributes heavily to the binding between the Fc region and FcγRIIb, while in the CH2 domain B The Fc region at positions 234 to 238 (EU numbering) contributes greatly to the binding between the Fc region and FcγRIIaR.

[Example 15] Fc variants for which sites of change were determined based on the crystal structure

As described in Example 14, it was proposed that Asp at position 268 (EU numbering) interacted electrostatically with Arg at position 292 (EU numbering) (FIG. 39) as a result of a local structural change due to the introduction of a P271G change in the B domain of the enhanced binding variant. FcγRIIb (P208). There is a possibility that the loop structure at positions 266-271 (EU numbering) stabilizes when the interaction is formed, resulting in increased FcγRIIb binding. Thus, the present inventors evaluated whether it is possible to enhance FcγRIIb binding by further stabilizing its loop structure due to enhanced electrostatic interaction by replacing Asp with Glu at position 268 (EU numbering) in the variant. On the other hand, as shown in FIG. 38, Tyr at position 160 (EU numbering) FcγRIIb interacts with the main chain Asp at position 237 (EU numbering) in the A domain of P208. Meanwhile, the Asp side chain at position 237 (EU numbering) is close to Ile at position 332, Glu at position 333, and Lys at position 334 (EU numbering) in the molecule without forming any particularly significant interaction. Thus, the present inventors also evaluated whether the interaction with Tyr at position 160 in FcγRIIb could be enhanced by stabilizing the loop structure at positions 266-271 (EU numbering) due to increased interaction with the Asp side chain at position 237 (EU numbering) by replacing hydrophilic amino acid residues at the positions described above.

Variants containing each of the H268E, I332T, I332S, I332E, I332K, E333K, E333R, E333S, E333T, K334S, K334T, and K334E changes that were identified based on structural information were generated using IL6R-BP230 as a template. IL6R-L, obtained as described in example 13, which showed excellent selectivity for FcγRIIb. IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and the heavy chain variants described above were expressed and purified by the method described in Reference Examples 1 and 2. Purified antibodies were evaluated for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) method described in Reference Example 25.

The KD of each variant for FcγR is shown in Table 22. In the table, "change" refers to the change in IL6R-BP3 (SEQ ID NO: 144). IL6R-B3/IL6R-L, which was used as a template to obtain IL6R-BP230, is indicated by an asterisk (*). The KD (IIb) of the original polypeptide/KD (IIb) of the modified polypeptide in the table shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant in relation to FcγRIIb. Meanwhile, the KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγR IIaR by the KD value of each variant in relation to FcγRIIaR. KD(IIaR)/KD(IIb) shows the value obtained by dividing the KD of each FcγRIIaR variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb. In Table 22, the number in gray boxes indicates that it was concluded that FcγR binding to IgG was too weak to be correctly analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

Both FcγRIIb binding activity and FcγRIIb selectivity for IL6R-BP264/IL6R-L, IL6R-BP465/IL6R-L, IL6R-BP466/IL6R-L and IL6R-BP470 obtained by amending H268E, E333K, E333R and E333T , respectively, in IL6R-BP230/IL6R-L were increased compared to IL6R-BP230/IL6R-L. The selectivity for FcγRIIb of IL6R-BP391/IL6R-L modified with I332T was reduced while its FcγRIIb binding activity was increased compared to IL6R-BP230/IL6R-L.

[Example 16] Exhaustive modification of amino acid residues at position 271 (EU numbering)

In a structural comparison between Fc (P208) and FcγRIIb and Fc (P238D)/FcγRIIb, the most significant difference was found in the structure at position 271 (EU numbering) in the CH2 domain of the Fc region (FIG. 39). As described in Example 11, it was hypothesized that when in Fc (P238D) Asp at position 270 (EU numbering) forms a strong electrostatic interaction with Arg at position 131 in FcγRIIb, the interaction can induce a stereochemical strain at Pro at position 271 (EU numbering). ). In the Fc (P208)/FcγRIIb structure, due to the replacement of Pro with Gly at position 271 (EU numbering), a positional deviation occurred at the level of the main chain, eliminating structural distortion, which led to a significant structural change in the region of position 271. There is a possibility that additional stabilization of the structure changed at position 271 will further reduce the loss of entropy energy caused by binding when forming an electrostatic interaction with Arg at position 131 in FcγRIIb. Thus, a search was made for changes that enhance the binding of the Fc region to FcγRIIb or increase its selectivity for FcγRIIb by exhaustively amending the amino acid residues at position 271 (EU numbering).

IL6R-BP267 was designed as a template for exhaustive modification by amending E233D, G237D, P238D, H268E and P271G to IL6R-B3 (SEQ ID NO: 144). IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and the heavy chain variants described above were expressed and purified according to the method described in Reference Examples 1 and 2. Purified antibodies were evaluated for binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV ) in the manner described in Reference Example 25. The amino acids at positions 264, 265, 266, 267, 269 and 272 (EU numbering) in IL6R-BP267 were replaced with each of the 18 amino acid types except for Cys and the amino acid before the change. IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and the heavy chain variants described above were expressed and purified according to the method described in Reference Example 1 and 2. Antibodies purified by the method of Reference Example 25 were evaluated for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV) in the manner described in Reference Example 25. Variants whose FcγRIIb binding was enhanced or whose FcγRIIb selectivity was increased relative to FcγRIIb binding or FcγRIIb selectivity of IL6R-BP267/IL6R-L before changes are presented in Table 23.

The KD value of each variant for each FcγR is shown in Table 23. In the table, "change" refers to the change made to IL6R-BP3, which was used as a template. IL6R-B3/IL6R-L, which was used as a template to obtain IL6R-BP267, is indicated by an asterisk (*). In Table KD (IIb) of the original polypeptide/KD (IIb) of the modified polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant in relation to FcγRIIb. Meanwhile, the KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD of IL6R-B3/IL6R-L in relation to FcγRIIaR by the KD of each variant in relation to FcγR IIaR. KD (IIaR)/KD (IIb) shows the value obtained by dividing the KD value of each variant in relation to FcγRIIaR by the KD value of the variant in relation to FcγRIIb. The larger the value, the higher the selectivity for FcγRIIb. In Table 23, the number in gray boxes indicates that it was concluded that FcγR binding to IgG was too weak to be correctly analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

The binding activity of all variants shown in Table 23 for FcγRIa, FcγRIIaH, and FcγRIIIaV was comparable or reduced compared to IL6R-B3/IL6R-L. Meanwhile, the binding activity of FcγRIIb variants obtained by adding changes S267A, V264I, E269D, S267E, V266F, S267G and V266M, respectively, to IL6R-BP267/IL6R-L was increased compared to IL6R-BP267/IL6R-L before the introduction changes. Meanwhile, the KD(IIaR)/KD(IIb) values of the variants obtained by modifying S267A, S267G, E272M, E272Q, D265E, E272D, E272N, V266L, E272I and E272F, respectively, in IL6R-BP267/IL6R-L were increased compared to IL6R-BP267/IL6R-L before the change. This demonstrates that changes in S267A, S267G, E272M, E272Q, D265E, E272D, E272N, V266L, E272I and E272F have the effect of increasing selectivity for FcγRIIb.

[Example 17] Enhancement of FcγRIIb Binding by Modifying the CH3 Region

Change by replacing Pro with Leu at position 396 (EU numbering) has been reported to enhance FcγRIIb binding (Cancer Res. (2007) 67, 8882-8890). The amino acid at position 396 (EU numbering) is present at a position that is not directly involved in the interaction with FcγR. However, it can be assumed that the amino acid has an effect on interaction with FcγR by changing the structure of the antibody. Thus, the present inventors assessed whether the binding of the Fc region to FcγRIIb is enhanced or whether its selectivity for FcγRIIb is increased by exhaustively making amino acid changes at position 396 (EU numbering) in the Fc region.

In exhaustive modification, IL6R-BP423 was designed as a template by modifying E233D, G237D, P238D, S267A, H268E, P271G, and A330R into IL6R-B3 (SEQ ID NO: 144). Variants were constructed in which the amino acid at position 396 (EU numbering) in IL6R-BP423 was replaced with each of the 18 amino acid types except for cysteine and the amino acid prior to the change. IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and the heavy chain variants described above were expressed and purified by the method described in Reference Example 1. Purified antibodies were evaluated for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) by the method described in Reference Example 25. The association of the resulting variants with each FcγR is shown in Table 24.

In the table, "change made to IL6R-BP423" refers to the change made to IL6R-BP423, which was used as a template. IL6R-B3/IL6R-L, which was used as a template to obtain IL6R-BP423, is indicated by an asterisk (*). In Table KD (IIb) of the original polypeptide/KD (IIb) of the modified polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant in relation to FcγRIIb. Meanwhile, the KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγR IIaR by the KD value of each variant in relation to FcγR IIaR. KD(IIaR)/KD(IIb) shows the value obtained by dividing the KD of each FcγRIIaR variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb. In Table 24, the number in gray boxes indicates that it was concluded that FcγR binding to IgG was too weak to be correctly analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in example 25.

The result presented in Table 24 demonstrates that: FcγRIIb binding activity in

IL6R-BP456/IL6R-L obtained by amending P396M to IL6R-BP42 3/IL6R-L,

IL6R-BP455/IL6R-L obtained by amending P396L to IL6R-BP423/IL6R-L,

IL6R-BP464/IL6R-L obtained by amending P396Y to IL6R-BP423/IL6R-L,

IL6R-BP450/IL6R-L obtained by amending P396F to IL6R-BP423/IL6R-L,

IL6R-BP448/IL6R-L obtained by amending P396D to IL6R-BP423/IL6R-L,

IL6R-BP458/IL6R-L obtained by amending P396Q to IL6R-BP423/IL6R-L,

IL6R-BP453/IL6R-L obtained by amending P396I to IL6R-BP423/IL6R-L,

IL6R-BP449/IL6R-L obtained by amending P396E to IL6R-BP423/IL6R-L,

IL6R-BP454/IL6R-L obtained by amending P396K to IL6R-BP423/IL6R-L and

The IL6R-BP459/IL6R-L obtained by changing P396R to IL6R-BP423/IL6R-L was increased in all cases compared to IL6R-BP423/IL6R-L before the change. Meanwhile, the KD(IIaR)/KD(IIb) value for IL6R-BP456/IL6R-L obtained by introducing the P396M change into IL6R-BP423/IL6R-L was higher compared to IL6R-BP423/IL6R-L before the addition changes, showing increased selectivity for FcγRIIb. As can be seen from table 24, the binding activity of the resulting variants with FcγRIa, FcγRIIaH and FcγRIIIaV in all cases was lower than that of IL6R-B3/IL6R-L, which was the original polypeptide.

[Example 18] Generation of FcγRIIb Enhanced Binding Variants Using Subclass Sequences

The binding profile of FcγR varies depending on the subclass of human IgG. The present inventors evaluated whether the binding activity of each FcγR between IgG1 and IgG4 could be used to increase FcγRIIb binding activity and/or increase selectivity. First, IgG1 and IgG4 were analyzed for their binding activity to each FcγR. IL6R-G4d (SEQ ID NO: 164) containing G4d was designed as the H chain of the antibody. G4d is an Fc region that lacks C-terminal Gly and Lys and contains a Ser to Pro substitution at position 228 (EU numbering) in human IgG4. IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the light chain of IL6R-L and IL6R-G1d/IL6R-L or IL6R-G4d/IL6R-L were expressed and purified according to the method described in Reference Examples 1 and 2. Purified antibodies were evaluated for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV) in the manner described in Reference Example 25. The association of the resulting variants to each FcγR is summarized in Table 25.

It was demonstrated that the binding of IL6R-G4d/IL6R-L to FcγRIIb was 1.5 times stronger than that of IL6R-G1d/IL6R-L, while the binding of IL6R-G4d/IL6R-L to FcγRIIaR was 2 .2 times weaker than IL6R-G1d/IL6R-L. Meanwhile, the binding activity of IL6R-G4d/IL6R-L to FcγRIa, FcγRIIaH and FcγRIIIaV is lower than that of IL6R-G1d/IL6R-L. The result described above indicated that IL6R-G4d had superior performance over IL6R-G1d in terms of both FcγRIIb binding activity and selectivity.

In FIG. 44 shows an alignment for comparing the CH1 G1d and G4d sequences up to the C-terminus (positions 118 to 445 (EU numbering)). In FIG. 44, amino acid residues that differ between G1d and G4d are enclosed in a bold box. The present inventors evaluated whether FcγRIIb binding could be further increased and/or whether FcγRIIb selectivity could be further increased by selecting, from the above differing amino acids, some portions predicted to be involved in FcγR interaction and transplanting at least at least one or more amino acid residues from the G4d sequence that confers a property advantageous in terms of both binding activity and FcγRIIb selectivity to the FcγRIIb enhanced binding variant.

In particular, the authors of the present invention obtained:

IL6R-BP473 obtained by amending A327G to IL6R-BP230;

IL6R-BP472 obtained by amending A330S to IL6R-BP230;

IL6R-BP471 obtained by amending P331S to IL6R-BP230;

IL6R-BP474 obtained by amending A330S and P331S to IL6R-BP230;

IL6R-BP475 obtained by amending A327G and A330S to IL6R-BP230;

IL6R-BP476 obtained by amending A327G, A330S and P331S to IL6R-BP230; and

IL6R-BP477 obtained by amending A327G and P331S to IL6R-BP230. Moreover, to construct IL6R-BP478, the amino acids Ala at position 118 to Thr at position 225 (EU numbering) in IL6R-BP230 were changed to amino acids in the G4d sequence from Ala at position 118 to Pro at position 222 (EU numbering). IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and heavy chain variants described above were purified according to the method described in Reference Examples 1 and 2. Purified antibodies were evaluated for their binding activity to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) in the manner described in Reference Example 25.

The KD value of each variant against each FcγR is shown in Table 26. The KD(IIb) of the original polypeptide/KD(IIb) of the modified polypeptide in the table shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L against FcγRIIb by the KD value of each variant for FcγRIIb. In the table, "change made to IL6R-BP230" refers to the change made to IL6R-BP230. IL6R-B3/IL6R-L used as a template for obtaining IL6R-BP230 is indicated by *1. Meanwhile, IL6R-BP478, in which the G4d sequence from Ala at position 118 to Pro at position 222 (EU numbering) is replaced by a segment from Ala at position 118 to Thr at position 225 (EU numbering) in IL6R-BP230, is indicated by *2 . The KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγR IIaR by the KD value of the variant in relation to FcγR IIaR. KD(IIaR)/KD(IIb) shows the value obtained by dividing the KD of each FcγRIIaR variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb. In Table 2-6, the number in the shaded boxes indicates that it was concluded that FcγR binding to IgG was too weak to be correctly analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

Among the variants shown in Table 2-6, IL6R-BP473/IL6R-L modified with A327G showed 1.2-fold enhanced FcγRIIb binding compared to IL6R-BP230/IL6R-L. Meanwhile, IL6R-BP478/IL6R-L, in which the amino acids Ala at position 118 to Thr at position 225 (EU numbering) in IL6R-BP230 are replaced by the amino acids Ala at position 118 to Pro at position 222 (EU numbering) in amino acid sequence G4d showed FcγRIIb and FcγRIIaR binding enhanced 1.1-fold compared to IL6R-BP230/IL6R-L. All of the variants showed lower FcγRIa, FcγRIIaH and FcγRIIIaV binding activity compared to the parent IL6R-B3/IL6R-L polypeptide.

Variants with modifications in other regions, shown in FIG. 44. In particular, K274Q was introduced into the H chain of the IL6R-BP230 antibody to obtain IL6R-BP541; Y296F was made to obtain IL6R-BP542; H2 68Q contributed to obtain IL6R-BP543; R355Q was added to give IL6R-BP544; D356E was made to obtain IL6R-BP545; L358M contributed to obtain IL6R-BP546; K409R was made to obtain IL6R-BP547; and Q419E were added to give IL6R-BP548. Meanwhile, IL6R-L was used as the L chain of a conventional antibody. Antibodies that contain the heavy chain variant described above and the IL6R-L light chain were purified by the methods described in Reference Examples 1 and 2. Purified antibodies were evaluated for binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) by the method of Reference Example 25 .

The KD of each variant for each FcγR is shown in Table 27. In Table 27, "KD (IIb) parent polypeptide/KD (IIb) altered polypeptide" corresponds to the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant for FcγRIIb. In the table, "IL6R-BP230 change" refers to a modification made to IL6R-BP230. IL6R-B3/IL6R-L used as a template for obtaining IL6R-BP230 is indicated as *1. "KD (IIaR) of the original polypeptide/KD (IIaR) of the modified polypeptide" corresponds to the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIaR by the KD value of the same variant in relation to FcγRIIaR. KD(IIaR)/KD(IIb) correspond to the value obtained by dividing the KD of each variant for FcγRIIaR by the KD of the same variant for FcγRIIb. The larger the value, the higher the selectivity for FcγRIIb. In Table 27, the number in the gray box indicates that FcγR binding to IgG was weak and it was determined that the assay could not be correctly performed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

As shown in Table 27, IL6R-BP541/IL6R-L obtained by adding K274Q to IL6R-BP230/IL6R-L, IL6R-BP544/IL6R-L obtained by adding R355Q to IL6R-BP230/IL6R-L, IL6R-BP545/ IL6R-L generated by insertion of D356E into IL6R-BP230/IL6R-L and IL6R-BP546/IL6R-L obtained by insertion of L358M into IL6R-BP230/IL6R-L showed enhanced FcγRIIb binding compared to IL6R-BP230/IL6R-L before making the change. Among them, it was shown that IL6R-BP544/IL6R-L obtained by introducing R355Q into IL6R-BP230/IL6R-L, IL6R-BP545/IL6R-L obtained by introducing D356E into IL6R-BP230/IL6R-L and IL6R-BP546/ IL6R-L made by incorporating L358M into IL6R-BP230/IL6R-L has increased KD(IIaR)/KD(IIb) and increased selectivity for FcγRIIb compared to IL6R-BP230/IL6R-L before modification.

[Example 19] Evaluation of combinations of changes that enhance binding to FcγRIIb or increase selectivity for FcγRIIb

Additional combinations of changes described herein in sections up to and including Example 18 were evaluated to be effective in enhancing FcγRIIb binding or increasing FcγRIIb selectivity. In particular, changes judged to be effective in terms of enhancing FcγRIIb binding and/or increasing FcγRIIb selectivity were made in combination in IL6R-B3 (SEQ ID NO: 144). Moreover, the known changes S267E and L328F were introduced that enhance the binding of FcγRIIb (Seung et al., (Mol. Immunol. (2008) 45, 3926-3933)) to IL6R-B3 to give IL6R-BP253 as a control for comparison. IL6R-L (SEQ ID NO: 135) was used as the L chain of the antibody. Antibodies containing the IL6R-L light chain and the heavy chain variants described above were expressed and purified according to the method described in Reference Examples 1 and 2. Purified antibodies were evaluated for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb or FcγRIIIaV) in the manner described in Reference Example 25.

The KD of each variant for each FcγR is shown in Table 28. In the table, "change" refers to the change made to IL6R-B3 (SEQ ID NO: 144). IL6R-B3/IL6R-L, which was used as a template for obtaining each option, indicated by an asterisk (*). The KD (IIb) of the original polypeptide/KD (IIb) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIb by the KD value of each variant in relation to FcγRIIb. Meanwhile, the KD (IIaR) of the original polypeptide/KD (IIaR) of the altered polypeptide shows the value obtained by dividing the KD value of IL6R-B3/IL6R-L in relation to FcγRIIaR by the KD of the variant in relation to FcγRIIaR. KD(IIaR)/KD(IIb) shows the value obtained by dividing the KD of each FcγRIIaR variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb over FcγRIIaR. Meanwhile, KD(IIaH)/KD(IIb) shows the value obtained by dividing the KD of each FcγRIIaH variant by the FcγRIIb KD of each variant. The larger the value, the higher the selectivity for FcγRIIb over FcγRIIaH. In Table 28, the number in gray boxes indicates that it was concluded that FcγR binding to IgG was too weak to be correctly analyzed by kinetic analysis and thus was calculated using:

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C,

which is described in Reference Example 25.

Among the variants shown in Table 28, IL6R-BP253/IL6R-L, which had known modifications that enhance FcγRIIb binding, exhibited FcγRIIb and FcγRIIaR binding activity increased 277-fold and 529-fold relative to IL6R-B3/IL6R-L before the change, respectively. Moreover, the FcγRIa binding activity of IL6R-BP253/IL6R-L was also higher than that of IL6R-B3/IL6R-L. Meanwhile, FcγRIIaH binding and FcγRIIIaV binding in IL6R-BP253/IL6R-L were reduced compared to IL6R-B3/IL6R-L. Among other variants, IL6R-BP436/IL6R-L and IL6R-BP438/IL6R-L showed FcγRIa binding slightly enhanced compared to pre-change IL6R-B3/IL6R-L. All other variants showed reduced FcγRIa binding. In addition, all variants showed reduced FcγRIIaH binding and FcγRIIIaV binding compared to IL6R-B3/IL6R-L.

Variants prepared as described in this section were compared with IL6R-BP253/IL6R-L, which is a known FcγRIIb enhanced binding variant. The KD(IIaH)/KD(IIb) value of IL6R-BP480/IL6R-L, which is the lowest, is 107.7, while the value of IL6R-BP480/IL6R-L, which is the highest, was 8362. Thus thus, the values for these two variants exceeded the value of 107.1 for IL6R-BP253/IL6R-L. Meanwhile, the KD(IIaR)/KD(IIb) value of IL6R-BP479/IL6R-L, which was the lowest, was 16.1, while the value of IL6R-BP559/IL6R-L, which was the highest, was 58.4. Thus, the values of these two variants exceeded the value of 0.2 for IL6R-BP253/IL6R-L. These results demonstrate that the selectivity for FcγRIIb of the variants shown in Table 28 is increased compared to the selectivity of variants with known modifications that enhance FcγRIIb binding. In particular, all of IL6R-BP559/IL6R-L, IL6R-BP493/IL6R-L, IL6R-BP557/IL6R-L, IL6R-BP492/IL6R-L and IL6R-BP500/IL6R-L have FcγRIIb binding activity, increased by 100 times or more, while their FcγRIIaR binding was 1.5 times or less increased compared to IL6R-B3/IL6R-L. Thus, the effect of enhanced FcγRIIb binding is expected to be achieved while avoiding side effects due to increased FcγRIIaR binding.

As for IL6R-BP489/IL6R-L, IL6R-BP487/IL6R-L, IL6R-BP499/IL6R-L, IL6R-BP498/IL6R-L, IL6R-BP503/IL6R-L, IL6R-BP488/IL6R-L, IL6R-BP490/IL6R-L, IL6R-BP445/IL6R-L, IL6R-BP552/IL6R-L, IL6R-BP507/IL6R-L, IL6R-BP536/IL6R-L, IL6R-BP534/IL6R-L, IL6R- 491/IL6R-L, IL6R-BP553/IL6R-L, IL6R-BP532/IL6R-L, IL6R-BP506/IL6R-L, IL6R-BP511/IL6R-L, IL6R-BP502/IL6R-L, IL6R-BP531/ IL6R-L, IL6R-BP510/IL6R-L, IL6R-BP535/IL6R-L, IL6R-BP497/IL6R-L, IL6R-BP533/IL6R-L, IL6R-BP555/IL6R-L, IL6R-BP554/IL6R- L, IL6R-BP436/IL6R-L, IL6R-BP423/IL6R-L, IL6R-BP440/IL6R-L, IL6R-BP538/IL6R-L, IL6R-BP429/IL6R-L, IL6R-BP438/IL6R-L, IL6R-BP565/IL6R-L, IL6R-BP540/IL6R-L, BP426/IL6R-L, IL6R-BP437/IL6R-L, IL6R-BP439/IL6R-L, IL6R-BP551/IL6R-L, IL6R-BP494/ IL6R-L, IL6R-BP537/IL6R-L, IL6R-BP550/IL6R-L, IL6R-BP556/IL6R-L, IL6R-BP539/IL6R-L, IL6R-BP558/IL6R-L, IL6R-BP425/IL6R- L and IL6R-BP495/IL6R-L, their FcγRIIb binding was higher than that of IL6R-BP253/IL6R-L, which had a known modification that enhances binding FcγRIIb. From the above, the enhancement of FcγRIIb binding ranges from 321-fold (lowest) to 3100-fold (highest), compared to IL6R-B3/IL6R-L binding (which is defined as 1), from IL6R-BP495/IL6R- L to IL6R-BP489/IL6R-L, respectively. Thus, these variants can be said to be superior in terms of both the level and selectivity of enhancing FcγRIIb binding activity compared to the prior art.

[Example 20] Production of antibodies that bind to human IgA in a calcium dependent manner

(20-1) Production of human IgA (hIgA)

Examples 2-4 demonstrate that molecules that have enhanced mouse FcγR binding and that bind in a pH dependent manner to the human IL-6 receptor as an antigen can significantly reduce the plasma concentration of the antigen. An additional test was then performed using anti-human IgA antibodies as the antigen to evaluate whether there was a similar effect in eliminating soluble antigens from plasma in a living body injected with antibodies that have enhanced mouse FcγR binding and that bind in a pH-dependent manner to antigens other than from the human IL-6 receptor. The antigen, human IgA (hereinafter also referred to as hIgA) (its variable region is derived from an anti-human IL6R antibody) was obtained using the following recombinant method. hIgA was expressed by culturing host cells containing recombinant vectors containing H (WT)-IgAl (SEQ ID NO: 145) and L (WT) (SEQ ID NO: 42) and purified in a manner known to those skilled in the art, with using ion exchange chromatography and gel filtration chromatography.

(20-2) Expression and purification of an antibody that binds to hIgA

GA2-IgG1 (heavy chain, SEQ ID NO: 146; light chain, SEQ ID NO: 147) is an antibody that binds to hIgA. The DNA sequence encoding GA2-IgG1 (heavy chain, SEQ ID NO: 146; light chain, SEQ ID NO: 147) was inserted into animal cell expression plasmids in a manner known to those skilled in the art. The antibody was expressed and purified by the method described below. Cells derived from Freestyle 293-F human embryonic kidney cells (Invitrogen) were suspended in Freestyle 293 Expression Medium (Invitrogen). The cell suspension was seeded at a cell density of 1.33×10 ⁶ cells/ml in 3 ml in each well of a 6-well plate. The resulting plasmid was then introduced into the cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for 4 days. From the resulting culture supernatant, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance (wavelength: 280 nm) of the purified antibody solution was measured using a spectrophotometer. The antibody concentration was determined using the extinction coefficient calculated from the measured value by the PACE method (Protein Science (1995) 4, 2411-2423).

(20-3) Evaluation of an isolated antibody for its calcium dependent hIgA binding capacity

The antibody isolated as described in Example (20-2) was evaluated for its hIgA binding activity (dissociation constant, KD(M)) using Biacore T200 (GE Healthcare). Binding activity was measured using 0.05% tween20, 20 mmol/l ACES, 150 mmol/l NaCl (pH 7.4 or pH 5.8) containing 3 μM or 1.2 mM CaCl ₂ as running buffer. An appropriate amount of recombinant A/G protein (Thermo Scientific) was immobilized on a CM5 sensor chip (GE Healthcare) by the amine addition method and the antibody was allowed to bind to it. Then, an appropriate concentration of hIgA (described in (Al-1)) was injected as an analyte and allowed to interact with the antibody on the sensor chip. The measurements were carried out at 37°C. After measurement, 10 mmol/l glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. From the measurement result, the dissociation constant KD (M) was calculated by curve fitting analysis and equilibrium analysis using Biacore T200 Evaluation Software (GE Healthcare). The result is shown in Table 29. GA2-IgG1 strongly bound to hIgA at a Ca ²⁺ concentration of 1.2 mM and weakly bound to hIgA at a Ca ²⁺ concentration of 3 μM. Meanwhile, at a Ca ²⁺ concentration of 1.2 mM, GA2-IgG1 strongly bound to human IgA at pH 7.4 and weakly bound to human IgA at pH 5.8. Overall, GA2-IgG1 has been shown to bind to human IgA in a pH- and calcium-dependent manner.

[Example 21] Preparation of antibody variants that bind to hIgA in a calcium-dependent manner

Further, to further accelerate the clearance of antigen (hIgA) from plasma, GA2-F1087 (heavy chain, SEQ ID NO: 148) was generated by replacing Lys with Asp at position 326 (EU numbering) in GA2-IgG1 to enhance binding to mouse FcγR GA2 -IgG1, which binds to hIgA in a calcium dependent manner. The DNA sequence encoding GA2-F1087 (heavy chain, SEQ ID NO: 148; light chain, SEQ ID NO: 147) was inserted into an animal expression plasmid in a manner known to those skilled in the art. Antibody variants were expressed as described above using a plasmid. Variant concentrations were measured after purification. Antibodies containing the modification described above exhibited significantly enhanced mouse FcγR binding as shown in Example (4-3).

[Example 22] Evaluation of the effect on plasma antigen retention time in normal mice administered antibodies with Ca-dependent hIgA binding

(22-1) In vivo tests using normal mice

hIgA (human IgA prepared as described in Example (20-1)) was administered alone or in combination with an anti-hIgA antibody to normal mice (C57BL/6J mouse, Charles River Japan). After administration, the in vivo dynamics of hIgA and anti-hIgA antibodies were evaluated. An hIgA solution (80 μg/ml) or a mixed solution of hIgA and anti-hIgA antibodies was injected once at a dose of 10 ml/kg into the tail vein. The anti-hIgA antibodies used were GA2-IgG1 and GA2-F1087 described above.

In all mixed solutions, the hIgA concentration was 80 μg/ml and the anti-hIgA antibody concentration was 2.69 mg/ml. In this experiment, anti-hIgA antibodies were present in significant excess relative to hIgA and thus it was believed that the majority of hIgA bound to the antibodies. In the GA-IgG1-administered group, mice were bled five minutes, seven hours, one day, two days, three days and seven days after administration. Meanwhile, in the GA-F1087-administered group, mice were bled at five minutes, 30 minutes, one hour, two hours, one day, three days, and seven days after administration. The collected blood was immediately centrifuged at 12,000 rpm. and 4°C for 15 minutes to release plasma. The isolated plasma was stored in a freezer at -20° C. or below until use.

(22-2) Determination of anti-hIgA antibody concentration in plasma in normal mice by ELISA method

Mouse plasma concentrations of anti-hIgA antibody were measured by ELISA. First, to prepare a plate immobilized with anti-human IgG antibody, anti-IgG F(ab')2 fragment (γ chain specific) (SIGMA) was aliquoted into each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International ) and the plate was allowed to stand at 4° C. overnight. Anti-hIgA calibration curve samples prepared as standard dilutions for plasma concentrations (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0.007813 µg/mL) and plasma samples Assay mice diluted 100-fold or more were aliquoted into the above-mentioned plate immobilized with anti-IgG antibody. After incubating the plate for one hour at 25°C, aliquots of goat anti-human IgG (γ-chain specific) biotin conjugate (BIOT) (Southern Biotechnology Associates Inc.) were dispensed into each well of the plate. Then the tablet was incubated at 25°C for one hour. Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was then aliquoted into each well of the plate. Then the tablet was incubated at 25°C for one hour. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After completion of the reaction with 1 H sulfuric acid (Showa Chemical), the absorbance of the reaction solution in each well at 450 nm was measured in a microplate reader. Mouse plasma concentrations of anti-hIgA antibody were determined from a standard absorbance curve using SOFTmax PRO analysis software (Molecular Devices). The concentrations of GA2-IgG1 and GA2-F1087 antibodies over time in the plasma of normal mice after intravenous administration, which were measured by the method described above, are shown in FIG. 45. The results demonstrate that, with respect to clone GA2-IgG1, which has a pH-dependent potent hIgA binding activity, the plasma concentration of the antibody did not decrease significantly even though FcγR binding increased.

(22-3) Determination of plasma hIgA concentration by ELISA method Mouse plasma hIgA concentrations were measured by ELISA method. First, to prepare an anti-human IgA immobilized plate, goat anti-human IgA (BETHYL) was aliquoted into each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) and the plate was allowed to stand at 4° C. overnight. The hIgA samples for the calibration curve were prepared as plasma concentration standard solutions (0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 μg/mL) and used. 100 μl of each of the standard curve samples and mouse plasma samples for analysis diluted 100 times or more were combined with 200 μl of 500 ng/ml hsL6R. They were mixed and incubated at room temperature for one hour. Then 100 μl of the mixtures were aliquoted into a plate with immobilized anti-human IgA antibody. The tablet was allowed to stand at room temperature for one hour. Next, biotinylated anti-human IL-6 R (R&D) was aliquoted into each well of the plate. After incubation for one hour at room temperature, streptavidin-PolyHRPSO (Stereospecific Detection Technologies) was aliquoted into each well of the plate. The tablet was incubated at room temperature for one hour. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After completion of the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction solution in each well at 450 nm was measured using a microplate reader. Mouse plasma concentrations were determined from an absorbance standard curve using SOFTmax PRO analysis software (Molecular Devices). Plasma hIgA concentration in normal mice over time after intravenous administration, which was measured as described above, is shown in FIG. 46.

The result showed that in mice administered with hIgA in combination with GA2-IgG1 having Ca-dependent hIgA binding activity increased by 100 times or more, hIgA elimination was accelerated compared with administration of hIgA alone. Meanwhile, in the plasma of mice injected with GA2-F1087 with enhanced binding to hIgA and FcγR, the concentration of hIgA was reduced below the measurable range (0.006 μg/mL or more) one day after administration, and thus the elimination of hIgA was significantly accelerated compared to the plasma of mice injected with GA-IgG1. The data described in Examples 2-7 above demonstrate an increased antigen-clearing effect on antibodies with enhanced FcγR binding in mice treated with IL6R and anti-IL6R antibody. Similarly, it has also been shown to achieve a similar effect in mice injected with hIgA antigen and anti-hIgA antibody.

[Example 23] Production of pH-dependent anti-IgE antibody

(23-1) Production of anti-human IgE antibody

To generate a pH dependent anti-human IgE antibody, human IgE (heavy chain, SEQ ID NO: 149; light chain, SEQ ID NO: 150) (its variable region is derived from human anti-glypican 3 antibody) was expressed as antigen using FreeStyle293 (Life Technologies).

Expressed human IgE was obtained and purified by a common chromatography method known to those skilled in the art. An antibody that binds to human IgE in a pH dependent manner was selected from a variety of isolated antibodies. The variable regions of the selected anti-IgE antibody were fused to a human IgG1 heavy chain constant region and a human IgG1 light chain constant region, and the resulting antibody gene was inserted into a vector. Using a vector, a recombinant anti-human IgE antibody was expressed and purified. The resulting antibody was named clone 278 (hereinafter referred to as 278-IgG1; heavy chain, SEQ ID NO: 151, light chain, SEQ ID NO: 152).

(23-2) Evaluation of anti-human IgE antibody for IgE binding activity and pH dependent binding activity

Antibodies capable of dissociating from antigens in the endosome can be prepared in such a way that they bind to antigens not only in a pH-dependent manner, but also in a Ca-dependent manner. Thus, 278-IgG1 and the control human IgG1 antibody Xolair (omalizumab, Novartis) without pH/Ca-dependent IgE binding capacity were evaluated for pH-dependent binding capacity and pH/Ca-dependent binding capacity of human IgE ( hIgE). In particular, 278-IgG1 and Xolair were evaluated for their hIgE binding activity (dissociation constant, KD(M)) using Biacore T200 (GE Healthcare). The measurements were carried out using the following three types of moving buffers:

1.2 mmol/L CaCl ₂ , 0.05% tween20, 20 mmol/L ACES, 150 mmol/L NaCl, pH 7.4,

1.2 mmol/L CaCl ₂ , 0.05% tween20, 20 mmol/L ACES, 150 mmol/L NaCl, pH 5.8,

3 µmol/l CaCl ₂ , 0.05% tween20, 20 mmol/l ACES, 150 mmol/l NaCl, pH 5.8.

An appropriate amount of a peptide (hereinafter referred to as "biotinylated GPC3 peptide") obtained by attaching biotin to the C-terminal Lys of a chemically synthesized sequence derived from human glypican 3 protein (SEQ ID NO: 153) was applied to an SA sensor chip (GE Healthcare) and immobilized on a SA sensor chip based on biotin/streptavidin affinity. The appropriate concentration of human IgE was injected and captured with biotinylated GPC3 peptide to immobilize human IgE onto the chip. The appropriate concentration of 278-IgG1 was injected as an analyte and allowed to interact with human IgE on the sensor chip. Then 10 mmol/l glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. All interaction analysis was performed at 37°C. Using Biacore T200 Evaluation Software (GE Healthcare), the assay results were analyzed by curve fitting to determine the binding rate constant ka (1/Ms) and the dissociation rate constant kd (1/s). From the above constants, the dissociation constant KD (M) was calculated. The pH dependent binding was then evaluated by calculating the KD ratio of each antibody between the conditions of pH 5.8/1.2 mM Ca and pH 7.4/1.2 mM Ca. pH/Ca-dependent binding was assessed by calculating the KD ratio for each antibody between the conditions of pH 5.8/3 μM Ca and pH 7.4/1.2 mm Ca. The results are presented in table 30.

[Example 24] Preparation of an antibody variant that binds to human IgE in a pH dependent manner

Further, to further accelerate the elimination of the antigen (human IgE) from plasma, the DNA sequence encoding 278-F1087 (heavy chain, SEQ ID NO: 154; light chain, SEQ ID NO: 152) with the change of Lys to Asp at position 326 (numbering EU) in 278-IgG1 was inserted into an animal expression plasmid in a manner known to those skilled in the art to enhance binding of the mouse 278-IgG1 FcγR, which binds to human IgE in a pH dependent manner. Antibody variants were expressed in the manner mentioned above using animal cells into which the plasmid was introduced. After purification, the concentrations of antibody variants were determined.

[Example 25] In vivo evaluation of 278-IgG1

(25-1) Production of human IgE (hIgE (Asp6)) for in vivo evaluation

hIgE (Asp6) (its variable region is derived from anti-glypican 3 antibody), which is a human IgE for in vivo evaluation, containing a heavy chain (SEQ ID NO: 155) and a light chain (SEQ ID NO: 150), was obtained by the same in the manner described in example (23-1). hIgE (Asp6) is a molecule in which asparagine is replaced by aspartic acid at six N-glycosylation sites in human IgE, such that time-dependent changes in human IgE concentration as an antigen are not affected by the heterogeneity of N-linked human IgE sugar chains.

(25-2) Evaluation of the effect of accelerating the elimination of human IgE from the plasma of normal mice injected with 278

As described in Examples 2-4 and 22, it was demonstrated that the antigen concentration was significantly reduced in the plasma of mice administered with molecules that bind in a pH-dependent manner to the human IL-6 receptor or human IgA as an antigen, and whose binding to The mouse FcγR was amplified. An additional test was performed using antibodies to human IgE as antigen to assess whether a similar effect of eliminating soluble antigens from the plasma of a living organism, which was injected with antibodies with enhanced binding of mouse FcγR, which bind in a pH-dependent manner to antigens other than the receptor, is achieved. Human IL-6 and human IgA when FcγR binding is enhanced.

hIgE (Asp6) and anti-human IgE antibodies were evaluated for their dynamics in vivo after administration of hIgE (Asp6) alone or hIgE (Asp6) in combination with anti-hIgE antibodies (278-IgG1 and 278-F1087) to C57BL/6J mice (Charles river Japan). hIgE (Asp6) (20 µg/mL) or a mixture of hIgE (Asp6), and anti-IgE antibody was administered once at 10 ml/kg in the tail vein (as described in Table 31, all antibodies were obtained at the same concentration) . In this case, each antibody was present in significant excess relative to hIgE (Asp6) and thus, it was believed that almost all hIgE (Asp6) was associated with the antibody. In the group in which clone 278 (278-IgG1) was administered, blood was taken from mice five minutes, two hours, seven hours, one day, two days, four days, five days, seven days, 14 days and 21 days after introductions. In the 278-F1087-administered group, mice were bled at five minutes, 30 minutes, one hour, two hours, one day, three days, seven days, 14 days, and 21 days after administration. The collected blood was immediately centrifuged at 15,000 rpm. and 4°C for 5 minutes to release plasma. The isolated plasma was stored in a freezer at -20° C. or below until use.

(25-3) Determination of anti-human IgE antibody concentration in normal mice

Mouse plasma hIgE antibody concentrations were measured by ELISA. Standard curve samples were prepared for plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 μg/mL. To ensure homogeneity of the immune complex between hIgE (Asp6) and anti-hIgE antibody, hIgE (Asp6) was added at a concentration of 1 μg/ml to standard curve samples and mouse plasma samples for analysis. Samples of administration group 2 7 8-hIgG1 and samples for the corresponding standard curve were allowed to stand at room temperature for 30 minutes. Meanwhile, the samples of the injection group 278-F1087 and the samples for the corresponding standard curve were stirred at 37° C. overnight. After incubation or mixing, standard curve samples and mouse plasma samples for analysis were aliquoted into an Immunoplate (Nunc-Immuno Plate, MaxiSorp (Nalge nunc International)), on which anti-human kappa light chain antibody (Bethyl Laboratories) was immobilized and allowed to stand. /stir at room temperature for two hours (278-F1087 injection group samples and 278-F1087 standard curve samples) or allowed to stand at 4°C overnight (278-hIgG1 administration group samples and 278-hIgG1 standard curve samples ). Then, each of a rabbit anti-human IgG (Fc) secondary antibody conjugated with biotin (Pierce Biotechnology) and streptavidin-PolyHRPSO (Stereospecific Detection Technologies) was reacted sequentially for one hour. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After completion of the reaction with 1 H sulfuric acid (Showa Chemical), mouse plasma concentrations were determined based on color development by the absorbance measurement method at 450 nm on a microplate reader. Mouse plasma concentrations were determined from an absorbance standard curve using SOFTmax PRO analysis software (Molecular Devices). Plasma antibody concentrations over time following intravenous administration, as determined by the method described above, are shown in FIG. 47. This result demonstrates that in mice administered variants derived from enhanced binding of 278-IgG1 to FcγR with pH-dependent potent human IgE binding activity, plasma antibody concentration in mice was not significantly reduced compared to 278-IgG1.

(25-4) Determination of hIgE (Asp6) concentration in plasma in normal mice mice

Mouse plasma hIgE antibody concentrations were measured by ELISA. Standard curve samples were prepared for plasma concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. To ensure homogeneity of the immune complex between hIgE (Asp6) and anti-hIgE antibody, in the 278-hIgG1 administration group, Xolair (Novartis) at 10 µg/mL was added to standard curve samples and mouse plasma samples for analysis, and the mixtures were allowed to stand at room temperature for 30 minutes. In the group in which 278-F1087 was administered, 278-F1022 (heavy chain, SEQ ID NO: 156; light chain, SEQ ID NO: 152; prepared analogously to Example 24) or 278-F760 (heavy chain, SEQ ID NO: 157 ; light chain, SEQ ID NO: 152; obtained according to example 24) was added at a concentration of 20 μg/ml and the mixture was stirred at 37°C for 60 hours. Mouse plasma samples for analysis were aliquoted into an Immunoplate (MABTECH) onto which an anti-IgE antibody was immobilized, or Immunoplate (Nunc F96 MicroWell Plate (Nalge nunc International)) onto which an anti-human IgE antibody (clone 107, MAVTESCH) was immobilized. and they were allowed to stand and stir at room temperature for two hours or allowed to stand at 4° C. overnight. Then, human core protein GPC3 (SEQ ID NO: 158), anti-GPC3 antibody (produced in-house) biotinylated NHS-PEG4-6hothhom (Thermo Fisher Scientific), and streptavidin-PolyHKP80 (Stereospecific Detection Technologies) were each reacted sequentially for one hours. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After completion of the reaction with 1 H sulfuric acid (Showa Chemical), mouse plasma concentrations were determined based on color development by the absorbance measurement method at 450 nm on a microplate reader. Alternatively, the chromogenic reaction was performed using SuperSignal(r) ELISA Pico Chemiluminesrent Substrate (Thermo Fisher Scientific) as the substrate and plasma concentrations were determined by the luminescence intensity measurement method using a microplate reader. Mouse plasma concentrations were determined from absorbance or luminescence intensity of a standard curve using SOFTmax PRO analysis software (Molecular Devices). Plasma concentrations of hIgE (Asp6) over time after intravenous administration, which were determined as described above, are shown in FIG. 48.

Regarding the elimination of human IgE alone, the result shows that in mice administered with human IgE in combination with 278-IgG1 having a pH-dependent potent binding activity, the elimination of human IgE was accelerated compared with administration of human IgE alone. Moreover, in mice administered with human IgE in combination with 278-F1087, obtained by enhancing the binding of 278-IgG1 to FcγR, it was demonstrated that the elimination of human IgE is significantly accelerated compared to mice administered with human IgE alone or with human IgE in combinations with 278-IgG1. Therefore, it was shown that antigen clearance was accelerated not only in mice administered with the above-mentioned anti-IL6R antibody and anti-IgA antibody with enhanced FcγR binding, but also in mice administered with anti-IgE antibody with enhanced FcγR binding.

[Reference Example 1] Construction of expression vectors for IgG antibodies with amino acid substitution

Mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described in the accompanying instructions. Plasmid fragments containing the mutants were inserted into animal cell expression vectors to construct the desired H chain and L chain expression vectors. The nucleotide sequences of the resulting expression vectors were determined by methods known to those skilled in the art.

[Reference Example 2] Expression and purification of IgG antibodies

Antibodies were expressed using the following method. Cells of a cell line derived from human embryonic kidney cancer HEK293H (Invitrogen) were suspended in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). Cells were plated at 10 ml per dish in non-adherent cell dishes (10 cm diameter; CORNING) at a cell density of 5-6×10 ⁵ cells/ml and cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) throughout the day and night. The medium was then removed by aspiration and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The resulting plasmid was introduced into cells by lipofection. The resulting culture supernatants were collected, centrifuged (approximately 2000×g, 5 min, room temperature) to remove cells, and sterilized by filtration through a 0.22 µm MILLEX (registered trademark)-GV (Millipore) filter to obtain supernatants. Antibodies were purified from purified culture supernatants in a manner known to those skilled in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine the concentration of purified antibody, absorbance at 280 nm was measured using a spectrophotometer. From the determined values, antibody concentrations were calculated using the absorbance calculated by the method described in Protein Science (1995) 4: 2411-2423.

[Reference Example 3] Preparation of Soluble Human IL-6 Receptor (hsIL-6R)

The recombinant human IL-6 receptor, which is an antigen, was prepared as follows. A CHO cell line was produced constitutively expressing the soluble human IL-6 receptor (hereinafter hsIL-6R) having an amino acid sequence consisting of positions 1 to 357 from the N-terminus as described in J. Immunol. 152: 4958-4968 (1994), in a manner known to those skilled in the art. human hsIL-6R was expressed by culturing this CHO line. human hsIL-6R was purified from the culture supernatant of the resulting CHO line using two steps: Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the last step was obtained as the final purification product.

[Reference Example 4] Production of Low-Fucose Antibodies

Low fucose antibodies were expressed as follows. Fucose transporter-deficient CHO cells (patent document WO 2006/067913) suspended in α-MEM medium (Invitrogen) supplemented with 10% fetal bovine serum (CBS) were seeded at a concentration of 2E+6 cells/10 ml per attachment plates. cells (diameter: 10 cm; CORNING). After culturing in a CO ₂ incubator (37°C, 5% CO ₂ ) for one day and one night, the medium was removed by aspiration and then 7 ml of CHO-S-SFM-II medium (Invitrogen) was added thereto. 7 ml of CHO-S-SFM-II medium (Invitrogen) was added to cells that were obtained separately and into which plasmids were introduced by the lipofection method. After 72 hours, culture supernatants were collected by centrifugation (approximately 2000 g, 5 minutes, room temperature) and sterilized by filtration through a 0.22 μm MILLEX(R)-GV filter (Millipore). From the resulting culture supernatants, antibodies were purified in a manner known to those skilled in the art using rProtein A SepharoseTM Fast Flow (Amersham Biosciences). Purified antibody concentrations were calculated using the extinction coefficient calculated by the method described in Protein Science (1995) 4, 2411-2423, based on absorbance at 280 nm measured with a spectrophotometer.

[Reference Example 5] Obtaining antibodies that bind to the IL-6 receptor in a Ca-dependent manner from an antibody library using phage display technology

(5-1) Obtaining a phage display library for naive human antibodies

A phage display library for human antibodies, consisting of a plurality of phages representing the Fab domains of mutually distinct human antibody sequences, was constructed according to a method known to those skilled in the art using poly-A RNA derived from human PBMC and a commercial poly- And human RNA as a template.

(5-2) Obtaining antibody fragments that bind to an antigen in a Ca-dependent manner from a library by panning beads

The engineered phage display library for naïve human antibodies was subjected to initial selection by concentrating only those antibody fragments that have antigen-binding ability (IL-6 receptor) or concentrating antibody fragments using Ca concentration-dependent antigen-binding ability (IL-6 receptor) in as an indicator. Concentration of antibody fragments using Ca concentration-dependent antigen binding capacity (IL-6 receptor) as an indicator was performed by eluting phage library phages bound to the IL-6 receptor in the presence of Ca ions with EDTA, which chelates Ca ions. The biotinylated IL-6 receptor was used as an antigen.

Phages were obtained from Escherichia coli containing the engineered phage display phagemid. The phage library solution was prepared by diluting with TBS a population of phages precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture solution in which the phages were produced. Then BSA and CaCl ₂ were added to the phage library solution at a final concentration of 4% BSA and a calcium ion concentration of 1.2 mM. The conventional panning method using an antigen immobilized on magnetic beads has been called the panning method (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1- 2), 191-203, Biotechnol Prog (2002) 18(2) 212-20, Mol Cell Proteomics (2003) 2(2), 61-9. Neutravidin-coated beads (Sera-Mag SpeedBeads Neutravidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

Specifically, 250 pmol of biotin labeled antigen was added to the resulting phage library solution to allow said phage library solution to be contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added to bind to antigen-phage complexes at room temperature for 15 minutes. The beads were washed once with 1 ml of 1.2 mM CaCl ₂ /TBS (TBS containing 1.2 mM CaCl ₂ ). The phage solution was then isolated using the general elution method to concentrate the antibody fragment having the ability to bind the IL-β receptor, or by elution from beads suspended in 2 mM EDTA/TBS (TBS containing 2 mM EDTA) to concentrate the antibody fragment with using the ability to bind the IL-6 receptor in a Ca concentration dependent manner as an index. The isolated phage solution was added to 10 ml of E. coli TG1 strain in logarithmic growth phase (OD600 0.4-0.7). E. coli were cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coli. Infected E. coli were inoculated into a 225 mm×225 mm dish. The phages were then isolated from the E. coli culture medium after inoculation to obtain a phage library solution.

In the second and subsequent panning, phages were increased in number using Ca-dependent binding capacity as an indicator. Specifically, 40 pmol of biotin labeled antibody was added to the resulting phage library solution to allow the phage library to contact the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added to bind to antigen-phage complexes at room temperature for 15 minutes. The beads were washed once with 1 ml of 1.2 mM CaCl ₂ /TBST and 1.2 mM CaCl ₂ /TBS. Then, the beads, to which 0.1 ml of 2 mM EDTA/TBS was added, were suspended at room temperature. Immediately after that, the beads were separated using a magnetic stand to collect the phage solution. The isolated phage solution was added to 10 ml of E. coli TG1 strain in logarithmic growth phase (OD600 0.4-0.7). E. coli were cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coli. Infected E. coli were inoculated into a 225 mm×225 mm dish. The phages were then isolated from the E. coli culture medium after inoculation to obtain a phage library solution. Panning using Ca-dependent binding capacity as an index was repeated several times.

(5-3) ELISA study of phages

The phage containing the culture supernatant was harvested in a conventional manner (Methods Mol. Biol. (2002) 178, 133-145) from a single E. coli colony prepared as described above.

Culture supernatant containing phages to which BSA and CaCl ₂ were added to a final concentration of 4% BSA and a calcium ion concentration of 1.2 mM was subjected to ELISA as described below. A 96-well StreptaWell microtiter plate (Roche) was loaded overnight with 100 μl of PBS containing biotin labeled antigen. Each well of said plate was washed with PBST to remove antigen, and then the wells were blocked with 250 μl of 4% BSA-TBS for 1 hour or longer. This plate, with the resulting culture supernatant added to each well from which 4% BSA-TBS had been removed, was allowed to stand intact at 37° C. for 1 hour, allowing the phage-presenting antibody to bind to the antigen present in each well. To each well washed with 1.2 mM CaCl ₂ /TBST was added 1.2 mM CaCl ₂ /TBS or 1 mM EDTA/TBS. The tablet was allowed to stand intact for 30 minutes at 37°C for incubation. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS at a final concentration of 4% BSA and 1.2 mM ionized calcium was added to each well, and the plate was incubated for 1 hours. After washing with 1.2 mM CaCl ₂ /TBST, the chromogenic reaction of the solution in each well, to which only TMB solution (ZYMED) was added, was stopped by adding sulfuric acid. Then the specified color was determined by measuring the absorbance at 450 nm.

As a result of the above phage ELISA, the base sequence of the gene amplified with the specific primers and the antibody fragment identified as having the ability of Ca-dependent antigen binding as a template was analyzed.

(5-4) Expression and purification of antibodies

As a result of the phage ELISA described above, a clone identified as having a Ca-dependent antigen-binding ability was introduced into an expression plasmid for animal cells. Antibodies were expressed as described below. Freestyle 293-F (Invitrogen), derived from human embryonic kidney cells, was suspended in Freestyle 293 Expression Medium (Invitrogen) and then inoculated at 3 ml into each well of a 6-well plate at a cell density of 1.33×10 ⁶ cells /ml The resulting plasmid was introduced into cells by lipofection. Cells were cultured for 4 days in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm). Antibodies were purified from the culture supernatant prepared as described above by a method known in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). The absorbance of the purified antibody solution was measured at 280 nm using a spectrophotometer. The antibody concentration was calculated from the measurement data obtained using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[Reference Example 6] Investigation of Ca-Dependent Binding Ability of Obtained Human IL-6 Receptor Antibodies

To investigate whether the antibody binding activity is 6RL#9-IgG1 [heavy chain (SEQ ID NO: 44; sequence in which the IgG1-derived constant region is linked to SEQ ID NO: 9); light chain SEQ ID NO: 45] and FH4-IgG1 [heavy chain, SEQ ID NO: 46; light chain SEQ ID NO: 47] obtained according to Reference Example 5 with Ca-dependent human IL-6 receptor, kinetic analysis of antigen-antibody reactions for these antibodies with human IL-6 receptor was performed using Biacore T100 (GE Healthcare). H54/L28-IgG1 [heavy chain SEQ ID NO: 36; light chain SEQ ID NO: 37] described in WO 2009125825 was used as a control antibody that has a Ca-dependent binding activity to the human IL-6 receptor. Kinetic analysis of antigen-antibody reactions was performed in solutions with calcium ion concentrations of 2 mM and 3 μM as high and low calcium ion conditions, respectively. The antibody of interest was immobilized on a CM4 sensor chip (GE Healthcare) on which an appropriate amount of protein A (Invitrogen) was immobilized by the amine coupling method. Two buffers were used as running buffers [10 mM ACES, 150 mM NaCl, 0.05% (w/v) Tween 20, and 2 mM CaCl ₂ (pH 7.4) or 10 mM ACES, 150 mM NaCl, 0.05% (w/v) Tween 20, and 3 µmol/l CaCl ₂ (pH 7.4)]. These buffers were used to dilute the human IL-6 receptor. All measurements were carried out at 37°C.

In an antigen-antibody kinetic assay using the H54L28-IgG1 antibody, the H54L28-IgG1 antibody captured on the sensor chip was allowed to interact with the IL-6 receptor by injecting IL-6 receptor diluent and running buffer (blank sample) at a flow rate of 20 µl /min for 3 minutes. Then, after detecting dissociation of the IL-6 receptor using running buffer at a flow rate of 20 μl/min for 10 minutes, the sensor chip was regenerated by injecting 10 mM glycine-HCl (pH 1.5) at a flow rate of 30 μl/min for 30 seconds. From the sensorgrams obtained during the measurement, the kinetic parameters were calculated: the binding constant (ka) (1/Ms) and the dissociation rate constant (kd) (1/s). These values were used to calculate the dissociation constant (KD) (M) of the H54L28-IgG1 antibody against the human IL-6 receptor. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare).

In an antigen-antibody kinetic assay using FH4-IgG1 and 6RL#9-IgG1 antibodies, an FH4-IgG1 or 6RL#9-IgG1 antibody captured on a sensor chip was allowed to interact with the IL-6 receptor by injection of an IL-6 receptor diluent and running buffer (blank sample) at a flow rate of 5 µl/min for 15 minutes. The sensor chip was then regenerated by injecting 10 mM glycine-HCl (pH 1.5) at a flow rate of 30 μl/min for 30 seconds. Dissociation constants (KD) (M) were calculated from sensograms when measured using a stationary affinity model. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare). The dissociation constants (KD) between each antibody and the IL-6 receptor in the presence of 2 mM CaCl ₂ determined as described above are shown in Table 32.

The KD value for the H54/L28-IgG1 antibody under conditions of 3 μM Ca concentration can be calculated in the same way as in the presence of 2 mM Ca concentration. Under conditions of Ca concentration of 3 μM, FH4-IgG1 and 6RL#9-IgG1 antibodies were observed to hardly bind to the IL-6 receptor, thus calculating KD values by the method described above is difficult. However, the KD values for these antibodies under 3 μM Ca concentration conditions can be estimated using Equation 3 (Biacore T100 Software Handbook, BR-1006-48, AE 01/2007) described below.

[equation 3]

R _eq =C×R _max /(KD+C)+RI

The meaning of each parameter in [equation 3] mentioned above is as follows:

Req (RU): Steady State Binding Level

Rmax (RU): Surface binding capacity of the analyte

RI (RU): Cumulative contribution of the refractive index in the sample

C (M): Concentration of the analyzed compound

KD(M): Equilibrium dissociation constant

Approximate results for the dissociation constant KD values for antibodies and IL-6 receptor at Ca 3 μmol/L concentration estimated using Equation 3 above are presented in Table 33. In Table 33, the _Req , R _max , RI and C values are estimated based on the result of the analysis.

Based on the data described above, it was predicted that the K _D between the IL-6 receptor and the FH4-IgG1 antibody or the 6RL#9-IgG1 antibody increased by approximately 60 or 120-fold (affinity decreased by 60 or 120-fold or more) when the concentration of CaCl ₂ in the buffer was reduced from 2 mm to 3 μm.

Table 34 summarizes the K _D values at CaCl ₂ concentrations of 2 mM and 3 μM and the dependence on Ca for the three types of antibodies H54/L28-IgG1, FH4-IgG1 and 6RL#9-IgG1.

There were no differences in the binding of the H54/L28-IgG1 antibody to the IL-6 receptor due to differences in Ca concentration. On the other hand, it was found that the binding of antibodies FH4-IgG1 and 6RL#9-IgG1 to the IL-6 receptor was significantly attenuated under conditions of low Ca concentration (table 34).

[Reference Example 7] Calcium ion binding study of the resulting antibody

Then, the intermediate temperature of thermal denaturation (Tm value) was measured using differential scanning calorimetry (DSC) as an indicator for the study of calcium ion binding to the antibody (MicroCal VP-Capillary DSC, MicroCal). The intermediate thermal denaturation temperature (Tm value) is an indicator of stability. The intermediate temperature of thermal denaturation (Tm value) becomes higher when the protein is stabilized by binding to calcium ions, compared with no binding of calcium ions (J. Biol. Chem. (2008) 283, 37, 25140-25149). The activity of calcium ion binding to the antibody was investigated by examining changes in the Tm value of the antibody, depending on changes in the concentration of calcium ions in the antibody solution. The purified antibody was dialyzed (EasySEP, TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl ₂ (pH 7.4), or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl ₂ (pH 7.4). DSC measurement was performed at a heating rate of 240°C/h from 20 to 115°C using an antibody solution prepared at a concentration of approximately 0.1 mg/ml with dialysate as the test substance. The intermediate thermal denaturation temperatures (Tm values) of the Fab domains of each antibody, calculated from the DSC denaturation curve, are shown in Table 35.

The results presented in Table 35 show that the Tm values for FH4-IgG1 and 6RL#9-IgG1 Fab, which exhibit calcium-dependent binding ability, varied with calcium ion concentration, while the Tm values for H54 Fab /L28-IgG1, which does not exhibit calcium-dependent binding ability, does not change with changes in the concentration of calcium ions. The variation in Tm values for the Fab of the FH4-IgG1 and 6RL#9-IgG1 antibodies demonstrates that the calcium ions bound to these antibodies stabilize the Fab portions. The results described above show that calcium ions bound to the FH4-IgG1 and 6RL#9-IgG1 antibodies, while calcium ions did not bind to the H54/L28-IgG1 antibody.

[Reference Example 8] Identification of the calcium ion binding site in 6RL#9 antibody by X-ray diffraction analysis

(8-1) X-ray diffraction analysis

As described in Reference Example 7, measurement of the thermal denaturation temperature Tm showed that the 6RL#9 antibody binds to calcium ions. However, it was unpredictable how much of the 6RL#9 antibody binds to the calcium ion. Therefore, using the method of X-ray diffraction analysis identified the remains of the antibody 6RL#9, which interact with the calcium ion.

(8-2) Expression and purification of 6RL#9 antibody

The 6RL#9 antibody was expressed and purified for x-ray analysis. Specifically, animal expression plasmids engineered to be capable of expressing the heavy chain (SEQ ID NO: 44) and light chain (SEQ ID NO: 45) of the 6RL#9 antibody were transiently introduced into animal cells. The engineered plasmids were introduced by lipofection into a FreeStyle 293-F human embryonic kidney cell line (Invitrogen) suspended in 800 ml of FreeStyle 293 Expression Medium (Invitrogen) (final cell density: 1×10 ⁶ cells/ml) . Cells with the introduced plasmid were cultured in a CO ₂ incubator (37°C, 8% CO ₂ , 90 rpm) for five days. From the culture supernatant obtained as described above, antibodies were purified in a manner known to those skilled in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). The absorbance at 280 nm of the purified antibody solutions was measured using a spectrophotometer. From the measured values, antibody concentrations were calculated using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

(8-3) Purification of the Fab fragment of the 6RL#9 antibody

The 6RL#9 antibody was concentrated to 21 mg/ml using a 10,000 MWCO ultrafilter. An antibody sample at a concentration of 5 mg/ml (2.5 ml) was prepared by diluting the antibody solution using 4 mM L-cysteine/5 mM EDTA/20 mM sodium phosphate (pH 6.5) buffer. 0.125 mg papain (Roche Applied Science) was added to the sample. After mixing, the sample was incubated at 35°C for two hours. After incubation, an EDTA-free Protease Inhibitor Cocktail Mini tablet (Roche Applied Science) was dissolved in 10 ml of 25 mM MES buffer, pH 6, and added to the sample. The sample was incubated on ice to stop proteolytic cleavage with papain. The sample was then applied to a 1 ml HiTrap SP HP cation exchange column (GE Healthcare) equilibrated with 25 mM MES buffer (pH 6), below which a 1 ml HiTrap MabSelect Sure Protein A column (GE Healthcare) was connected in series. A purified fraction of the Fab fragment of the 6RL#9 antibody was obtained by eluting with a linear concentration gradient of NaCl up to 300 mm in the buffer described above. The resulting purified fraction was then concentrated to approximately 0.8 ml using a 5000 MWCO ultrafilter. The concentrate was applied to a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with 100 mM HEPES buffer (pH 8) containing 50 mM NaCl. The purified Fab fragment of the 6RL#9 antibody for crystallization was eluted from the column using the same buffer. All column treatments described above were performed at a low temperature of 6 to 7.5°C.

(8-4) Crystallization of the Fab fragment of the antibody 6RL#9 in the presence of Ca

Seed crystals of the 6RL#9 Fab fragment were prepared in advance according to general conditions. The purified Fab fragment of the 6RL#9 antibody in 5 mM CaCl ₂ was then concentrated to 12 mg/ml using a 5000 MWCO ultrafilter. Next, the sample concentrated as described above was crystallized by hanging drop method by vapor diffusion using 100 mM HEPES buffer (pH 7.5) containing 20%-29% PEG4000 as a reservoir solution. The seed crystals described above were crushed in 100 mM HEPES buffer (pH 7.5) containing 29% PEG4000 and 5 mM CaCl ₂ and serially diluted from 100 to 10,000 times. Then 0.2 µl of the diluted solutions were combined with a mixture of 0.8 µl of the reservoir solution and 0.8 µl of the concentrated sample to obtain crystallization droplets on a coverslip. The crystal droplets were allowed to stand at 20° C. for two to three days until flat crystals were obtained. Using crystals, X-ray diffraction data were obtained.

(8-5) Crystallization of the Fab fragment of the 6RL#9 antibody in the absence of Ca

The purified Fab fragment of the 6RL#9 antibody was concentrated to 15 mg/ml using a 5000 MWCO ultrafilter. Next, the sample concentrated as described above was crystallized by hanging drop method by vapor diffusion using 100 mM HEPES buffer (pH 7.5) containing 18%-25% PEG4000 as a reservoir solution. The seed crystals described above were crushed in 100 mM HEPES buffer (pH 7.5) containing 25% PEG4000 and serially diluted from 100 to 10,000 times. Then 0.2 µl of the diluted solutions were combined with a mixture of 0.8 µl of the reservoir solution and 0.8 µl of the concentrated sample to obtain crystallization droplets on a coverslip. The crystal droplets were allowed to stand at 20° C. for two to three days until flat crystals were obtained. Using crystals, X-ray diffraction data were obtained.

(8-6) Measurement of X-ray diffraction data of a Fab fragment crystal from 6RL#9 antibody in the presence of Ca

. Кристалл принадлежит к группе симметрии Р212121 с константой решетки а=45,47

, b=79,86

, с=116,25

, α=90°, β=90° и γ=90°.A single crystal of the Fab fragment of the 6RL#9 antibody obtained in the presence of Ca was wetted with 100 mM HEPES buffer (pH 7.5) containing 35% PEG4000 and 5 mM CaCl ₂ . The single crystal was fished out of solution using a pin with a thin nylon loop attached and frozen in liquid nitrogen. Frozen crystal X-ray diffraction data were obtained from a BL-17A Photon Factory beamline at the High Energy Accelerator Research Organization. During the measurement, the crystal was constantly placed in a stream of nitrogen at -178°C to maintain it in a frozen state. A total of 180 diffraction images were acquired using a Quantum315r CCD detector (ADSC) attached to the beamline while rotating the crystal 1° at a time. Determination of the lattice constant, indexing of diffraction spots, and analysis of diffraction data were performed using Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensity data were obtained with resolutions up to 2.2

. The crystal belongs to the P212121 symmetry group with a lattice constant a=45.47

, b=79.86

, s=116.25

, α=90°, β=90° and γ=90°.

(8-7) Measurement of X-ray diffraction data of a Fab fragment crystal from 6RL#9 antibody in the absence of Ca

. Кристалл принадлежит к группе симметрии Р212121 с константой решетки а=45,40

, b=79,63

, с=116,07

, α=90°, β=90° и γ=90°, и, таким образом, он структурно идентичен кристаллу, полученному в присутствии Са.Crystals of the Fab fragment of the 6RL#9 antibody obtained in the absence of Ca were wetted with 100 mM HEPES buffer (pH 7.5) containing 35% PEG4000. After removing the external solution from the surface of the single crystal with a pin, with a thin nylon loop attached, the single crystal was frozen in liquid nitrogen. Frozen crystal X-ray diffraction data were obtained from a BL-5A Photon Factory beam line at the High Energy Accelerator Research Organization. During the measurement, the crystal was constantly placed in a stream of nitrogen at -178°C to maintain it in a frozen state. A total of 180 diffraction images were acquired using a Quantum315r CCD detector (ADSC) attached to the beamline while rotating the crystal 1° at a time. Determination of the lattice constant, indexing of diffraction spots, and analysis of diffraction data were performed using Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensity data were obtained with resolutions up to 2.3

. The crystal belongs to the P212121 symmetry group with a lattice constant a=45.40

, b=79.63

, s=116.07

, α=90°, β=90° and γ=90°, and thus it is structurally identical to the crystal obtained in the presence of Ca.

(8-8) Analysis of the crystal structure of the Fab fragment from the antibody 6RL#9 in the presence of Ca

составлял 46,9% и свободный R составлял 48,6% после детализации жесткого каркаса, где каждому из VH-, VL-, СН1- и CL-доменов позволяли отклоняться от первоначальной структурной модели. Затем детализации модели достигали путем повторения структурной детализации с использованием программы Refmac5 (ССР4 Software Suite) с последующей проверкой модели, проводимой с использованием программы Coot (Paul Emsley) на основании карт электронной плотности Fo-Fc и 2Fo-Fc, где коэффициенты Fo-Fc и 2Fo-Fc вычисляли с использованием экспериментально определенного структурного фактора Fo, структурного фактора Fc, вычисленного на основе этой модели, и фаз. Конечную детализацию проводили с использованием программы Refmac5 (ССР4 Software Suite) на основе карт электронной плотности Fo-Fc и 2Fo-Fc путем добавления молекулы воды и иона Са в модель. С помощью 21020 данных отражения при разрешении от 25 до 2,2

, в конечном итоге кристаллографический фактор достоверности R составил 20,0% и свободный R составил 27,9% для модели, состоящей из 3440 атомов.The crystal structure of the Fab fragment of the 6RL#9 antibody in the presence of Ca was determined by the molecular displacement method using the Phaser program (CCP4 Software Suite). The number of molecules in the asymmetric element was estimated based on the size of the crystal lattice and the molecular weight of the Fab fragment of the antibody 6RL#9. Based on the homology of the primary sequence, part of amino acid positions 112-220 of the A chain and part of amino acid positions 116-218 of the B chain in the conformational coordinate of the PDB code 1ZA6 were used as model molecules for the analysis of the CL and CH1 regions. Then, a portion of amino acid positions 1-115 of the B chain in the conformational coordinate of the PDB code 1ZA6 was used as a model molecule for analysis of the VH region. Finally, a portion of amino acid positions 3-147 of the light chain in the conformational coordinate of the PDB code 2A9M was used as a model molecule for analysis of the VL region. From this order, an initial structural model of the 6RL#9 antibody Fab fragment was generated by determining positions and orientations of the model molecules for lattice analysis from the shear and rotation functions. Crystallographic confidence factor R for reflectance data at 25 to 3.0 resolution

was 46.9% and free R was 48.6% after rigid framework detailing where each of the VH, VL, CH1 and CL domains was allowed to deviate from the original structural model. Model refinement was then achieved by repeating the structural refinement using Refmac5 (CCP4 Software Suite) followed by model validation using Coot (Paul Emsley) based on Fo-Fc and 2Fo-Fc electron density maps, where the coefficients Fo-Fc and 2Fo-Fc was calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated from this model, and the phases. The final detailing was performed using the Refmac5 program (ССР4 Software Suite) based on the Fo-Fc and 2Fo-Fc electron density maps by adding a water molecule and a Ca ion to the model. Using 21020 reflection data at 25 to 2.2 resolution

, the final crystallographic confidence factor R was 20.0% and the free R was 27.9% for the 3440-atom model.

(8-9) Determination of X-ray diffraction data of the 6RL#9 antibody Fab fragment crystal in the absence of Ca

составил 30,3% и свободный R составил 31,7% после детализации жесткого каркаса, где каждому из VH-, VL-, СН1- и CL-доменов позволяли отклоняться. Затем детализации модели достигали путем повторения структурной детализации с использованием программы Refmac5 (ССР4 Software Suite) с последующей проверкой модели, проводимой с использованием программы Coot (Paul Emsley) на основании карт электронной плотности Fo-Fc и 2Fo-Fc, где коэффициенты Fo-Fc и 2Fo-Fc вычисляли с использованием экспериментально определенного структурного фактора Fo, структурного фактора Fc, вычисленного на основе этой модели, и фаз. Конечную детализацию проводили с использованием программы Refmac5 (ССР4 Software Suite) на основе карт электронной плотности Fo-Fc и 2Fo-F путем добавления молекулы воды в модель. С помощью 18357 данных отражения при разрешении от 25 до 2,3

, в конечном итоге кристаллографический фактор достоверности R составил 20,9% и свободный R составил 27,7% для модели, состоящей из 3351 атомов.The crystal structure of the Fab fragment of the 6RL#9 antibody in the absence of Ca was determined based on the structure of the crystal obtained in the presence of Ca. Water molecules and Ca ions were neglected in the conformational coordinates of the crystal of the Fab fragment of the 6RL#9 antibody obtained in the presence of Ca. Crystallographic confidence factor R for reflectance data at 25 to 3.0 resolution

was 30.3% and free R was 31.7% after rigid framework detailing where each of the VH, VL, CH1 and CL domains was allowed to deviate. Model refinement was then achieved by repeating the structural refinement using Refmac5 (CCP4 Software Suite) followed by model validation using Coot (Paul Emsley) based on Fo-Fc and 2Fo-Fc electron density maps, where the coefficients Fo-Fc and 2Fo-Fc was calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated from this model, and the phases. The final detailing was performed using the Refmac5 program (ССР4 Software Suite) based on Fo-Fc and 2Fo-F electron density maps by adding a water molecule to the model. Using 18357 reflection data at 25 to 2.3 resolution

, the final crystallographic confidence factor R was 20.9% and the free R was 27.7% for a model consisting of 3351 atoms.

(8-10) Comparison of X-ray crystallographic diffraction data of Fab fragments of the 6RL#9 antibody in the presence and absence of Ca

When comparing the crystal structures of the Fab fragments of the 6RL#9 antibody in the presence and absence of Ca, significant changes in the CDR3 of the heavy chain were revealed. The structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody, determined by X-ray diffraction analysis, is shown in FIG. 49. In particular, the calcium ion was located in the center of the CDR3 loop region of the heavy chain of the Fab fragment of the 6RL#9 antibody obtained in the presence of Ca. It has been suggested that the calcium ion interacts with positions 95, 96 and 100a (Kabat numbering) of the heavy chain CDR3. It was proposed that the heavy chain CDR3 loop, which is important for antigen binding, was stabilized by calcium binding in the presence of Ca, and became the optimal structure for antigen binding. There are no reports demonstrating that calcium binds to the heavy chain CDR3 of the antibody. Thus, the calcium-linked structure of the antibody heavy chain CDR3 is a novel structure.

The calcium-binding motif present in the heavy chain CDR3 identified in the structure of the Fab fragment of the 6RL#9 antibody could also be a novel design element for the Ca library to generate an antigen-binding domain included in the antigen-binding molecule of the present invention that is directed against an antigen in depending on the concentration of ions. The calcium binding motif was inserted into the variable region of Reference Examples 18 and 19 described below and, for example, a library containing the heavy chain CDR3 of the 6RL#9 antibody and non-strict residues in other CDRs, including the light chain, is considered to be possible.

[Reference Example 9] Obtaining Antibodies That Bind to IL-6 Ca-Dependently from a Human Antibody Library Using Phage Display Methods

(9-1) Construction of a phage display library of naive human antibodies

A human antibody phage display library containing a plurality of phages that display different human antibody Fab sequences was constructed in a manner known to those skilled in the art using, as a template, a poly-A-RNA derived from human PBMC, a commercially available poly- human A-RNA, and the like.

(9-2) Obtaining antibody fragments that bind to antigen in a Ca-dependent manner from a library using bead panning

Primary selection from engineered phage display libraries of naïve human antibodies was performed by enrichment with antibody fragments that have antigen binding activity (IL-6). The antigen used was biotin labeled IL-6.

Phages were generated using E. coli bearing phage display phagemids. 2.5 M NaCl/10% PEG was added to the sediment of phages obtained using E. coli in the culture medium for E. coli. The phage fraction was diluted with TBS to obtain a solution with a phage library. BSA and CaCl ₂ were then added to the phage library solution to final concentrations of 4% BSA and 1.2 mM calcium ions, respectively. The panning method used was the conventional panning method using antigen-immobilized magnetic beads (J Immunol Methods. 2008 Mar 20, 332(1-2): 2-9; J Immunol Methods. 2001 Jan 1, 247(1-2 ): 191-203; Biotechnol Prog. 2002 Mar-Apr, 18(2): 212-20; Mol Cell Proteomics 2003 Feb, 2(2): 61-9). The magnetic beads used were neutravidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and streptavidin-coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotinylated antigen was added to the resulting phage library solution. Thus, the solution was contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added and the antigen-phage complex was allowed to bind to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with 1 ml 1.2 mM CaCl ₂ /TBS (TBS containing 1.2 mM CaCl ₂ ). After that, 0.5 ml of trypsin at a concentration of 1 mg/ml was added to the granules. After dispersing for 15 minutes at room temperature, the beads were immediately separated using a magnetic stand to collect the phage suspension. The resulting phage suspension was added to 10 ml of E. coli strain TG1 in the logarithmic growth phase (OD600=0.4-0.7). E. coli were incubated with gentle agitation at 37°C for one hour for infection with phages. Infected E. coli were plated (225 mm x 225 mm). Phages were then collected from the culture medium of the seeded E. coli to obtain a solution with a phage library.

At the second and subsequent pannings, phages were enriched using Ca-dependent binding capacity as an indicator. Specifically, 40 pmol of biotin labeled antigen was added to the resulting phage library solution. Thus, the phage library was contacted with the antigen at room temperature for 60 minutes. The BSA-blocked magnetic beads were added and the antigen-phage complex was allowed to bind to the magnetic beads at room temperature for 15 minutes. The beads were washed once with 1 ml 1.2 mM CaCl ₂ /TBST (TBS containing 1.2 mM CaCl ₂ and 0.1% Tween-20) and 1.2 mM CaCl ₂ /TBS. Then 0.1 ml of 2 mM EDTA/TBS was added to the beads. After dispersion at room temperature, the beads were immediately separated using a magnetic stand to collect the phage suspension. The pIII protein (pIII protein derived from helper phage) was cleaved from phages that did not exhibit Fab by adding 5 μl of trypsin at a concentration of 100 mg/ml to the harvested phage suspension to eliminate the ability of phages not exhibiting Fab to infect E. coli. Phages collected from the stock solution with phage after trypsin treatment were added to 10 ml of E. coli strain TG1 cells in the logarithmic growth phase (OD600=0.4-0.7). E. coli were incubated with gentle agitation at 37°C for one hour for infection with phages. Infected E. coli were plated (225 mm x 225 mm). Phages were then collected from the culture medium of the seeded E. coli to obtain a liquid stock solution with a phage library. Panning was performed three times using Ca-dependent binding activity as an indicator.

(9-3) Evaluation by ELISA of phages

Phage-containing culture supernatants were collected from single colonies of E. coli obtained by the method described above, according to the conventional method (Methods Mol. Biol. (2002) 178, 133-145). BSA and CaCl ₂ were added to phage-containing culture supernatants to final concentrations of 4% BSA and 1.2 mM calcium ions, respectively. The supernatants were subjected to ELISA according to the following method. StreptaWell 96-well microtiter plates (Roche) were coated overnight with 100 μl of PBS containing biotin labeled antigen. The antigen was removed from the well by washing each well of the PBST plate. The wells were then blocked with 250 μl of 4% BSA-TBS for one hour or more. After removal of 4% BSA-TBS, the resulting culture supernatants were added to each well. The plate was incubated at 37° C. for one hour so as to allow the antibody-expressing phages to bind to the antigen on each well. After washing each well with 1.2 mM CaCl ₂ /TBST, 1.2 mM CaCl ₂ /TBS or 1 mM EDTA/TBS was added. The tablet was left to incubate at 37°C for 30 minutes. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS containing BSA and calcium ions at final concentrations of 4% and 1.2 mM calcium ions was added to each well, and a plate incubated for one hour. After washing with 1.2 mM CaCl ₂ /TBST, only TMB solution (ZYMED) was added to each well. The chromogenic reaction in the solution in each well was stopped by adding sulfuric acid. The developed color was then evaluated by measuring the absorbance at 450 nm.

From 96 isolated clones, the 6KC4-1#85 antibody was obtained by phage ELISA, having a Ca-dependent IL-6 binding activity. Using antibody fragments predicted to have Ca-dependent antigen-binding activity based on the phage ELISA result described above as a template, the genes were amplified with specific primers and their sequences analyzed.

The sequences of the heavy chain and light chain variable regions of the 6KC4-1#85 antibody are shown in SEQ ID NOs: 10 and 48, respectively. A polynucleotide encoding the heavy chain variable region of the antibody 6KC4-1#85 (SEQ ID NO: 49) was linked to a polynucleotide encoding a sequence derived from IgG1 by PCR. The resulting DNA fragment was inserted into an expression vector of animal cells to construct an expression vector for the heavy chain of SEQ ID NO: 48. The polynucleotide encoding the light chain variable region of the antibody 6KC4-1#85 (SEQ ID NO: 50) was linked to the polynucleotide encoding the constant region natural kappa chain (SEQ ID NO: 100) by PCR. The linked DNA fragment was inserted into an animal cell expression vector. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art.

(9-4) Expression and purification of antibodies

Clone 6KC4-1#85, which was evaluated as having the ability to Ca-dependent binding using phage ELISA, was introduced into the expression plasmids of animal cells. Antibodies were expressed using the following method. FreeStyle 293-F embryonic kidney derived cells (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and 3 ml aliquots plated in each well of 6-well plates at a cell density of 1.33 x 10 ⁶ cells/ml. The resulting plasmids were introduced into cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for four days. From the resulting culture supernatants, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. Purified antibody concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. Antibody concentrations were calculated from established values based on the extinction coefficient determined by the PACE method (Protein Science 1995; 4: 2411-2423).

[Reference Example 10] Calcium ion binding evaluation of 6KC4-1#85 antibody

The calcium-dependent binding antibody 6KC4-1#85, which was isolated from a human antibody library, was evaluated for its calcium binding. Whether the measured Tm value varies depending on the concentration of ionized calcium was judged by the method described in Reference Example 7.

The Tm values for the Fab domain of the 6KC4-1#85 antibody are shown in Table 36. As shown in Table 36, the Tm value for the Fab domain of the 6KC4-1#85 antibody varied, depending on the concentration of calcium ions. This demonstrates that the 6KC4-1#85 antibody binds to calcium.

[Reference Example 11] Identification of the calcium ion binding site in 6KC4-1#85 antibody

As shown in Reference Example 10, the 6KC4-1#85 antibody binds to calcium ion. However, 6KC4-1#85 does not have a calcium binding motif, such as the hVk5-2 sequence, which has been evaluated to have a calcium binding motif. Then, confirmation that the calcium ion binds to either or both of the heavy chain and light chain of the 6KC4-1#85 antibody was performed by evaluating the calcium ion binding of the altered antibodies due to the replacement of the heavy chain and light chain of 6KC4-1#85, respectively, with the heavy chain. chain and light chain of anti-glypican 3 antibody (heavy chain sequence GC_H (SEQ ID NO: 51), light chain sequence GC_L (SEQ ID NO: 52)), which does not bind calcium ion. The Tm values of the altered antibodies measured by the method described in Reference Example 7 are shown in Table 37. The result indicates that the heavy chain of the 6KC4-1#85 antibody binds to calcium because the Tm values of the altered antibody having the heavy chain of the 6KC4-1 antibody #85, changed depending on the concentration of calcium ions.

Thus, to further identify the residues responsible for calcium ion binding by the 6KC4-1#85 antibody, altered heavy chains (6_H1-11 (SEQ ID NO: 53), 6_H1-12 (SEQ ID NO: 54), 6_H1-13 (SEQ ID NO: 55), 6_H1-14 (SEQ ID NO: 56), 6_H1-15 (SEQ ID NO: 57)) and altered light chains (6_L1-5 (SEQ ID NO: 58) and 6_L1-6 ( SEQ ID NO: 59)) by replacing the Asp (D) residue in the CDR of antibody 6KC4-1#85 with an Ala (A) residue that does not participate in calcium ion binding or chelation. By the method described in Reference Example 9, the altered antibodies were purified from the culture supernatants of animal cells into which expression vectors carrying genes of the altered antibodies were introduced. Purified modified antibodies were evaluated for their calcium binding in the manner described in Example 7. The result of the measurement is shown in Table 38. As shown in Table 38, substitution of an Ala residue at position 95 or 101 (Kabat numbering) in the heavy chain CDR3 of the 6KC4-1 antibody #85 resulted in loss of activity of the 6KC4-1#85 antibody in relation to calcium binding. This indicates that these residues are responsible for calcium binding.

The calcium-binding motif located at the base of the CDR3 loop in the heavy chain of the 6KC4-1#85 antibody, which was found based on the ability to bind calcium in an antibody altered from 6KC4-1#85, may represent a new factor for constructing Ca libraries, which are used to generate antigen-binding domains against an antigen depending on the concentration of ions that are contained in the antigen-binding molecules of the present invention. In Reference Examples 18 and 19 below, calcium binding motifs were introduced into the light chain variable region. Meanwhile, such libraries include, for example, libraries containing the heavy chain CDR3 from the 6KC4-1#85 antibody and non-strict residues in the CDRs other than the heavy chain CDR3 but including the light chain CDRs.

[Reference Example 12] Investigation of the Effects of Ca-Dependent Binding Antibody on Antigen Plasma Retention Using Normal Mice

(12-1) In vivo study using normal mice

A normal mouse (C57BL/6J mouse, Charles River Japan) was injected with hsIL-6R (soluble human IL-6 receptor prepared according to Reference Example 3) alone, or injected with hsIL-6R and anti-human IL-6 receptor antibody simultaneously to study the kinetics hsIL-6R and anti-human IL-6 receptor antibodies in vivo. A single dose (10 ml/kg) of a solution of hsIL-6R (5 μg/ml) or hsIL-6R and antibodies against human IL-6 receptor was injected into the tail vein. The above H54/L28-IgG1, 6RL#9-IgG1 and FH4-IgG1 were used as antibodies against human IL-6 receptor.

The concentration of hsIL-6R in all mixtures is 5 µg/ml. Anti-human IL-6 receptor antibody concentrations vary for antibodies: 0.1 mg/ml for H54/L28-IgG1 and 10 mg/ml for 6RL#9-IgG1 and FH4-IgG1. At this time, most of the hsIL-6Rs were believed to bind to the antibody because the anti-human hsIL-6R antibody is present in sufficient or excessive amounts. Blood samples were taken 15 minutes, 7 hours and 1, 2, 4, 7, 14, 21 and 28 days after administration. The obtained blood samples were immediately centrifuged for 15 minutes at 4°C and 12000 rpm. for plasma separation. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement.

(12-2) Plasma concentration determination of anti-human IL-6 receptor in normal mice by ELISA

The concentration of anti-human IL-6 receptor antibody in mouse plasma was determined by ELISA. First, anti-human IgG (γ-chain specific) F(ab')2 fragment (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) and allowed to stand overnight at 4°C to give tablets with anti-human IgG antibody in solid phase. Calibration curve samples having plasma concentrations of 0.64, 0.32, 0.16, 0.08, 0.04, 0.02, or 0.01 µg/mL and mouse plasma samples for measurement diluted 100 times or more, dispensed into a solid phase anti-human IgG antibody plate, and then incubated for 1 hour at 25°C. The plate was then allowed to react with a biotinylated anti-human IL-6 R (R&D) antibody for 1 hour at 25°C and then reacted with streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 0.5 hour at 25°C. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as substrate. After stopping the chromogenic reaction with 1N sulfuric acid (Showa Chemical), the absorbance of the color solution at 450 nm was measured on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices). Changes in plasma concentrations of H54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 antibodies in normal mice after intravenous administration, measured as described above, are shown in FIG. fifty.

(12-3) Plasma concentration determination of hsIL-6R by electrochemiluminescence method

The concentration of hsIL-6R in mouse plasma was determined by the method of electrochemiluminescence. An hsIL-6R calibration curve sample prepared at a concentration of 2000, 1000, 500, 250, 125, 62.5, or 31.25 pg/mL and a mouse plasma sample for measurement diluted 50 times or more were mixed with the monoclonal antibody anti-human IL-6R (R&D), rutenated with SULFO-TAG NHS Ester (Meso Scale Discovery) and biotinylated anti-human IL-6R (R&D), and tocilizumab (heavy chain SEQ ID NO: 60, light chain SEQ ID NO : 61), followed by overnight reaction at 4°C. By this time, the assay buffer contained 10 mM EDTA to reduce the concentration of free Ca in the sample and dissociate virtually all hsIL-6R in the sample from 6RL#9-IgGl or FH4-IgG1 to bind to the added tocilizumab. This reaction liquid was then dispensed into a MA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, after washing each well of the plate, which was allowed to react for 1 hour at 25°C, reading buffer T (×4) (Meso Scale Discovery) was dispensed into each well. Immediately thereafter, the reaction liquid was measured using a SECTOR PR 400 (Meso Scale Discovery) reader. The hsIL-6R concentration was calculated from the standard curve response using SOFTmax PRO analysis software (Molecular Devices). Changes in plasma concentration of hsIL-6R in normal mice following intravenous administration, determined as described above, are shown in FIG. 51.

As a result, the disappearance of hsIL-6R was very rapid when hsIL-6R was administered alone, while the disappearance of hsIL-6R was significantly delayed when hsIL-6R was administered simultaneously with H54/L28-IgGl, a conventional antibody lacking binding activity. Ca with hsIL-6R. On the contrary, the disappearance of hsIL-6R was significantly accelerated when hsIL-6R was administered simultaneously with 6RL#9-IgG1 or FH4-IgG1, which has a 100-fold or higher Ca-dependent binding capacity to hsIL-6R. Plasma concentrations of hsIL-6R one day after administration of hsIL-6R simultaneously with 6RL#9-IgG1 and FH4-IgG1 were reduced by 39-fold and 2-fold, respectively, compared with simultaneous administration with H54/L28-IgG1. Thus, it was confirmed that antibodies with calcium-dependent binding are able to accelerate the disappearance of antigen from plasma.

[Reference Example 13] Investigation of human germline sequences that bind to calcium ions

(13-1) An antibody that binds to an antigen in a calcium dependent manner

Antibodies that bind to an antigen in a calcium-dependent manner (antibodies with calcium-dependent antigen binding) are antibodies whose interactions with an antigen change depending on the concentration of calcium. An antibody with calcium-dependent binding is believed to bind to an antigen via a calcium ion. Thus, the amino acids that form an epitope on one side of the antigen are negatively charged amino acids that can chelate calcium ions, or amino acids that can be a hydrogen bond acceptor. These properties of the amino acids that form the epitope allow targeting of an epitope other than binding molecules, which are obtained by introducing histidine residues and which bind to the antigen in a pH-dependent manner. Moreover, it is believed that the use of antigen-binding molecules having calcium- and pH-dependent binding properties will allow the formation of antigen-binding molecules that can be individually targeted to various epitopes having broad properties. Thus, if a pool of molecules containing a calcium-binding motif (Ca-library) is constructed from which antigen-binding molecules are derived, it is believed that antibodies with calcium-dependent antigen binding will be generated in an efficient manner.

(13-2) Obtaining Human Germline Sequences

An example of a population of molecules containing a calcium-binding motif is one in which said molecules are antibodies. In other words, a library of antibodies containing a calcium-binding motif may be a Ca-library.

Antibodies with calcium ion dependent binding containing human germline sequences have not been described. Thus, germline antibody sequences having human germline sequences were cloned using cDNA derived from embryonic spleen polyRNA (Clontech) as a template to assess whether antibodies having human germline sequences bind to calcium ion. The cloned DNA fragments were inserted into expression vectors of animal cells. The nucleotide sequences of the constructed expression vectors were determined in a manner known to those skilled in the art. SEQ IDs are shown in Table 39. By PCR, polynucleotides encoding SEQ ID NO: 5 (Vkl), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), SEQ ID NO: 8 (Vk4), and SEQ ID NO: 62 (Vk5-2) was linked to a polynucleotide encoding a natural kappa chain constant region (SEQ ID NO: 50). Linked DNA fragments were inserted into animal cell expression vectors. Moreover, the heavy chain variable region polynucleotides encoding SEQ ID NO: 63 (Vk1), SEQ ID NO: 64 (Vk2), SEQ ID NO: 65 (Vk3), and SEQ ID NO: 66 (Vk4) were linked by PCR to a polynucleotide encoding IgG1 SEQ ID NO: 163 (having a deletion of two amino acids at the C-terminus of IgG1). The resulting DNA fragments were inserted into expression vectors of animal cells. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art.

(13-3) Expression and purification of antibodies

Constructed expression vectors of animal cells into which DNA fragments having five types of human germline sequences are inserted were introduced into animal cells. The expression of the antibody was carried out in the following way. Cells from the FreeStyle 293-F human embryonic kidney cell line (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and seeded at a cell density of 1.33×10 ⁶ cells/mL (3 mL) into each well of a plate with 6 cells. The resulting plasmids were introduced into cells by lipofection. Cells were cultured for four days in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm). From culture supernatants prepared as described above, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance of purified antibody solutions at 280 was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(13-4) Evaluation of antibodies having human germline sequences for their calcium ion binding activity

Purified antibodies were evaluated for their calcium ion binding activity. The thermal denaturation intermediate temperature (Tm value) was measured by differential scanning calorimetry (DSC) as an indicator for the study of calcium ion binding to an antibody (MicroCal VP-Capillary DSC, MicroCal). The intermediate thermal denaturation temperature (Tm value) is an indicator of stability. The Tm value becomes higher when the protein is stabilized by binding to calcium ions, compared with no binding of calcium ions (J. Biol. Chem. (2008) 283, 37, 25140-25149). The activity of calcium ion binding to the antibody was investigated by examining changes in the Tm value of the antibody, depending on changes in the concentration of calcium ions in the antibody solution. The purified antibody was dialyzed (EasySEP, TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl ₂ (pH 7.4), or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl ₂ (pH 7.4). DSC measurement was performed at a heating rate of 240°C/h from 20 to 115°C using an antibody solution prepared at a concentration of approximately 0.1 mg/ml with dialysate as the test substance. The intermediate thermal denaturation temperatures (Tm values) of the Fab domains of each antibody, calculated from the DSC denaturation curve, are shown in Table 40.

The result showed that the Tm values of the Fab domains of antibodies having the sequence hVk1, hVk2, hVk3 or hVk4 did not vary depending on the concentration of calcium ions in solutions containing the Fab domain. Meanwhile, the Tm value for the Fab domain of an antibody having the sequence hVk5 varied depending on the concentration of calcium ions in the solution containing the Fab domain. This demonstrates that the hVk5 sequence binds to calcium ions.

(13-5) Sequence evaluation of hVk5-2 for calcium binding

In (5-2) of Reference Example 5, Vk5-2 variant 1 (SEQ ID NO: 68) and Vk5-2 variant 2 (SEQ ID NO: 69) were obtained in addition to Vk5-2 (SEQ ID NO: 4), all of which are classified as Vk5-2. These variants were evaluated for their calcium binding. The DNA fragments for Vk5-2, Vk5-2 variant 1, and Vk5-2 variant 2 were each inserted into animal cell expression vectors. The nucleotide sequences of the constructed expression vectors were determined in a manner known to those skilled in the art. Using the method described in (12-3) of Reference Example 12, animal cell expression vectors into which the DNA fragments for Vk5-2, Vk5-2 variant 1, and Vk5-2 variant 2 were inserted were administered in combination with the expression vector containing the CIM_H expression insert (SEQ ID NO: 67) as the heavy chain into animal cells and the antibodies were purified. Purified antibodies were evaluated for their calcium ion binding activity. Purified antibodies were dialyzed (EasySEP, TOMY) against 20 mM Tris-HCl/150 mM NaCl (pH 7.5) (in Table 41, indicated as 0 mM calcium ion concentration) or 20 mM Tris-HCl/150 mM NaCl/2 mM CaCl ₂ (pH 7.5). DSC measurement was performed at a temperature increase rate of 240°C/h from 20 to 115°C using antibody solutions prepared at a concentration of approximately 0.1 mg/ml using the same solution used for dialysis. Based on the obtained DSC denaturation curves, the thermal denaturation intermediate temperature (Tm value) was calculated for the Fab domain of each antibody. The Tm values are presented in Table 41.

The result showed that the Tm value for Fab domains of antibodies having the sequence Vk5-2, Vk5-2 variant 1 or Vk5-2 variant 2 varied depending on the concentration of calcium ions in solutions containing antibodies having Fab domains. This demonstrates that antibodies having a sequence classified as Vk5-2 bind to the calcium ion.

[Reference Example 14] Human Vk5 sequence evaluation (hVk5)

(14-1) Sequence hVk5

The only hVk5 sequence registered in the Kabat database is the hVk5-2 sequence. In the following, hVk5 and hVk5-2 are used as synonyms.

WO 2010/136598 describes that the relative abundance of the hVk5-2 sequence in the germline sequence is 0.4%. There are also other reports that the relative abundance of the hVk5-2 sequence in the germline sequence is 0-0.06% (J. Mol. Biol. (2000) 296, 57-86; Proc. Natl. Acad. Sci. (2009 ) 106, 48, 20216-20221). As described above, since the hVk5-2 sequence is a sequence with a low frequency of occurrence in the germline sequence, it was believed to be ineffective for generating a calcium-dependent binding antibody from an antibody library composed of human germline sequences or B cells. obtained by immunizing a mouse expressing human antibodies. Thus, it is possible to construct Ca-libraries containing the human hVk5-2 sequence. Meanwhile, the described synthetic antibody libraries (WO 2010/105256 and WO 2010/136598) did not contain the hVk5 sequence. In addition, the feasibility is unknown because there are no published reports on the physical properties of the hVk5-2 sequence.

(14-2) Construction, expression and purification of the non-glycosylated form of the hVk5-2 sequence

The hVk5-2 sequence has an N-type glycosylation sequence at amino acid position 20 (Kabat numbering). Sugar chains attached to proteins exhibit heterogeneity. Thus, it is desirable to eliminate glycosylation from the point of view of the homogeneity of the substance. In this context, a variant of hVk5-2_L65 (SEQ ID NO: 70) was constructed in which the Asn (N) residue at position 2 0 (Kabat numbering) was replaced by Thr (T). Amino acid substitution was performed in a manner known to those skilled in the art using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). DNA encoding the hVk5-2_L65 variant was inserted into an animal expression vector. An animal expression vector into which engineered DNA encoding the hVk5-2_L65 variant is inserted, in combination with an animal expression vector having an insert for CIM_H expression (SEQ ID NO: 67) as a heavy chain, was inserted into animal cells by the method described in Reference Example 5. An antibody containing hVk5-2_L65 and CIM_H, which were expressed in animal cells into which the vectors were introduced, was purified by the method described in Reference Example 13.

(14-3) Evaluation of an antibody having a non-glycosylated hVk5-2 sequence for physical properties

The isolated antibody having the modified hVk5-2_L65 sequence was analyzed by ion exchange chromatography to examine whether it is less heterogeneous than the antibody having the original hVk5-2 sequence before the change. The ion exchange chromatography procedure is shown in Table 42. The result of the analysis showed that hVk5-2_L65 modified at the glycosylation site was less heterogeneous than the original hVk5-2 sequence, as shown in FIG. 52.

Further, evaluation of whether an antibody containing the less heterogeneous hVk5-2_L65 sequence binds to calcium ion was evaluated by the method described in Reference Example 13. The result showed that the Tm value for the Fab domain of an antibody having hVk5-2_L65 with an altered glycosylation site, also varied depending on the concentration of calcium ions in antibody solutions, as shown in Table 43. In particular, it was demonstrated that the Fab domain of an antibody having hVk5-2_L65 with an altered glycosylation site binds to a calcium ion.

[Reference Example 15] Evaluation of calcium ion binding activity of antibody molecules having a CDR sequence of hVk5-2 sequence

(15-1) Construction, expression and purification of modified antibodies having a CDR sequence from the hVk5-2 sequence

The hVk5-2_L65 sequence is a sequence with altered amino acids in the glycosylation site of the framework region of the human Vk5-2 sequence. As described in Reference Example 14, it was demonstrated that calcium ion binding occurred even after the glycosylation site was changed. Meanwhile, from the viewpoint of immunogenicity, it is generally desirable that the framework sequence is a germline sequence. Thus, the present inventors evaluated whether the framework sequence of an antibody could be replaced with a framework sequence of a non-glycosylated germline sequence while maintaining the calcium ion binding activity of the antibody.

Polynucleotides encoding chemically synthesized sequences that contain an altered framework sequence from hVk5-2, hVk1, hVk2, hVk3 or hVk4 (CaVk1 (SEQ ID NO: 71), CaVk2 (SEQ ID NO: 72), CaVk3 (SEQ ID NO : 73) or CaVk4 (SEQ ID NO: 74), respectively) were linked by PCR to a polynucleotide encoding the constant region (SEQ ID NO: 50) of the natural kappa chain. Linked DNA fragments were inserted into animal cell expression vectors. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art. Each plasmid constructed as described above was inserted into animal cells in combination with a plasmid into which a polynucleotide encoding CIM_H (SEQ ID NO: 67) was inserted in the manner described in Example 13. Expressed antibody molecules of interest were purified from the cell culture medium. animals in which plasmids were introduced.

(15-2) Evaluation of altered antibodies having a CDR sequence from the hVk5-2 sequence for their calcium ion binding activity

The assessment of whether calcium ions bind to altered antibodies having a CDR sequence from the hVK5-2 sequence and framework sequences from germline sequences other than hVk5-2 (hVk1, hVk2, hVk3 and hVk4) was assessed using the method described in the reference Example 5. The evaluation result is shown in Table 44. It was found that the value of the Tm Fab domain of each of the altered antibodies varied, depending on the concentration of calcium ions in the antibody solutions. This demonstrates that antibodies having a framework sequence different from the framework sequence from the hVk5-2 sequence also bind to calcium ions.

It has been demonstrated that the thermal denaturation temperature (Tm value), as an indicator of the thermal stability of the Fab domain of each antibody modified to have a CDR sequence from the hVK5-2 sequence and a germline backbone sequence other than the hVk5-2 sequence (hVk1 , hVk2, hVk3, or hVk4) is higher than the thermal denaturation temperature of the Fab domain of the parent antibody having the hVk5-2 sequence. This result shows that antibodies having a CDR sequence from the hVk5-2 sequence and an hVk1, hVk2, hVk3 or hVk4 framework sequence not only have calcium ion binding activity, but are also excellent molecules in terms of thermal stability.

[Reference Example 16] Identification of the calcium ion binding site in the human germline hVk5-2 sequence

(16-1) Construction of a mutation site in the CDR sequence from the hVk5-2 sequence

As described in Reference Example 15, it was also shown that antibodies having a light chain obtained by introducing a CDR region of the hVk5-2 sequence into a framework sequence of a different germline sequence bind to a calcium ion. This result indicates that in hVk5-2, the calcium ion binding site is located in its CDR. Amino acids that bind to the calcium ion, i.e. chelate the calcium ion, include negatively charged amino acids and amino acids that can be a hydrogen bond acceptor. Thus, it was investigated whether antibodies having a mutant hVk5-2 sequence with an Ala (A) substitution of an Asp (D) or Glu (E) residue in the CDR sequence of the hVk5-2 sequence bind to calcium ion.

(16-2) Construction of hVk5-2 variant sequences with Ala substitution, and antibody expression and purification

Antibody molecules were prepared to contain a light chain with an Asp and/or Glu residue replaced by an Ala residue in the CDR sequence from the hVk5-2 sequence. As described in Reference Example 14, the non-glycosylated hVk5-2_L65 variant exhibited calcium ion binding and was assumed to be equivalent to the hVk5-2 sequence in terms of calcium ion binding. In this example, amino acid substitutions were made in hVk5-2_L65 as a template sequence. The engineered variants are shown in Table 45. Amino acid substitutions were performed by methods known to those skilled in the art, such as using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or the Infusion Advantage PCR Cloning Kit (TAKARA) for constructing expression vectors for altered light chains having an amino acid substitution.

The nucleotide sequences of the constructed expression vectors were verified in a manner known to those skilled in the art. Expression vectors designed for altered light chains were transiently introduced, in combination with the heavy chain expression vector CIM_H (SEQ ID NO: 67), into human embryonic kidney HEK293H (Invitrogen) or FreeStyle293 (Invitrogen) cells for expression. antibodies. From the resulting culture supernatants, antibodies were purified using rProtein A SepharoseTM Fast Flow (GE Healthcare) in a manner known to those skilled in the art. The absorbance of purified antibody solutions at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(16-3) Evaluation of antibodies having an Ala substitution in the hVk5-2 sequence for calcium ion binding activity

It was investigated whether the obtained purified antibodies bind to calcium ions by the method described in Reference Example 13. The result is shown in Table 46. It was found that some antibodies having the replacement of the Asp or Glu residue in the CDR sequence of the hVk5-2 sequence with the Ala residue , which cannot be involved in the binding or chelation of calcium ions, have a Fab domain whose Tm did not vary depending on the concentration of calcium ions in antibody solutions. It was shown that the substitution sites in which the change to Ala did not change Tm (positions 32 and 92 (Kabat numbering)) are significantly important for the binding of calcium ions by the antibody.

[Reference Example 17] Evaluation of antibodies having an hVk1 sequence with a calcium ion binding motif

(17-1) Construction of hVk1 sequence with calcium ion binding motif, and expression and purification of antibodies

The result described in Reference Example 16 for calcium binding activity in the case of Ala substitution demonstrates that Asp or Glu residues in the CDR sequence of the hVk5-2 sequence were important for calcium binding. Thus, the present inventors evaluated whether the antibody could bind to calcium ion when the residues at positions 30, 31, 32, 50 and 92 (Kabat numbering) were separately introduced into a different germline variable region sequence. Specifically, the LfVk1_Ca variant (SEQ ID NO: 83) was constructed by substituting residues at positions 30, 31, 32, 50 and 92 (Kabat numbering) in the hVk5-2 sequence for residues at positions 30, 31, 32, 50 and 92 ( Kabat numbering) hVkl sequence (human germline sequence). In particular, it was investigated whether antibodies having the hVk1 sequence in which only 5 residues from the hVk5-2 sequence are inserted can bind calcium. Variants were generated in the same manner as described in Reference Example 16. The resulting LfVk1_Ca and LfVk1 light chain variants having the hVk1 light chain sequence (SEQ ID NO: 84) were co-expressed with the CIM_H heavy chain (SEQ ID NO: 67). Antibodies were expressed and purified in the same manner as described in Reference Example 16.

(17-2) Evaluation of antibodies having a human hVk1 sequence with a calcium ion binding motif for calcium ion binding activity

Whether the purified antibody prepared as described above binds calcium ions was evaluated by the method described in Reference Example 13. The results are shown in Table 47. The Tm value for the Fab domain of an antibody having LfVk1 with the hVk1 sequence did not vary, depending on the calcium concentration in antibody solutions. Meanwhile, the Tm of an antibody having the LfVk1_Ca sequence shifted by 1° C. or more when the calcium concentration in the antibody solutions changed. Thus, an antibody having LfVk1_Ca was shown to bind to calcium. The results described above demonstrate that the complete hVk5-2 CDR sequence is not required, while the residues introduced to construct the LfVk1_Ca sequence alone are sufficient for calcium ion binding.

(17-3) Construction, expression and purification of the degradation-resistant LfVk1_Ca sequence

As described in (17-1) of Reference Example 15, the LfVk1_Ca variant (SEQ ID NO: 66) was designed to have substitutions by residues at positions 30, 31, 32, 50, and 92 (Kabat numbering) in the sequence hVk5-2 residues at positions 30, 31, 32, 50 and 92 (Kabat numbering) in the hVk1 sequence (human germline sequence). The variant has been shown to bind to the calcium ion.

Thus, it is possible to construct Ca-libraries containing the LfVk1_Ca sequence. Meanwhile, there are no reports on the properties of the LfVk1_Ca sequence, and thus its feasibility was unknown. The LfVk1_Ca sequence has Asp at positions 30, 31, and 32 (Kabat numbering). Thus, its CDR1 sequence contains an Asp-Asp sequence that has been described to degrade under acidic conditions (J. Pharm. Biomed. Anal. (2008) 47(1): 23-30). It is desirable to avoid degradation under acidic conditions in terms of storage stability. Then, LfVk1_Cal (SEQ ID NO: 85), LfVk1_Ca2 (SEQ ID NO: 86), and LfVk1_Ca3 (SEQ ID NO: 87) variants were designed to have Ala (A) substitutions for three Asp (D) residues, which possibly are susceptible to degradation. Amino acid substitution was performed in a manner known to those skilled in the art using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The DNA encoding the variants was inserted into animal expression vectors. In combination with an animal expression vector having an insert for GC_H expression (SEQ ID NO: 51) as a heavy chain, engineered animal expression vectors carrying DNA inserts for the variants were introduced into animal cells by the method described in Reference Example 13. Antibodies expressed in animal cells into which the vectors were introduced were purified by the method described in Reference Example 13.

(17-4) Evaluation of the stability of antibodies having a degradation-resistant LfVk1_Ca sequence

Whether the antibodies prepared as described in (17-3) of Reference Example 17 were more resistant to degradation in solutions at pH 6.0 than the original antibodies having the LfVk1_Ca sequence to be modified was judged by comparing the heterogeneity between corresponding antibodies after thermal acceleration. Each antibody was subjected to dialysis against a solution of 20 mm histidine-HCl, 150 mm NaCl (pH 6.0) at 4°C during the night. The dialyzed antibodies were adjusted to 0.5 mg/ml and stored at 5°C or 50°C for three days. Each antibody after storage was subjected to ion exchange chromatography using the method described in Reference Example 14. As shown in FIG. 53, the result of the analysis demonstrates that LfVk1_Ca1 with a change in the degradation site was less heterogeneous and significantly more resistant to thermal degradation than the original LfVk1_Ca sequence. In particular, degradation was shown to occur at the Asp(D) residue at position 30 of the LfVk1_Ca sequence, but could be prevented by changing the amino acid.

(17-5) Construction of an LfVk1_Ca light chain sequence resistant to degradation at the Asp residue at position 30, and antibody expression and purification

The result described in (17-4) of Reference Example 17 regarding degradation resistance of the Ala-substituted form demonstrates that, under acidic conditions, the LfVk1_Ca sequence degraded at the Asp(D) residue at position 30 (Kabat numbering) in its CDR sequence and degraded could have been prevented by substitution of a different amino acid (at (17-4), by substitution of an A1a (A)) residue at position 30 (Kabat numbering). Next, the inventors of the present invention investigated whether a stable sequence with substitution even for Ser (S), a calcium ion-chelating residue, of the residue at position 30 (Kabat numbering) (in relation to LfVk1_Ca6; SEQ ID NO: 88) is resistant to degradation at maintaining calcium binding activity. Variants were generated by the method described in Reference Example 9. Altered light chains of the LfVk1 Ca6 and LfVk1_Ca sequences were expressed in combination with the GC_H heavy chain (SEQ ID NO: 51). Antibodies were expressed and purified in the same manner as described in Example 16.

(17-6) Evaluation of the LfVk1_Ca light chain sequence resistant to degradation at the Asp residue at position 30

The purified antibodies prepared as described above were evaluated for their storage stability under acidic conditions by the method described in (17-4) of Reference Example 17. The result demonstrates that antibodies having the LfVk1_Ca6 sequence are more resistant to degradation than antibodies , having the original LfVk1_Ca sequence as shown in FIG. eighteen.

A study was then made as to whether antibodies having the sequence LfVk1_Ca and antibodies having the sequence LfVk1_Ca6 bind calcium ions in the manner described in Example 13. The result is shown in Table 48. Tm values of the Fab domains of antibodies having the sequence LfVk1_Ca and antibodies having the degradation-resistant LfVk1_Ca6 sequence shifted by 1°C or more with a change in the calcium concentration in antibody solutions.

[Reference Example 20] Construction of an antibody array (Ca library) with a calcium ion-binding motif embedded in the variable region to efficiently produce antibodies that bind to an antigen in a Ca concentration-dependent manner

Preferred calcium binding motifs include, for example, the hVk5-2 sequence and the CDR sequence, as well as residues at positions 30, 31, 32, 50 and 92 (Kabat numbering). Other calcium-binding motifs include the EF-hand motif, which is possessed by calcium-binding proteins (eg, calmodulin) and C-type lectin (eg, ASGPR).

The Ca-library consists of heavy and light chain variable regions. The human antibody sequence was used as the variable region and the calcium binding motif was introduced into the light chain variable region. The hVk1 sequence was chosen as the light chain variable region template for the insertion of the calcium binding motif. An antibody containing the LfVk1_Ca sequence obtained by inserting the hVk5-2 CDR sequence (one of the calcium-binding motifs) into the hVk1 sequence was shown to bind to calcium ions as shown in Reference Example 16. A plurality of amino acids were allowed to be in the template sequence, to diversify the antigen-binding molecules that make up the library. Positions exposed on the surface of the variable region that are likely to interact with the antigen were chosen as positions where a plurality of amino acids were allowed to be. In particular, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94, and 96 (Kabat numbering) were chosen as nonstrict residues.

The type and frequency of occurrence of amino acid residues were determined and subsequently allowed to appear. The frequency of occurrence of amino acid residues in the non-strict residues of the hVk1 and hVk3 sequences registered in the Kabat database (KABAT, EA ET AL.: "Sequence of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION) was analyzed. Based on the results of the analysis, the type of amino acids that were allowed to appear in the Ca-library was selected from the amino acids with a higher frequency of occurrence at each position. At this time, the amino acids that were determined to be low in frequency based on the analysis results were also selected to avoid deviating the properties of the amino acids. The frequency of occurrence of the selected amino acids was determined by referring to the results of the analysis in the Kabat database.

A Ca library containing a calcium binding motif with emphasis on sequence diversity such that it contains multiple amino acids at each non-motif residue was designed as a Ca library based on amino acids and frequency of occurrence as described above. Detailed designs of the Ca-library are presented in tables 1 and 2 (where the positions in each table correspond to Kabat numbering). In addition, with respect to the amino acids shown in Tables 1 and 2, if position 92 corresponding to Kabat numbering is Asn (N), position 94 may be Leu (L) instead of Ser (S).

[Reference Example 19] Obtaining a Ca Library

The antibody heavy chain variable region gene library was amplified by PCR using poly-A RNA derived from human PBMC and commercial human poly-A RNA, etc. as a matrix. As described in Reference Example 18, for the antibody light chain variable region, antibody light chain variable regions were engineered to increase the frequency of antibodies that retain the calcium binding motif and can bind to the antigen in a calcium concentration dependent manner. In addition, among the non-strict residues for amino acid residues other than the residues with the introduced calcium-binding motif, a library of antibody light chain variable regions with uniformly distributed amino acids with a high frequency of occurrence in natural human antibodies was constructed, referring to the information on the frequency of occurrence of amino acids in natural antibodies human (KABAT, E. A. ET AL.: "Seqeuences of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION). A combination of antibody heavy chain and light chain variable region gene libraries prepared as described above was inserted into a phagemid vector to construct a human antibody phage display library that presents Fab domains composed of human antibody sequences (Methods Mol Biol. (2002) 178, 87-100). The sequences of parts of antibody genes isolated from E. coli into which antibody gene libraries were introduced according to the method described in Reference Example 23 below were determined. The distribution of amino acids in the sequences of the isolated 290 types of clones and the model distribution of amino acids are shown in FIG. 55.

[Reference Example 20] Investigation of the calcium ion binding activity of the molecules contained in the Ca library

(20-1) Calcium ion binding activity of molecules contained in the Ca-library

As described in Reference Example 14, the hVk5-2 sequence that was shown to bind calcium ions is a sequence with a low frequency of occurrence in the germline sequence. Thus, it was considered insufficient to obtain a calcium-binding antibody from an antibody library consisting of human germline sequences or from B cells obtained by immunizing a mouse expressing human antibodies. As a result, a Ca-library was constructed. The presence or absence of a clone showing calcium binding was examined in the constructed Ca library.

(20-2) Expression and purification of antibodies

Clones of the Ca-library were inserted into expression plasmids of animal cells. Antibodies were expressed using the method described below. Cells derived from Freestyle 293-F human embryonic kidney cells (Invitrogen) were suspended in Freestyle 293 Expression Medium (Invitrogen) and plated at a cell density of 1.33×10 ⁶ cells/mL (3 mL) in each well of a plate with 6 cells. The resulting plasmids were introduced into cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for four days. In a manner known to those skilled in the art, antibodies were purified from culture supernatants prepared as described above using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). The absorbance of purified antibody solutions at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the absorbance determined by the PACE method (Protein Science (1995) 4: 2411-2423).

(20-3) Evaluation of the obtained antibodies in relation to their binding of calcium ions

Antibodies purified as described above were evaluated for their calcium ion binding in the manner described in Reference Example 6. The result is shown in Table 49. that the library contains molecules that bind to the calcium ion.

[Reference Example 21] Isolation of antibodies that bind to the IL-6 receptor in a Ca-dependent manner

(21-1) Obtaining antibody fragments that bind to an antigen in a Ca-dependent manner from a library by bead panning

The first selection from the engineered library of antibodies that bind to the IL-6 receptor in a Ca-dependent manner was carried out by enrichment with only antibody fragments having the ability to bind to the antigen (IL-6 receptor).

Phages were obtained from E. coli containing an engineered phagemid for phage display. For phage precipitation, 2.5 M NaCl/10% PEG was added to the culture medium of E. coli producing phages. The precipitated population of phages was diluted with TBS to obtain a solution with a phage library. Then BSA and CaCl ₂ were added to the solution with the phage library to bring the final concentration of BSA to 4% and the final concentration of calcium ions to 1.2 mM. With regard to the panning method, the present inventors refer to general panning methods using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001 ) 247 (1-2), 191-203 Biotechnol Prog (2002) 18(2) 212-20 Mol Cell Proteomics (2003) 2 (2) 61-9). The magnetic beads used were neutravidin-coated beads (Sera-Mag SpeedBeads Neutravidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labelled antigen was added to the resulting phage library solution to allow said phage library solution to be contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added and allowed to bind to antigen-phage complexes at room temperature for 15 minutes. The beads were washed once with 1 ml 1.2 mM CaCl ₂ /TBST (TBST containing 1.2 mM CaCl ₂ ) and then twice with 1 ml 1.2 mM CaCl ₂ /TBS (TBST containing 1.2 mM CaCl ₂ ). Then, the beads combined with 0.5 ml of 1 mg/ml trypsin were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand to collect the solution with phage. The collected solution with phage was added to 10 ml of E. coil strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). E. coli were infected with phages by culturing them with gentle agitation at 37° C. for one hour. Infected E. coli were plated in a 225 mm x 225 mm dish. Then, phages were collected from the culture medium of the seeded E. coli, obtaining a solution with a phage library.

In the second round of panning, phage enrichment was performed using antigen binding activity or Ca dependent binding activity as an index. Specifically, when enrichment was performed using antigen-binding capacity as an index, 40 pmol of biotin-labelled antigen was added to the resulting phage library solution to allow the phage library solution and antigen to be contacted at room temperature for 60 minutes. BSA-blocked magnetic beads were added and allowed to bind to antigen/phage complexes at room temperature for 15 minutes. The beads were washed three times with 1 ml 1.2 mM CaCl ₂ /TBST and then twice with 1.2 mM CaCl ₂ /TBS. Then, the beads, to which 0.5 ml of 1 mg/ml trypsin was added, were suspended at room temperature for 15 minutes. Immediately after that, the beads were separated using a magnetic stand to collect the solution with phage. To eliminate the ability of Fab-exposing phages to infect E. coil, the pIII protein (derived from the helper phage pIII protein) of non-Fab-expressing phages was digested by adding 5 μl of 100 mg/ml trypsin to the collected solution with phages. The collected solution with phages was added to 10 ml of E. coil strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). E. coli was cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coll. E. coli infected were inoculated into a 225 mm x 225 mm dish. Phages were then harvested from the culture medium of E. coli after inoculation to prepare a solution with a phage library.

When enrichment was performed using the Ca-dependent binding capacity as an index, 40 mol of biotin-labeled antigen was added to the resulting phage library solution to allow the phage library solution and antigen to be contacted at room temperature for 60 minutes. BSA-blocked magnetic beads were added and allowed to bind to antigen/phage complexes at room temperature for 15 minutes. The beads were washed with 1 ml 1.2 mM CaCl ₂ /TBST and 1.2 mM CaCl ₂ /TBS. Then, the beads to which 0.1 ml of 2 mM EDTA/TBS (TBS containing 2 mM EDTA) was added were suspended at room temperature. Immediately after that, the beads were separated using a magnetic stand to collect the solution with phage. To eliminate the ability of Fab-exposing phages to infect E. coil, the pIII protein (derived from the helper phage pIII protein) of non-Fab-expressing phages was digested by adding 5 μl of 100 mg/ml trypsin to the collected solution with phages. The collected solution with phages was added to 10 ml of E. coil strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). E. coli was cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coll. E. coli infected were inoculated into a 225 mm x 225 mm dish. Phages were then harvested from the culture medium of E. coli after inoculation to prepare a solution with a phage library.

(21-2) Evaluation by ELISA of phages

Phage-containing culture supernatants were harvested from single E. coli colonies prepared as described above according to the conventional method (Methods Mol. Biol. (2002) 178, 133-145).

Culture supernatant containing phages to which BSA and CaCl ₂ were added / subjected to ELISA as described below. A 96-well StreptaWell microtiter plate (Roche) was coated overnight with 100 μl of PBS containing biotin labeled antigen. Each well of said plate was washed with PBST to remove antigen, and then the wells were blocked with 250 μl of 4% BSA-TBS for 1 hour or longer. This plate, with the resulting culture supernatant added to each well from which 4% BSA-TBS had been removed, was allowed to stand intact at 37°C for 1 hour, allowing the phage-presenting antibody to bind to the antigen present in each well. To each well washed with 1.2 mM CaCl ₂ /TBST was added 1.2 mM CaCl ₂ /TBS or 1 mM EDTA/TBS. The tablet was allowed to stand intact for 30 minutes at 37°C for incubation. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS at 1.2 mM ionized calcium was added to each well and the plate was incubated for 1 hour. After washing with 1.2 mm CaCl ₂ /TBST chromogenic reaction of the solution in each well, which was added only solution of TMB (ZYMED), was stopped by adding sulfuric acid. Then the specified color was determined by measuring the absorbance at 450 nm.

For clones subjected to phage ELISA, the base sequences of genes amplified with specific primers were analyzed.

The result of phage ELISA and sequence analysis is shown in Table 50.

Clones that were determined to have Ca-dependent binding capability by phage ELISA were introduced into expression plasmids of animal cells. Antibodies were expressed using the following method. FreeStyle 293-F embryonic kidney-derived cells (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and seeded in each well of 6-well plates at a cell density of 1.33×10 ⁶ cells/ml (3 ml). The resulting plasmids were introduced into cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ / 90 rpm) for four days. From culture supernatants prepared as described above, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance at 280 nm of the purified antibody solutions was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(21-4) Evaluation of the ability of obtained antibodies to Ca-dependent binding to the human IL-6 receptor

Antibodies 6RC1IgG_010 (heavy chain SEQ ID NO: 111; light chain SEQ ID NO: 112), 6RC1IgG_012 (heavy chain SEQ ID NO: 113; light chain SEQ ID NO: 114) and 6RClIgG_019 (heavy chain, SEQ ID NO: 115; light chain, SEQ ID NO: 116) isolated as described above were evaluated for Ca dependency of their human IL-6 receptor binding activity by antibody-human IL-6 receptor interaction assay using Biacore T100 (GE Healthcare). Tocilizumab (heavy chain SEQ ID NO: 60; light chain SEQ ID NO: 61) was used as a control antibody that does not have Ca-dependent binding activity to the human IL-6 receptor. The interaction was analyzed in solutions at a calcium ion concentration of 1.2 mM and 3 μM, corresponding to high and low calcium ion concentration conditions, respectively. An appropriate amount of A/G protein (Invitrogen) was immobilized onto a CM5 sensor chip (GE Healthcare) by the amine addition method and antibodies of interest were captured on the chip. The two types of running buffers used were: 20 mM ACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl ₂ (pH 7.4); and 20 mM ACES/150 mM NaCl/0.05% (w/v) Tween20/3 μM CaCl ₂ (pH 7.4). Each of these buffers was used to dilute the human IL-6 receptor. All measurements were carried out at 37°C.

In an antigen-antibody interaction assay using the tocilizumab antibody as control antibody and 6RC1IgG_010 and 6RC1IgG_012 and 6RC1IgG_019 antibodies, a diluted IL-6 receptor solution and running buffer as blank were injected at a flow rate of 5 μl/min for three minutes, to allow the IL-6 receptor to interact with the antibodies tocilizumab, 6RC1IgG 010 and 6RC1IgG 012 and 6RC1IgG 019 captured on the sensor chip. Then, 10 mM glycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30 seconds to regenerate the sensor chip.

Sensograms at a high concentration of calcium ions, obtained by measuring using the method described above, are shown in FIG. 56.

Under conditions of low concentration of calcium ions, sensograms of antibodies tocilizumab, 6RC1IgG_010 and 6RC1IgG_012 and 6RC1IgG_019 were also obtained in a similar way. Sensorgrams at low concentration of calcium ions are presented in Fig. 57.

The results described above demonstrate that the ability of the antibodies 6RC1IgG_010 and 6RC1IgG_012 and 6RC1IgG_019 to bind the IL6 receptor was significantly reduced when the calcium ion concentration in the buffer was changed from 1.2 mM to 3 μM.

[Reference Example 22] Construction of an antibody library with pH-dependent binding

(22-1) Method for producing antibodies with pH-dependent binding

WO2009/125825 describes an antibody with pH-dependent antigen binding, the properties of which change in the region of neutral and acidic pH values, due to the introduction of histidine into the antigen-binding molecule. The disclosed pH dependent binding antibody is prepared by modifying to replace a portion of the amino acid sequence of the antigen binding molecule of interest with histidine. To more efficiently produce an antibody with pH-dependent binding without first obtaining the antigen-binding molecule of interest to be modified, one way may be to obtain an antigen-binding molecule that binds to the desired antigen from a pool of antigen-binding molecules (referred to as a His-library) with histidine added into the variable region (more preferably, the region potentially involved in antigen binding). It may be possible to efficiently obtain an antigen-binding molecule having the desired properties from a His library, since histidine occurs more frequently in antigen-binding molecules from a His library than from conventional antibody libraries.

(22-2) Construction of a population of antibody molecules (His libraries) with a histidine residue introduced into their variable region to efficiently obtain binding antibodies that bind to the antigen in a pH-dependent manner

First, the insertion positions of histidine in the His library were chosen. WO 2009/125825 describes the production of antibodies with pH-dependent antigen binding by replacing amino acid residues in the sequences of anti-IL-6 receptor antibody, anti-IL-6 antibody and anti-IL-31 receptor antibody with histidine. In addition, an antibody against egg white lysozyme (FEBS Letter 11483, 309, 1, 85-88) and an antibody against hepcidin (WO 2009/139822) with the ability of pH-dependent antigen binding were obtained by replacing the antigen-binding molecule in the amino acid sequence with histidine . The positions where histidine residues were added to anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-IL-31 receptor antibody, anti-egg white lysozyme antibody, and anti-hepcidin antibodies are shown in Table 51. The positions shown in Table 51 may be cited as candidate positions that control antigen-antibody binding.

In addition, in addition to the positions presented in table 51, positions that are likely to come into contact with the antigen were also considered suitable for introducing histidine residues.

In a His library consisting of heavy chain and light chain variable regions, a human antibody sequence was used as the heavy chain variable region, and histidine residues were introduced into the light chain variable region. The positions above and the positions that may be involved in antigen binding, ie. positions 30, 32, 50, 53, 91, 92, and 93 (numbered by Kabat, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) in the light chain were selected as positions for adding histidine residues to the His library. In addition, the Vk1 sequence was selected as a light chain variable region template sequence for the introduction of histidine residues. A variety of amino acids were allowed to appear in the template sequence to diversify the antigen-binding molecules that make up the library. Positions exposed on the surface of the variable region that are likely to interact with the antigen were selected as positions where multiple amino acids were allowed to appear. In particular, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94 and 96 of the light chain were chosen as non-strict residues (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest .NIH).

The type and frequency of occurrence of amino acid residues were determined and subsequently allowed to appear. The frequencies of amino acids in non-strict residues in the hVk1 and hVk3 sequences registered in the Kabat database (KABAT, E.A. ET AL.: "Sequences of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION) were analyzed. Based on the results of the analysis, the type of amino acids allowed to appear in the His library was selected from the amino acids with a higher frequency of occurrence at each position. At the same time, amino acids for which it was determined that the frequency of their occurrence is low, based on the results of the analysis, were also selected in order to avoid bias in the properties of amino acids. The frequency of occurrence of the selected amino acids was determined by referring to the results of the analysis in the Kabat database.

As His libraries, His library 1 was constructed, which is fixed to necessarily include one histidine residue in each CDR, and His library 2, which is more focused on sequence diversity than His library 1, considering amino acids and frequency of occurrence, as described above. Detailed constructions of His libraries 1 and 2 are presented in Tables 3 and 4 (where the positions in each table corresponded to the Kabat numbering). With respect to the frequency of occurrence of amino acids, as described in Tables 3 and 4, Ser (S) at position 94 can be omitted if Asn (N) is at position 92 corresponding to Kabat numbering.

[Reference Example 23] Preparation of a phage display library for human antibodies (His library 1) to obtain an antibody that binds to an antigen in a pH-dependent manner.

The antibody heavy chain variable region gene library was amplified by PCR using poly-A RNA derived from human PBMC and commercial human poly-A RNA as a template. An antibody light chain variable gene library constructed as His library 1 as described in Reference Example 22 was amplified using PCR. The combination of the antibody heavy chain and light chain variable region gene libraries prepared as described above was inserted into a phagemid vector to construct a human antibody phage display library that presents Fab domains composed of human antibody sequences. For the construction method, (Methods Mol Biol. (2002) 178, 87-100) were used as references. To construct the library, we used the linker part connecting the phagemid Fab with the phage pIII protein, and the sequences of the phage display library with a trypsin cleavage sequence inserted between the N2 and CT domains of the helper phage pIII protein gene. The sequences of portions of antibody genes isolated from E. coli into which the antibody gene libraries were introduced were identified and sequence information was obtained for 132 clones. The intended distribution of amino acids and the distribution of amino acids in the identified sequences is shown in FIG. 58. A library was constructed containing various sequences corresponding to the intended distribution of amino acids.

[Reference Example 24] Isolation of antibodies that bind to IL-6R in a pH dependent manner

(24-1) Isolation of antibody fragments that bind to antigens in a pH dependent manner from a library by bead panning

The first selection from the constructed His-library 1 was carried out by enrichment only with fragments of antibodies with the ability to bind antigen (IL-6R).

Phages were obtained using E. coil containing engineered phagemids for phage display. For phage precipitation, 2.5 M NaCl/10% PEG was added to the E. coli culture broth for phage production. The precipitated phage population was diluted with TBS to give a phage library solution. BSA and CaCl ₂ were added to the phage library solution to bring the final concentration of BSA to 4% and the final concentration of calcium ions to 1.2 mM. With regard to the panning method, the present inventors used general panning methods using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203 Biotechnol Prog (2002) 18(2) 212-20, Mol Cell Proteomics (2003) 2 (2), 61-9. The magnetic beads used were NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to the resulting phage library solution to ensure that said phage library solution was contacted with the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added and allowed to bind to antigen/phage complexes at room temperature for 15 minutes. Beads were washed three times with 1 ml 1.2 mM CaCl ₂ /TBST (TBS containing 1.2 mM CaCl ₂ and 0.1% Tween20) and then twice with 1 ml 1.2 mM CaCl ₂ /TBS (pH 7.6). Then, the beads, to which 0.5 ml of trypsin at a concentration of 1 mg/ml was added, were suspended at room temperature for 15 minutes, and then immediately separated using a magnetic stand to collect the solution with phage. The collected solution with phage was added to 10 ml of E. coil strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). E. coli were infected with phages by culturing them with gentle agitation at 37° C. for one hour. Infected E. coli were plated in a 225 mm x 225 mm dish. Then, phages were collected from the culture liquid of the seeded E. coli, obtaining a solution with a phage library.

For phage enrichment, the second and subsequent rounds of panning were performed using antigen-binding capacity or pH-dependent binding capacity as an indicator. Specifically, 40 pmol of biotin labeled antigen was added to the resulting phage library solution to allow the solution to contact the phage library and antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added and allowed to bind to the antigen/phage complexes at room temperature for 15 minutes. The beads were washed several times with 1 ml of 1.2 mM CaCl ₂ /TBST and 1.2 mM CaCl ₂ /TBS. Then, when enriched with phages using antigen-binding capacity as an indicator, the beads to which 0.5 ml of trypsin at a concentration of 1 mg/ml was added were suspended at room temperature for 15 minutes, and then immediately separated using a magnetic collection stand. solution with phages. Alternatively, when enriched with phages using pH-dependent antigen binding capacity as an index, beads to which 0.1 ml of 50 mM MES/1.2 mM CaCl ₂ /150 mM NaCl (pH 5.5) was added were suspended at room temperature, and then immediately separated using a magnetic stand to collect the solution with phage. To eliminate the ability of non-Fab-exposing phages to infect E. coli, the pIII protein (derived from the helper phage pIII protein) of non-Fab-expressing phages was digested by adding 5 μl of trypsin at a concentration of 100 mg/ml to the collected solution with phage. Harvested phages were added to 10 ml of E. coil strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). E. coli were infected with phages by culturing them with gentle agitation at 37° C. for one hour. Infected E. coli were plated in a 225 mm x 225 mm plate. Phages were then harvested from the culture medium of the seeded E. coli to collect the phage library solution. Panning using antigen-binding capacity or pH-dependent binding capacity as an index was repeated twice.

(24-2) Evaluation by ELISA of phages

The content of phage culture supernatants was collected according to the conventional method (Methods Mol. Biol. (2002) 178, 133-145) from individual E. coli colonies obtained by the method described above.

BSA and CaCl ₂ were added to culture supernatants containing phage at a final concentration of 4% BSA and a final concentration of calcium ions of 1.2 mM. These phage-containing culture supernatants were subjected to ELISA according to the following procedure. A StreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μl of PBS containing biotin labeled antigen. After washing each well of the plate with PBST (PBS containing 0.1% Tween 20) to remove the antigen, the wells were blocked with 250 μl of 4% BSA/TBS for one hour or more. After removal of 4% BSA/TBS, the resulting supernatants were added to each well. The antibodies on the phages were allowed to bind to the antigens on each well by incubating the plate at 37° C. for one hour. After washing with 1.2 mM CaCl ₂ /TBST, 12, mM CaCWTBS (pH 7.6) or 1.2 mM CaCl ₂ /TBS (pH 5.5) was added to each well. The tablet was incubated at 37°C for 30 minutes. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS containing 4% BSA and 1.2 mM ionized calcium was added to each well. The plate was incubated for one hour. After washing with 1.2 mM CaCl ₂ /TBST, a simple TMB solution (ZYMED) was added to each well. The chromogenic reaction in the solution of each well was stopped by adding sulfuric acid, and then the absorbance at 450 nm was measured to assess the development of color.

When enrichment was performed using antigen-binding capacity as an index, ELISA of the phages after two rounds of panning showed that 17 of 96 clones were ELISA positive in an antigen-specific manner. Thus, clones were analyzed after three rounds of panning. Meanwhile, when enrichment was performed using pH-dependent antigen-binding capacity as an index, ELISA of the phages after two rounds of panning showed that 70 out of 94 clones were ELISA positive. Thus, clones were analyzed after two rounds of panning.

For clones subjected to the phage ELISA described above, the base sequence of the gene amplified with specific primers was analyzed. The results of the phage ELISA and sequence analysis are shown in Table 52 below.

In the same way, antibodies with the ability to pH-dependent antigen binding were isolated from the phage display library of native human antibodies. When enrichment was performed using antigen-binding capacity as an index, 13 types of antibodies with pH-dependent binding were isolated from 88 clones examined. Meanwhile, when enrichment was carried out using pH-dependent antigen-binding ability as an index, 27 types of antibodies with pH-dependent binding were isolated from 83 examined clones.

The result described above demonstrated that the variation of clones with pH-dependent antigen binding capacity from His library 1 was higher compared to the naïve human antibody phage display library.

(24-3) Expression and purification of antibodies

Clones judged to have pH-dependent antigen-binding capability based on phage ELISA results were introduced into animal cell expression plasmids. Antibodies were expressed using the method described below. Freestyle 293-F embryonic kidney-derived cells (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and plated at a cell density of 1.33×10 ⁶ cells/ml (3 ml) in each well. tablet with 6 cells. The resulting plasmids were introduced into cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for four days. From culture supernatants prepared as described above, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance of purified antibody solutions was measured at 280 nm using a spectrophotometer. Antibody concentrations were calculated from the measured values using the absorbance determined by the PACE method (Protein Science (1995) 4, 2411-2423).

(24-4) Evaluation of isolated antibodies for their ability to bind pH-dependently to the human IL-6 receptor

Antibodies 6RpH#01 (heavy chain SEQ ID NO: 117; light chain SEQ ID NO: 118), 6RpH#02 (heavy chain SEQ ID NO: 119; light chain SEQ ID NO: 120), and 6RpH#03 (heavy chain SEQ ID NO: 121; light chain SEQ ID NO: 122) isolated as described in (24-3) were evaluated for pH dependence of their human IL-6 receptor binding activity by analyzing the interaction between antibodies and IL-6 receptor human using Biacore T100 (GE Healthcare). Tocilizumab (heavy chain SEQ ID NO: 60; light chain SEQ ID NO: 61) was used as a control antibody that does not have pH-dependent binding activity to the human IL-6 receptor. The interaction for the antigen-antibody reaction was analyzed in solutions at pH 7.4 and pH 6.0, corresponding to neutral pH and acidic pH conditions, respectively. An appropriate amount of A/G protein (Invitrogen) was immobilized onto a CM5 sensor chip (GE Healthcare) by the amine addition method and approximately 300 RU of each of the antibodies of interest were captured on the chip. The two types of running buffers used were: 20 mM ACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl ₂ (pH 7.4); and 20 mM ACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl ₂ (pH 6.0). Each of these buffers was used to dilute the human IL-6 receptor. All measurements were carried out at 37°C.

In an antigen-antibody interaction assay using tocilizumab as control antibody and antibodies 6RpH#01, and 6RpH#02, and 6RpH#03, diluted IL-6 receptor solution and running buffer as blank were injected at a flow rate of 5 µl /min for three minutes to allow the IL-6 receptor to interact with the tocilizumab, 6RpH#01, and 6RpH#02, and 6RpH#03 antibodies captured on the sensor chip. Then 10 mM glycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30 seconds to regenerate the sensor chip.

Sensorgrams at pH 7.4 measured using the method described above are shown in FIG. 59. Sensograms under pH 6.0 conditions obtained by the same method are presented in FIG. 60.

The result described above shows that the IL-6 receptor binding ability of antibodies 6RpH#01, 6RpH#02 and 6RpH#03 was significantly reduced when the pH of the buffer was changed from pH 7.4 to pH 6.0.

(Reference Example 25) Method for producing a human FcγR and method for analyzing the interaction between an altered antibody and a human FcγR

The extracellular domains of human FcγR were obtained by the following method. First, the FcγR extracellular domain gene was synthesized in a manner well known to those skilled in the art. At the same time, the sequence of each FcγR was obtained based on the information registered with the NCBI. Specifically, FcγRI was generated based on NCBI Accession #NM_000566 (Version #NM_000566.3), FcγRIIa was generated based on NCBI Accession #NM_001136219 (Version #NM 001136219.1), FcγRIIb was generated based on NCBI Accession #NM_000566.3. NM_004001 (Version #NM_004001.3), FcγRIIIa was generated based on the sequence with NCBI Accession #NM_001127593 (Version #NM_001127593.1) and FcγRIIIb was generated based on the sequence with NCBI Accession #NM_000570 (Version #NM 000570.3) and to the C-terminus attached His-tag. Moreover, the presence of polymorphism for FcγRIIa, FcγRIIIa and FcγRIIIb is known and polymorphic regions were obtained according to Warmerdam et al. (J. Exp. Med., 1990, 172: 19-25) for FcγRIIa; Wu et al. (J. Clin. Invest., 1997, 100 (5): 1059-1070) for FcγRIIIa; and Ory et al. (J. Clin. Invest., 1989, 84, 1688-1691) for FcγRIIIb.

Expression vectors were constructed by inserting the resulting gene fragments into expression vectors of animal cells. The engineered expression vectors were transiently introduced into cells derived from human embryonic kidney cancer cells, FreeStyle293 (Invitrogen) to express proteins of interest. FcγRIIb for use in crystallographic analysis was prepared so that the sugar chain associated with FcγRIIb was a high mannose sugar chain by expression of the protein of interest in the presence of kyfunesin at a final concentration of 10 μg/ml. Fluids obtained by filtering through a 0.22 μm filter of culture supernatants obtained from the culture medium of the above-described cells subjected to transient administration were purified mainly by the following four steps:

first step: cation exchange column chromatography (SP Sepharose FF);

second step: His tag affinity column chromatography (HisTrap HP);

third stage: gel filtration column chromatography (Superdex200); and

fourth stage: sterile filtration.

Anion exchange column chromatography with Q sepharose FF was used as a first step to purify FcγRI. The absorbance of the purified protein was measured at 280 nm using a spectrophotometer. Based on the measured values, the concentrations of purified proteins were measured using an extinction coefficient determined in a manner such as PACE (Protein Science (1995) 4, 2411-2423).

The interaction between each antibody variant and the Fcγ receptor prepared as described above was analyzed using Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100 and Biacore 4000. The running buffer used was HBS-EP+ (GE Healthcare ) and the measurement temperature was 25°C. The immobilized chip used was either a CM5 Series S sensor chip (GE Healthcare) or a CM4 Series S sensor chip (GE Healthcare) onto which an antigenic peptide, protein A (Thermo Scientific), protein A/G (Thermo Scientific) or protein was immobilized. L (ACTIGEN or BioVision) by any method of addition of amines. Alternatively, the immobilized antigen chip was: SA Series S (certified) sensor chip (GE Healthcare) onto which a prebiotinylated antigenic peptide was immobilized by peptide-chip interaction.

Antibodies of interest were captured on these sensor chips and the Fcγ receptor diluted with running buffer was allowed to interact with them. Antibody-binding amounts were measured and compared between antibodies. Since the Fcγ receptor binding amount depends on the amount of antibody captured, comparisons were made with normalized values obtained by dividing the Fcγ receptor binding amount by the amount of each antibody captured. Meanwhile, 10 mM glycine-HCl (pH 1.5) was subjected to a reaction to wash off the captured antibody from the sensor chip, and thus the sensor chip was regenerated for reuse.

Meanwhile, the KD value of each FcγR antibody variant was subjected to kinetic analysis by the following method. First, the antibodies of interest were captured on a sensor chip and the Fcγ receptor diluted with running buffer was allowed to react with them. The measurement results from the obtained sensograms were processed by a global fit according to the 1:1 Langmuir binding model using the Biacore Evaluation software. From the values of the binding rate constant ka (l/mol/s) and the dissociation rate constant kd (1/s), the dissociation constant KD (mol/l) was determined.

When the interaction between each of the altered antibodies and FcγR was weak and it was determined by the above kinetic analysis that a correct analysis was not possible, the KD for such interactions was calculated using the following 1:1 binding model equation described in the Biacore T100 Software Handbook BR1006-48 Edition AE.

The behavior of interacting molecules according to the 1:1 binding model on Biacore can be described using Equation 4 below.

[Equation 4]

R _eq = _C⋅Rmax /(KD+C)+RI,

Req: steady state binding levels versus analyte concentration

C: concentration

RI: cumulative contribution of the refractive index to the sample

Rmax: the ability of the surface to bind to the analyzed compound

After transforming this equation, KD can be expressed as Equation 2 below.

[Equation 2]

KD=C⋅R _max /(R _eq -RI)-C

KD can be calculated by assigning Rmax, RI and C in this equation. RI and C are determined from the measurement result on the sensorgram and the change conditions. Rmax was calculated in the following way. For a comparable antibody with a sufficiently strong interaction that is simultaneously assessed during the measurement period, the Rmax value obtained by a global approximation according to the 1:1 Langmuir binding model mentioned above was divided by the amount of the comparison antibody captured on the sensor chip, and then multiplied the amount of captured modified antibody to be evaluated. The resulting value was used as Rmax.

The extracellular domain of mouse FcγR was obtained by the following method.

First, genes for the extracellular domains of FcγR were synthesized in a manner known to those skilled in the art. Genes were designed based on the sequence information for each FcγR registered with the NCBI. In particular, mFcγRI was generated based on the NCBI reference sequence:

NP_034316.1; mFcγRII was generated based on the NCBI reference sequence: NP_034317.1; mFcγRIII was generated based on the NCBI reference sequence: NP 034318.2; and mFcγRIV were generated based on the NCBI reference sequence:

NP_653142.2; and a His tag was added to their C-terminus.

Culture supernatants obtained from the culture medium for the cells described above, subjected to temporary introduction, were filtered through a 0.22 μm filter. The resulting liquids were purified mainly by the following four steps:

first step: cation exchange column chromatography (SP Sepharose FF);

second step: His tag affinity column chromatography (HisTrap HP);

third stage: gel filtration column chromatography (Superdex200); and

fourth stage: sterile filtration.

Meanwhile, in the purification of FcγRI, anion exchange column chromatography with Q Sepharose FF was used in the first step. The absorbance of the purified proteins was measured at 280 nm using a spectrophotometer. Based on the measured values, the concentrations of purified proteins were calculated using an extinction coefficient by a method such as PACE (Protein Science (1995) 4, 2411-2423).

Expression vectors were constructed by inserting the obtained gene fragments into expression vectors of animal cells. The engineered expression vectors were transiently introduced into cells derived from a human embryonic kidney cancer cell, FreeStyle293 (Invitrogen) to express the proteins of interest. Culture supernatants obtained from the culture media of the cells described above, subjected to temporary introduction, were filtered through a 0.22 μm filter.

The resulting liquids were purified mainly by the following four steps:

first stage: ion-exchange column chromatography;

second step: His tag affinity chromatography (HisTrap HP);

third stage: gel filtration column chromatography (Superdex200); and

fourth stage: sterile filtration.

Meanwhile, for ion exchange column chromatography, Q Sepharose HP was used in the first step of mFcγRI purification; mFcγRII and mFc*yRIV were purified using SP Sepharose FF; and mFcγRIII was purified using SP Sepharose HP. In the third and subsequent steps D-PBS(-) was used as the solvent, and in the purification of mFcγRIII the solvent used was D-PBS(-) containing 0.1 M arginine. The absorbance of the purified proteins was measured at 280 nm using a spectrophotometer. Based on the measured values, the concentrations of purified proteins were calculated using an extinction coefficient determined by a method such as PACE (Protein Science (1995) 4, 2411-2423).

[Reference Example 27] Change to suppress binding to rheumatoid factor

(27-1) Inhibition of rheumatoid factor binding by Fv4-YTE, Fv4-N434H and LS variants

To suppress rheumatoid factor binding, Fv4-YTE, Fv4-N434H, and LS variants, in which plasma retention time was increased due to increased FcRn binding in the acidic pH range, were modified with Q438R/S440E or S424N. In particular, new Fc variants were generated as shown in Table 53. First, the variants were evaluated for their FcRn binding affinity at pH 6.0. The result is presented in table 53.

Variants (Fv4-F1166, F1167, F1172, F1173, F1170 and F1171) were then evaluated for their binding to rheumatoid factor. Rheumatoid factor binding assay was performed at pH 7.4 by electrochemiluminescence (ECL) method. Sera (Protogenex) from 15 or 30 patients with rheumatoid arthritis were used in this assay. Serum samples diluted 50-fold were mixed with biotin labeled test antibodies (1 μg/ml) and SULFO-TAG NHS-ester labeled test antibodies (1 μg/ml). The mixtures were incubated at room temperature for three hours. The mixtures were then aliquoted into each well of a streptavidin-coated 96-well MULTI-ARRAY plate (Meso Scale Discovery). It was incubated for two hours at room temperature, each well of the plate was washed and read buffer was aliquoted (×4). The plate was placed in a SECTOR imager 2400 Reader (Meso Scale Discovery) to measure chemiluminescence in each well.

The result is shown in Fig. 61. The rheumatoid factor binding of F1166 (Q438R/S440E) and F1167 (S424N) variants was significantly suppressed compared to the YTE variant, which exhibited high rheumatoid factor binding activity in the sera of 90216S and 90214S donors. With respect to the F1173 and F1171 variants containing the S424N modification, their rheumatoid factor binding, which occurred in the N434H and LS variants, respectively, was significantly suppressed. However, the Q438R/S440E change could not completely suppress rheumatoid factor binding provided by the N434H and LS variants, and rheumatoid factor binding was observed in the serum of one or two donors.

(27-2) Inhibition of binding to rheumatoid factor variant LS As shown in Table 54, Fc variants were generated by introducing a new change in Fv4-LS. Among them, variants (Fv4-F1380, F1384-F1386, F1388 and F1389) that retain FcRn binding at pH 6.0 were evaluated for their binding to rheumatoid factor according to the method described in Example (27-1). The result is shown in Fig. 62. These variants showed significantly suppressed binding to rheumatoid factor in donor sera. In particular, binding to rheumatoid factor Fv4-F1389 containing Y436T was comparable to native IgGl.

As shown above, it is possible to obtain antigen-binding molecules that do not have rheumatoid factor binding activity and whose binding activity to human FcRn is increased under acidic pH range conditions by introducing a modification in the Fc region region that reduces the binding activity to rheumatoid factor alone, without reducing FcRn binding activity under acidic pH range conditions.

Such changes that reduce rheumatoid factor binding activity include changes at positions 248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439 and 441-444 (EU numbering). ). Preferably, changes to provisions 387, 422, 424, 426, 433, 436, 438 and 440 (EU numbering) are used. Particularly preferably, a change by changing Val to Glu or Ser at position 422, a change by changing Ser to Arg at position 424, a change by changing His to Asp at position 433, a change by changing Tyr to Thr at position 436, a change by changing Gin to Arg or Lys at position 438 and change by replacing Ser with Glu or Asp at position 440 (EU numbering). These modifications can be used alone, or multiple sections can be used in combination.

Alternatively, an N-linked chain attachment sequence can be introduced to reduce rheumatoid factor binding activity. In particular, Asn-Xxx-Ser/Thr (Xxx corresponds to an arbitrary amino acid other than Pro) is known as the attachment sequence of the N-linked sugar chain, and this sequence can be introduced into the EU region to attach the N-linked sugar chain, steric hindrance N-linked chain sugars can inhibit RF binding. For changes to add an N-linked sugar chain, change by changing Lys to Asn at position 248, changing by changing Ser to Asn at position 424, changing by changing Tyr to Asn at position 436 and Gln to Thr at position 438, and changing by changing Qln to Asn at position 438 (EU numbering). Particularly preferably, a change is used by replacing Ser with Asn at position 424 (EU numbering). Industrial Applicability

The present invention relates to methods for enhancing the uptake of antigens into cells by antigen-binding molecules, methods for increasing the amount of antigens to which a single antigen-binding molecule can bind, and methods for reducing plasma antigen concentration by administering them. By enhancing the uptake of antigens into cells by antigen-binding molecules, it is possible to enhance the decrease in plasma antigen concentration by administering antigen-binding molecules, and also improve the pharmacodynamics of antigen-binding molecules and increase the number of antigens to which one antigen-binding molecule can bind. Thus, antigen-binding molecules can exhibit better in vivo effects than conventional antigen-binding molecules.

Claims (502)

1. An antigen-binding molecule for eliminating antigens from plasma, comprising an antigen-binding domain and a variant of the Fc region of human IgG, where the antigen-binding molecule has the activity of binding to human FcRn under conditions of an acidic pH range, where the antigen-binding domain is the variable region of an antibody with at least one histidine and the antigen-binding domain has an antigen-binding activity that varies with pH, where a KD (pH 5.8) / KD (pH 7.4) value of 2 or more is the ratio of the KD for an antigen under acidic pH range conditions to KD under neutral pH range conditions, and the Fc region variant has higher activity at at least one of the Fcγ receptors under conditions of the neutral pH range than the native human IgG Fc region in which the sugar chain attached at position 297 according to EU numbering is chain of sugars containing fucose, where an antigen is a soluble antigen whose plasma concentration must be reduced. 2. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule has neutralizing activity against the antigen. 3. The antigen-binding molecule according to claim 1, where the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 324, 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region. 4. The antigen-binding molecule according to claim 3, wherein the Fc region is an Fc region that contains at least one or more amino acids selected from the group consisting of: either Lys or Tyr at amino acid position 221; any of Phe, Trp, Glu and Tyr at amino acid position 222; any of Phe, Trp, Glu and Lys at amino acid position 223; any of Phe, Trp, Glu and Tyr at amino acid position 224; any of Glu, Lys and Trp at amino acid position 225; any of Glu, Gly, Lys and Tyr at amino acid position 227; any of Glu, Gly, Lys and Tyr at amino acid position 228; any of Ala, Glu, Gly and Tyr at amino acid position 230; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231; any of Glu, Gly, Lys and Tyr at amino acid position 232; any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239; any of Ala, Ile, Met and Thr at amino acid position 240; any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241; any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243; His at amino acid position 244; Ala at amino acid position 245; any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247; any of Glu, His, Gln and Tyr at amino acid position 249; either Glu or Gln at amino acid position 250; Phe at amino acid position 251; any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258; any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262; any of Ala, Ile, Met and Thr at amino acid position 263; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264; any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265; any of Ala, Ile, Met and Thr at amino acid position 266; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267; any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269; any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271; any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272; either Phe or Ile at amino acid position 273; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274; either Leu or Trp at amino acid position 275; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276; any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278; Ala at amino acid position 279; any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280; any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282; any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283; any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284; any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285; any of Glu, Gly, Pro and Tyr at amino acid position 286; any of Asn, Asp, Glu and Tyr at amino acid position 288; any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290; any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291; any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292; any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Ile at amino acid position 302; any of Asp, Gly and Tyr at amino acid position 303; any of Asp, His, Leu, Asn and Thr at amino acid position 304; any of Glu, Ile, Thr and Tyr at amino acid position 305; any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311; Phe at amino acid position 313; Leu at amino acid position 315; either Glu or Gln at amino acid position 317; any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318; any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320; any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322; Ile at amino acid position 323; any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325; any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329; any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330; any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331; any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334; any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335; any of Glu, Lys and Tyr at amino acid position 336; any of Glu, His and Asn at amino acid position 337; any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339; either Ala or Val at amino acid position 376; either Gly or Lys at amino acid position 377; Asp at amino acid position 378; Asn at amino acid position 379; any of Ala, Asn and Ser at amino acid position 380; either Ala or Ile at amino acid position 382; Glu at amino acid position 385; Thr at amino acid position 392; Leu at amino acid position 396; Lys at amino acid position 421; Asn at amino acid position 427; either Phe or Leu at amino acid position 428; Met at amino acid position 429; Trp at amino acid position 434; Ile at amino acid position 436; and any of Gly, His, Ile, Leu and Tyr at amino acid position 440, in the region of the Fc region according to the EU numbering. 5. Antigen-binding molecule according to any one of paragraphs. 1, 3, 4, where the Fc region of native human IgG, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose, is the Fc region of any native human IgG1, native human IgG2 , native human IgG3, and native human IgG4, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose. 6. Antigen-binding molecule according to any one of paragraphs. 1, 3, 4, wherein the human Fcγ receptor is at least one of the Fcγ receptors selected from the group consisting of FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), and FcγRIIIa(F) . 7. Antigen-binding molecule according to any one of paragraphs. 1, 3, 4, where the human Fcγ receptor is FcγRIIb. 8. A method of reducing total or free antigen plasma concentration, wherein the method comprises the step of contacting an antigen-binding molecule with an Fcγ receptor-expressing cell in vivo or ex vivo , wherein the antigen-binding molecule has human FcRn-binding activity under acidic pH range conditions, wherein the antigen-binding molecule the domain is the variable region of an antibody with at least one histidine, and the antigen-binding domain has an antigen-binding activity that varies with pH, where a KD (pH 5.8)/KD (pH 7.4) value of 2 or more represents is the ratio of the KD for an antigen under acidic pH range conditions to the KD at neutral pH range conditions, and an Fcγ receptor binding domain that contains a variant human IgG antibody Fc region has higher binding activity to at least one of the Fcγ receptors under neutral pH range conditions compared to the Fc region of a native Human IgG, in which the sugar chain attached at position 297 according to EU numbering, is a sugar chain containing fucose, where the antigen is a soluble antigen whose plasma concentration needs to be reduced. 9. The method of claim 8, wherein the antigen-binding molecule has neutralizing activity against the antigen. 10. The method of claim 8, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 , 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324 , 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region. 11. The method of claim 10, wherein the Fc region is an Fc region that contains at least one or more amino acids selected from the group consisting of: either Lys or Tyr at amino acid position 221; any of Phe, Trp, Glu and Tyr at amino acid position 222; any of Phe, Trp, Glu and Lys at amino acid position 223; any of Phe, Trp, Glu and Tyr at amino acid position 224; any of Glu, Lys and Trp at amino acid position 225; any of Glu, Gly, Lys and Tyr at amino acid position 227; any of Glu, Gly, Lys and Tyr at amino acid position 228; any of Ala, Glu, Gly and Tyr at amino acid position 230; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231; any of Glu, Gly, Lys and Tyr at amino acid position 232; any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239; any of Ala, Ile, Met and Thr at amino acid position 240; any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241; any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243; His at amino acid position 244; Ala at amino acid position 245; any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247; any of Glu, His, Gln and Tyr at amino acid position 249; either Glu or Gln at amino acid position 250; Phe at amino acid position 251; any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258; any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262; any of Ala, Ile, Met and Thr at amino acid position 263; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264; any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265; any of Ala, Ile, Met and Thr at amino acid position 266; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267; any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269; any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271; any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272; either Phe or Ile at amino acid position 273; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274; either Leu or Trp at amino acid position 275; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276; any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278; Ala at amino acid position 279; any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280; any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282; any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283; any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284; any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285; any of Glu, Gly, Pro and Tyr at amino acid position 286; any of Asn, Asp, Glu and Tyr at amino acid position 288; any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290; any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291; any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292; any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Ile at amino acid position 302; any of Asp, Gly and Tyr at amino acid position 303; any of Asp, His, Leu, Asn and Thr at amino acid position 304; any of Glu, Ile, Thr and Tyr at amino acid position 305; any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311; Phe at amino acid position 313; Leu at amino acid position 315; either Glu or Gln at amino acid position 317; any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318; any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320; any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322; Ile at amino acid position 323; any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325; any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329; any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330; any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331; any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334; any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335; any of Glu, Lys and Tyr at amino acid position 336; any of Glu, His and Asn at amino acid position 337; any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339; either Ala or Val at amino acid position 376; either Gly or Lys at amino acid position 377; Asp at amino acid position 378; Asn at amino acid position 379; any of Ala, Asn and Ser at amino acid position 380; either Ala or Ile at amino acid position 382; Glu at amino acid position 385; Thr at amino acid position 392; Leu at amino acid position 396; Lys at amino acid position 421; Asn at amino acid position 427; either Phe or Leu at amino acid position 428; Met at amino acid position 429; Trp at amino acid position 434; Ile at amino acid position 436; and any of Gly, His, Ile, Leu and Tyr at amino acid position 440, in the region of the Fc region according to the EU numbering. 12. The method according to any one of paragraphs. 8, 10, 11, where the Fc region of native human IgG, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose, is an Fc region according to any one of native human IgG1, native human IgG2, native human IgG3 and native human IgG4, in in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose. 13. The method according to any one of paragraphs. 8, 10, 11, wherein the human Fcγ receptor is at least one of the FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V) and FcγRIIIa(F) Fcγ receptors. 14. The method according to any one of paragraphs. 8, 10, 11, where the human Fcγ receptor is FcγRIIb. 15. A method of increasing the ability of an antigen-binding molecule to eliminate plasma antigens, wherein the method comprises the step of enhancing binding activity to at least one of the Fcγ receptors under conditions of a neutral pH range of the Fcγ receptor-binding domain in the antigen-binding molecule compared to the native Fc region of IgG- in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose, wherein the antigen-binding molecule has human FcRn binding activity under acidic pH range conditions and contains an Fcγ receptor-binding domain containing an Fc variant -regions of human IgG, where the antigen-binding domain is the variable region of an antibody with at least one histidine, and the antigen-binding domain has an antigen-binding activity that varies with pH, where the value of KD (pH 5.8) / KD (pH 7.4) equal to 2 or more is the ratio e KD for antigen under acidic pH range conditions to KD under neutral pH range conditions, where the antigen is a soluble antigen whose plasma concentration must be reduced. 16. The method of claim 15, wherein the antigen-binding molecule has neutralizing activity against the antigen. 17. The method of claim 15, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 , 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324 , 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differs from the amino acids in the corresponding regions in the native Fc region. 18. The method of claim 17, wherein the Fc region is an Fc region containing at least one or more amino acids selected from the group consisting of: either Lys or Tyr at amino acid position 221; any of Phe, Trp, Glu and Tyr at amino acid position 222; any of Phe, Trp, Glu and Lys at amino acid position 223; any of Phe, Trp, Glu and Tyr at amino acid position 224; any of Glu, Lys and Trp at amino acid position 225; any of Glu, Gly, Lys and Tyr at amino acid position 227; any of Glu, Gly, Lys and Tyr at amino acid position 228; any of Ala, Glu, Gly and Tyr at amino acid position 230; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231; any of Glu, Gly, Lys and Tyr at amino acid position 232; any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239; any of Ala, Ile, Met and Thr at amino acid position 240; any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241; any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243; His at amino acid position 244; Ala at amino acid position 245; any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247; any of Glu, His, Gln and Tyr at amino acid position 249; either Glu or Gln at amino acid position 250; Phe at amino acid position 251; any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258; any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262; any of Ala, Ile, Met and Thr at amino acid position 263; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264; any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265; any of Ala, Ile, Met and Thr at amino acid position 266; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267; any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269; any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271; any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272; either Phe or Ile at amino acid position 273; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274; either Leu or Trp at amino acid position 275; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276; any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278; Ala at amino acid position 279; any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280; any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282; any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283; any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284; any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285; any of Glu, Gly, Pro and Tyr at amino acid position 286; any of Asn, Asp, Glu and Tyr at amino acid position 288; any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290; any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291; any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292; any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Ile at amino acid position 302; any of Asp, Gly and Tyr at amino acid position 303; any of Asp, His, Leu, Asn and Thr at amino acid position 304; any of Glu, Ile, Thr and Tyr at amino acid position 305; any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311; Phe at amino acid position 313; Leu at amino acid position 315; either Glu or Gln at amino acid position 317; any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318; any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320; any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322; Ile at amino acid position 323; any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325; any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329; any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330; any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331; any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334; any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335; any of Glu, Lys and Tyr at amino acid position 336; any of Glu, His and Asn at amino acid position 337; any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339; either Ala or Val at amino acid position 376; either Gly or Lys at amino acid position 377; Asp at amino acid position 378; Asn at amino acid position 379; any of Ala, Asn and Ser at amino acid position 380; either Ala or Ile at amino acid position 382; Glu at amino acid position 385; Thr at amino acid position 392; Leu at amino acid position 396; Lys at amino acid position 421; Asn at amino acid position 427; either Phe or Leu at amino acid position 428; Met at amino acid position 429; Trp at amino acid position 434; Ile at amino acid position 436; and any of Gly, His, Ile, Leu and Tyr at amino acid position 440, in the region of the Fc region according to the EU numbering. 19. The method according to any one of paragraphs. 15, 17, 18, where the Fc region of native human IgG, in which the sugar chain attached at position 297 according to EU numbering, is a sugar chain containing fucose, is the Fc region of any native human IgG1, native human IgG2 , native human IgG3 and native human IgG4, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose. 20. The method according to any one of paragraphs. 15, 17, 18, wherein the human Fcγ receptor is at least one of the Fcγ receptors selected from the group consisting of FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), and FcγRIIIa(F ). 21. The method according to any one of paragraphs. 15, 17, 18, where the human Fcγ receptor is FcγRIIb. 22. A method for producing an antigen-binding molecule for reducing total antigen concentration or plasma free antigen concentration, wherein the method includes the steps of: (a) determining the antigen-binding activity of an antibody variable region that contains at least one histidine under neutral pH conditions; (b) determining the antigen-binding activity of the variable region of the antibody under acidic pH conditions; (c) selecting an antibody variable region for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b); (d) linking a polynucleotide encoding an antibody variable region selected according to (c) to a polynucleotide encoding an Fcγ receptor binding domain containing a human IgG Fc region variant and having human FcRn binding activity under acidic pH range conditions and in which activity binding to at least one of the Fcγ receptors under conditions of the neutral pH range is higher than that of the Fc region of native human IgG, in which the chain the sugar attached at position 297 according to EU numbering is a sugar chain containing fucose; (e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and (f) collecting antigen-binding molecules from the cell culture according to (e), where the antigen is a soluble antigen whose plasma concentration is to be reduced. 23. A method for producing an antigen-binding molecule to reduce the total concentration of antigen or the concentration of free antigen in plasma, where the method includes the steps: (a) determining the antigen-binding activity of an antibody whose variable region contains at least one histidine under neutral pH conditions; (b) determining the antigen-binding activity of the antibody under acidic pH conditions; (c) selecting an antibody for which the antigen-binding activity determined according to (a) is higher than the antigen-binding activity determined according to (b); (d) linking a polynucleotide encoding an antigen-binding domain of an antibody selected according to (c) to a polynucleotide encoding an Fcγ receptor-binding domain containing a variant human IgG Fc region and having a human FcRn binding activity under conditions of an acidic pH range, wherein the activity binding to at least one of the Fcγ receptors under conditions of the neutral pH range is higher than that of the Fc region of native human IgG, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose; (e) culturing cells into which a vector is introduced in which the polynucleotide obtained according to (d) is operably linked; and (f) collecting antigen-binding molecules from the cell culture according to (e), where the antigen is a soluble antigen whose plasma concentration is to be reduced. 24. The method of obtaining according to claim 23, where the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 311 313 315 317 318 320 322 323 324, 325, 326, 327, 328, 329, 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 in the region of the Fc region according to EU numbering differ from the amino acids in the corresponding regions in the native Fc region. 25. The production method according to claim 24, where the Fc region contains at least one or more amino acids selected from the group consisting of: either Lys or Tyr at amino acid position 221; any of Phe, Trp, Glu and Tyr at amino acid position 222; any of Phe, Trp, Glu and Lys at amino acid position 223; any of Phe, Trp, Glu and Tyr at amino acid position 224; any of Glu, Lys and Trp at amino acid position 225; any of Glu, Gly, Lys and Tyr at amino acid position 227; any of Glu, Gly, Lys and Tyr at amino acid position 228; any of Ala, Glu, Gly and Tyr at amino acid position 230; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 231; any of Glu, Gly, Lys and Tyr at amino acid position 232; any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239; any of Ala, Ile, Met and Thr at amino acid position 240; any of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241; any of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243; His at amino acid position 244; Ala at amino acid position 245; any of Asp, Glu, His and Tyr at amino acid position 246; any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247; any of Glu, His, Gln and Tyr at amino acid position 249; either Glu or Gln at amino acid position 250; Phe at amino acid position 251; any of Phe, Met and Tyr at amino acid position 254; any of Glu, Leu and Tyr at amino acid position 255; any of Ala, Met and Pro at amino acid position 256; any of Asp, Glu, His, Ser and Tyr at amino acid position 258; any of Asp, Glu, His and Tyr at amino acid position 260; any of Ala, Glu, Phe, Ile and Thr at amino acid position 262; any of Ala, Ile, Met and Thr at amino acid position 263; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264; any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265; any of Ala, Ile, Met and Thr at amino acid position 266; any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267; any of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269; any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271; any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272; either Phe or Ile at amino acid position 273; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274; either Leu or Trp at amino acid position 275; any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276; any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278; Ala at amino acid position 279; any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280; any of Asp, Lys, Pro and Tyr at amino acid position 281; any of Glu, Gly, Lys, Pro and Tyr at amino acid position 282; any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283; any of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284; any of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285; any of Glu, Gly, Pro and Tyr at amino acid position 286; any of Asn, Asp, Glu and Tyr at amino acid position 288; any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290; any of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291; any of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292; any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293; any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297; any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299; any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300; any of Asp, Glu, His and Tyr at amino acid position 301; Ile at amino acid position 302; any of Asp, Gly and Tyr at amino acid position 303; any of Asp, His, Leu, Asn and Thr at amino acid position 304; any of Glu, Ile, Thr and Tyr at amino acid position 305; any of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311; Phe at amino acid position 313; Leu at amino acid position 315; either Glu or Gln at amino acid position 317; any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318; any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320; any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322; Ile at amino acid position 323; any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325; any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328; any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329; any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330; any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331; any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332; any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334; any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335; any of Glu, Lys and Tyr at amino acid position 336; any of Glu, His and Asn at amino acid position 337; any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339; either Ala or Val at amino acid position 376; either Gly or Lys at amino acid position 377; Asp at amino acid position 378; Asn at amino acid position 379; any of Ala, Asn and Ser at amino acid position 380; either Ala or Ile at amino acid position 382; Glu at amino acid position 385; Thr at amino acid position 392; Leu at amino acid position 396; Lys at amino acid position 421; Asn at amino acid position 427; either Phe or Leu at amino acid position 428; Met at amino acid position 429; Trp at amino acid position 434; Ile at amino acid position 436; and any of Gly, His, Ile, Leu and Tyr at amino acid position 440, in the region of the Fc region according to the EU numbering. 26. The method of obtaining according to any one of paragraphs. 22-25, where the Fc region of native human IgG, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose, is the Fc region of any native human IgG1, native human IgG2, native human IgG3 and native human IgG4, in which the sugar chain attached at position 297 according to EU numbering is a sugar chain containing fucose. 27. The method of obtaining according to any one of paragraphs. 22-25, wherein the human Fcγ receptor is at least one of the Fcγ receptors selected from the group consisting of FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), and FcγRIIIa(F). 28. The method of obtaining according to any one of paragraphs. 22-25, where the human Fcγ receptor is FcγRIIb.

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