drug-containing cell vehicle for formation of an immune complex

专利摘要:
use of an antigen-binding molecule and screening methods and for the production of said molecule. the present inventors refer to the formation of a major immune complex comprising antigens containing two or more antigen-binding units (epitopes) and two or more antigen-binding molecules (eg, antibodies), the elimination from the plasma of the antigens containing two or more antigen-binding units can be accelerated. in addition, they found that by using this feature and the additional use of antigen-binding molecules having an ion-dependent antigen-binding activity, the elimination of antigens can still be accelerated and the above problem can be solved.
公开号:BR112014013081A2
申请号:R112014013081-7
申请日:2012-11-30
公开日:2020-10-20
发明作者:Tomoyuki Igawa；Naoka Hironiwa
申请人:Chugai Seiyaku Kabushiki Kaisha；
IPC主号:

专利说明:

[001] [001] The present invention relates to uses of antigen-binding molecules to eliminate antigens from plasma; methods for eliminating antigens from plasma, which include administration of antigen-binding molecules; pharmaceutical compositions comprising antigen-binding molecules that are capable of eliminating antigens from plasma; methods of tracking antigen-binding molecules to eliminate antigens from plasma; and methods for producing antigen-binding molecules to eliminate antigens from plasma. Background of the Technique
[002] [002] Antibodies are attracting attention as pharmaceutical products since they have a high stability in the plasma and have few side effects. At the moment, several IgG antibody pharmaceutical products are available on the market and many antibody pharmaceutical products are currently under development (Non-Patent Documents 1 and 2). However, several technologies applicable to second generation antibody pharmaceutical products have been reported, including those that increase effector function, antigen-binding capacity, pharmacokinetics and stability, and those that reduce the risk of immunogenicity (Document No Patent 3). In general, the required dose of pharmaceutical antibody products is very high. This in turn led to problems such as high cost of production, as well as the difficulty in producing subcutaneous formulations. In theory, the dose of pharmaceutical antibodies can be reduced by improving the pharmacokinetics of the antibody or by improving the affinity between antibodies and antigens.
[003] [003] The literature has reported methods to improve antibody pharmacokinetics using artificial amino acid substitution in constant regions (Non-Patent Documents 4 and 5). Similarly, affinity maturation has been reported as a technology to increase antigen-binding capacity or antigen neutralization activity (Non-Patent Document 6). This technology allows increased antigen-binding activity by introducing mutations of amino acids in the CDR region of a variable or similar region. The increase in the capacity of binding to the antigen allows the improvement of the biological activity in vitro or the reduction of the dosage, and still allows the improvement of the effectiveness in vivo (in the body) (Document 7 Non-Patent).
[004] [004] However, the antigen neutralization capacity of a single antibody molecule depends on its affinity. By increasing affinity, an antigen can be neutralized by a smaller amount of an antibody. Various methods can be used to increase the affinity of the antibody (Document 6 Non-patent). In addition, if the affinity can be made infinite by covalently binding the antibody to the antigen, a single antibody molecule can neutralize an antigen molecule (a divalent antibody can neutralize two antigen molecules). However, conventional methods have a limitation in which a single antibody molecule binds to a single antigen molecule (two antigens when it is bivalent). However, it has recently been reported that a single antigen-binding molecule can bind to a plurality of antigen molecules using an antigen-binding molecule that binds the antigen in a pH-dependent manner ( Patent Document 1 and Non-Patent Document 8). A pH-dependent molecule binding to the antigen binds strongly to an antigen under neutral condition in the plasma and releases the antigen under acidic condition in the endosome.
[005] [005] Furthermore, since pH-dependent antigen-binding molecules that have been modified to increase FcRn binding under a neutral condition (pH 7.4) have the advantage of being able to bind if repeatedly to antigens, and the effect of eliminating antigens from plasma, the administration of such antigen binding molecules has been reported to allow the elimination of antigen from plasma (Patent Document 2). Conventional pH-dependent antigen-binding molecules comprising an IgG antibody Fc region only show FcRn binding under neutral conditions. Therefore, the absorption of complexes formed between an antigen-binding molecule and an antigen in cells can be mainly due to non-specific absorption. According to this report, pH-dependent antigen-binding molecules that have been modified to increase FcRn binding under a neutral condition (pH 7.4) can accelerate antigen elimination more than molecules binding to conventional pH-dependent antigens comprising an IgG antibody Fc region (Patent Document 2).
[006] [006] Since antigens have very short plasma retentivity compared to antibodies having an FcRn-mediated recycling mechanism, the binding of an antigen in plasma to an antibody having a recycling mechanism (where binding does not is pH dependent) prolongs normal plasma retentivity and increases plasma antigen concentration. For example, when a plasma antigen has multiple types of physiological functions, even if one type of physiological activity is blocked by binding to the antibody, the plasma concentration of the antigen can exacerbate symptoms caused by other physiological functions due to binding to the antibody. From such a point of view, the elimination of plasma antigens is favorable at times, and methods similar to those described above for making modifications to the Fc region to increase the binding of FcRn for the purpose of accelerating antigen elimination have been reported, but other methods for accelerating antigen elimination have not been reported yet.
[007] [007] The prior art documents of the present invention are shown below. Prior Art Documents Patent Documents
[008] [008] Patent Document 1 International Publication WO2009 / 125825
[009] [009] Patent Document 2 International Publication WO2011 / 122011 Non-Patent Documents
[010] [010] Non-Patent Document 1 Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078
[011] [011] Non-Patent Document 2 Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396
[012] [012] Non-Patent Document 3 Kim SJ, Park Y, Hong HJ., Anti-body engineering for the development of therapeutic antibodies, Mol. Cells. (2005) 20 (1), 17-29
[013] [013] Non-Patent Document 4 Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N, J. Immunol. (2006) 176 (1), 346-356
[014] [014] Non-Patent Document 5 Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Nat. Biotechnol.
[015] [015] Non-Patent Document 6 Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., Proc. Natl. Acad. Sci. USA. (2005) 102 (24), 8466-8471
[016] [016] Non-Patent Document 7 Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA., J. Mol. Biol. (2007) 368, 652-665
[017] [017] Non-Patent Document 8 Igawa T, et al., Nat. Biotechnol. (2010) 28, 1203-1207 Summary of the invention Problems to be solved by the invention
[018] [018] The present invention has been achieved in view of the above circumstances. An object of the present invention is to provide uses of antigen-binding molecules to eliminate antigens from plasma; methods for eliminating antigens from plasma, which include administration of antigen-binding molecules; pharmaceutical compositions comprising antigen binding molecules that are capable of eliminating antigens from plasma; methods of tracking antigen-binding molecules to eliminate antigens from plasma; and methods for producing antigen-binding molecules to eliminate antigens from plasma. Troubleshooting Means
[019] [019] The present inventors have conducted dedicated studies to achieve the objectives described above. As a result, the present inventors have produced antigen-binding molecules that comprise (i) an Fc region and (ii) two or more antigen-binding domains where at least one of the domains is an antigen-binding domain whose antigen binding activity varies depending on the condition of ionic concentration, which are antigen binding molecules that can form an immune complex comprising
[020] [020] More specifically, the present invention provides the following:
[021] [021] [1] use of an antigen binding molecule comprising
[022] [022] a region of Fc and
[023] [023] two or more antigen-binding domains,
[024] [024] where at least one of the domains is an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition, and
[025] [025] in which the antigen-binding molecule can form an immune complex comprising
[026] [026] two or more of the antigen-binding molecules and
[027] [027] two or more antigens, in which the antigens comprise two or more antigenic binding units, to eliminate the antigens from the plasma;
[028] [028] [2] the use of [1], in which the ionic concentration condition is a calcium ion concentration condition;
[029] [029] [3] the use of [2], wherein the antigen-binding domain has an antigen-binding activity under a condition of low calcium ion concentration that is lower than the antigen-binding activity under a condition of high calcium ion concentration;
[030] [030] [4] the use of any of [1] to [3], where the ionic concentration condition is a pH condition;
[031] [031] [5] the use of [4], in which the antigen-binding domain has an antigen-binding activity in an acidic pH range that is lower than the antigen-binding activity in a neutral pH range;
[032] [032] [6] the use of any one of [1] to [5], in which antigens comprising two or more antigenic binding units are multimers;
[033] [033] [7] the use of [6], where the antigen is either 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), TNF, TNF-alpha , TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL Apo-2 linker, TL2), TNFSF11 (TRANCE / RANK linker ODF, OPG linker), TNFSF12 (TWEAK Apo-3 linker, DR3 linker), TNFSF13 (APRIL TALL2 ), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM linker, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR linker AITR, TL6), TNFSF1A (TNF-a Conectina, DIF, TNF2 ), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 CD154 ligand, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand , Apo-1 linker, APT1 linker), TNFSF7 (CD27 CD70 linker), TNFSF8 (CD30 CD153 linker), TNFSF9 (4- 1BB linker, CD137 linker), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF -alpha, TGF-beta, TGF-beta Pan Specific and IL-8;
[034] [034] [8] the use of any one of [1] to [5], in which antigens comprising two or more antigenic binding units are monomers;
[035] [035] [9] the use of any one of [1] to [8], in which the antigen-binding molecules are a multispecific or multiparticle antigen-binding molecule or cocktail of antigen-binding molecules;
[036] [036] [10] the use of any of [1] to [9], in which the Fc region is represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[037] [037] [11] the use of any one of [1] to [9], where the Fc region is an Fc region with an increased FcRn binding activity under an acidic pH range condition in comparison with that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[038] [038] [12] the use of [11], in which the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of em amino acids at positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) in the amino acid sequence the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[039] [039] [13] the use of [12], in which the Fc region comprises at least one or more amino acids selected from the group consisting of:
[040] [040] Read for the amino acid of position 238;
[041] [041] Read for the amino acid at position 244;
[042] [042] Arg for the amino acid at position 245;
[043] [043] Pro for the amino acid of position 249;
[044] [044] Gln or Glu for the amino acid of position 250;
[045] [045] Arg, Asp, Glu or Leu for the amino acid of position 251;
[046] [046] Phe, Ser, Thr or Tyr for the amino acid of position 252;
[047] [047] Ser or Thr for the amino acid of position 254;
[048] [048] Arg, Gly, Ile or Leu for the amino acid of position 255;
[049] [049] Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position 256;
[050] [050] Ala, Ile, Met, Asn, Ser or Val for the amino acid of position 257;
[051] [051] Asp for the amino acid of position 258;
[052] [052] Be for the amino acid of position 260;
[053] [053] Read for the amino acid of position 262;
[054] [054] Lys for the amino acid of position 270;
[055] [055] Leu or Arg for the amino acid of position 272;
[056] [056] Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 279;
[057] [057] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 283;
[058] [058] Asn for the amino acid of heading 285;
[059] [059] Phe for the amino acid of position 286;
[060] [060] Asn or Pro for the amino acid of position 288;
[061] [061] Val for the amino acid of position 293;
[062] [062] Ala, Glu, Gln, or Met for the amino acid of position 307;
[063] [063] Ala, Glu, Ile, Lys, Leu, Met, Ser, Val or Trp for the amino acid of position 311;
[064] [064] Pro for the amino acid of position 309;
[065] [065] Ala, Asp, or Pro for the amino acid of position 312;
[066] [066] Ala or Leu for the amino acid of position 314;
[067] [067] Lys for the amino acid of position 316;
[068] [068] Pro for the amino acid of position 317;
[069] [069] Asn or Thr for the amino acid of position 318;
[070] [070] Phe, His, Lys, Leu, Met, Arg, Ser or Trp for the amino acid of position 332;
[071] [071] Asn, Thr or Trp for the amino acid of position 339;
[072] [072] Pro for the amino acid of position 341;
[073] [073] Glu, His, Lys, Gln, Arg, Thr or Tyr for the amino acid of position 343;
[074] [074] Arg for the amino acid of position 375;
[075] [075] Gly, Ile, Met, Pro, Thr or Val for the amino acid of position 376;
[076] [076] Lys for the amino acid of position 377;
[077] [077] Asp, Asn or Val for the amino acid of position 378;
[078] [078] Ala, Asn, Ser or Thr for the amino acid of position 380;
[079] [079] Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 382;
[080] [080] Ala, Arg, Asp, Gly, His, Lys, Ser or Thr for the amino acid of position 385;
[081] [081] Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr for the amino acid of position 386;
[082] [082] Ala, Arg, His, Pro, Ser or Thr for the amino acid of position 387;
[083] [083] Asn, Pro, or Ser for the amino acid of heading 389;
[084] [084] Asn for the amino acid of position 423;
[085] [085] Asn for the amino acid of heading 427;
[086] [086] Leu, Met, Phe, Ser or Thr for the amino acid of position 428;
[087] [087] Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
[088] [088] His or Asn for the amino acid of position 431;
[089] [089] Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433;
[090] [090] Ala, Gly, His, Phe, Ser, Trp or Tyr for the amino acid of position 434;
[091] [091] Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid at position 436;
[092] [092] Lys, Leu, Thr or Trp for the amino acid of position 438;
[093] [093] Lys for the amino acid of position 440; and
[094] [094] Lys for the amino acid of position 442; Ile, Pro, or Thr for the amino acid of position 308;
[095] [095] as indicated by the EU numbering in the amino acid sequence of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[096] [096] [14] the use of any of [1] to [9], where the Fc region is an Fc region with increased FcRn binding activity under a neutral pH range condition in comparison with that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[097] [097] [15] the use of [14], where the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino acid sequence of the Fc region represented by any of SEQ ID NOS: 13, 14 , 15, and 16;
[098] [098] [16] the use of [15], in which the Fc region comprises at least one or more amino acids selected from the group consisting of
[099] [099] Met for the amino acid of position 237;
[0100] [0100] Ile for the amino acid of position 248;
[0101] [0101] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250;
[0102] [0102] Phe, Trp or Tyr for the amino acid of position 252;
[0103] [0103] Thr for the amino acid of position 254;
[0104] [0104] Glu for the amino acid of position 255;
[0105] [0105] Asp, Asn, Glu or Gln for the amino acid of position 256;
[0106] [0106] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257;
[0107] [0107] His for the amino acid of position 258;
[0108] [0108] Ala for the amino acid of position 265;
[0109] [0109] Ala or Glu for the amino acid of position 286;
[0110] [0110] His for the amino acid of position 289;
[0111] [0111] Wing for the amino acid of position 297;
[0112] [0112] Wing for the amino acid of position 303;
[0113] [0113] Wing for the amino acid of position 305;
[0114] [0114] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307;
[0115] [0115] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308;
[0116] [0116] Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309;
[0117] [0117] Ala, His or Ile for the amino acid of position 311;
[0118] [0118] Ala or His for the amino acid of position 312;
[0119] [0119] Lys or Arg for the amino acid of position 314;
[0120] [0120] Ala, Asp or His for the amino acid of position 315;
[0121] [0121] Wing for the amino acid of position 317;
[0122] [0122] Val for the amino acid of position 332;
[0123] [0123] Read for the amino acid of position 334;
[0124] [0124] His for the amino acid of position 360;
[0125] [0125] Wing for the amino acid of position 376;
[0126] [0126] Wing for the amino acid of position 380;
[0127] [0127] Ala for the amino acid of position 382;
[0128] [0128] Ala for the amino acid of position 384;
[0129] [0129] Asp or His for the amino acid of position 385;
[0130] [0130] Pro for the amino acid of position 386;
[0131] [0131] Glu for the amino acid of position 387;
[0132] [0132] Ala or Ser for the amino acid of position 389;
[0133] [0133] Ala for the amino acid of position 424;
[0134] [0134] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 428;
[0135] [0135] Lys for the amino acid of position 433;
[0136] [0136] Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and
[0137] [0137] His, Ile, Leu, Phe, Thr or Val for the amino acid of position 436;
[0138] [0138] as indicated by the EU numbering following amino acids of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0139] [0139] [17] the use of any one from [1] to [13], wherein the Fc region includes an Fc region having a higher Fcγ receptor binding activity than that of the Fc region of Native human IgG;
[0140] [0140] [18] the use of [17], wherein the Fc region comprises in its amino acid sequence at least one or more amino acids that are different from the amino acids of the Fc region of native human IgG selected from the group consisting of heading 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,
[0141] [0141] [19] the use of [18], in which the Fc region comprises in its amino acid sequence at least one or more amino acids selected from the group consisting of:
[0142] [0142] Lys or Tyr for the amino acid of position 221;
[0143] [0143] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[0144] [0144] Phe, Trp, Glu or Lys for the amino acid of position 223;
[0145] [0145] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[0146] [0146] Glu, Lys or Trp for the amino acid of position 225;
[0147] [0147] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[0148] [0148] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[0149] [0149] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[0150] [0150] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[0151] [0151] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[0152] [0152] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[0153] [0153] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[0154] [0154] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235;
[0155] [0155] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[0156] [0156] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
[0157] [0157] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[0158] [0158] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239;
[0159] [0159] Ala, Ile, Met, or Thr for the amino acid at position 240;
[0160] [0160] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[0161] [0161] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[0162] [0162] His for the amino acid at position 244;
[0163] [0163] Ala for the amino acid of position 245;
[0164] [0164] Asp, Glu, His or Tyr for the amino acid of position 246;
[0165] [0165] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[0166] [0166] Glu, His, Gln or Tyr for the amino acid of position 249;
[0167] [0167] Glu or Gln for the amino acid of position 250;
[0168] [0168] Phe for the amino acid of position 251;
[0169] [0169] Phe, Met, or Tyr for the amino acid of position 254;
[0170] [0170] Glu, Leu or Tyr for the amino acid of position 255;
[0171] [0171] Ala, Met, or Pro for the amino acid of position 256;
[0172] [0172] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[0173] [0173] Asp, Glu, His or Tyr for the amino acid of position 260;
[0174] [0174] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[0175] [0175] Ala, Ile, Met, or Thr for the amino acid of position 263;
[0176] [0176] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[0177] [0177] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[0178] [0178] Ala, Ile, Met, or Thr for the amino acid of position 266;
[0179] [0179] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[0180] [0180] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[0181] [0181] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[0182] [0182] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[0183] [0183] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[0184] [0184] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[0185] [0185] Phe or Ile for the amino acid of position 273;
[0186] [0186] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[0187] [0187] Leu or Trp for the amino acid of position 275;
[0188] [0188] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[0189] [0189] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 278;
[0190] [0190] Wing for the amino acid of position 279;
[0191] [0191] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[0192] [0192] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[0193] [0193] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282;
[0194] [0194] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[0195] [0195] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of the
[0196] [0196] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[0197] [0197] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[0198] [0198] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[0199] [0199] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[0200] [0200] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[0201] [0201] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[0202] [0202] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[0203] [0203] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[0204] [0204] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[0205] [0205] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[0206] [0206] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297;
[0207] [0207] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[0208] [0208] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 299;
[0209] [0209] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 300;
[0210] [0210] Asp, Glu, His or Tyr for the amino acid of position 301;
[0211] [0211] Ile for the amino acid of position 302;
[0212] [0212] Asp, Gly or Tyr for the amino acid of position 303;
[0213] [0213] Asp, His, Leu, Asn or Thr for the amino acid of position 304;
[0214] [0214] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[0215] [0215] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311;
[0216] [0216] Phe for the amino acid of position 313;
[0217] [0217] Read for the amino acid of position 315;
[0218] [0218] Glu or Gln for the amino acid of position 317;
[0219] [0219] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[0220] [0220] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid at position 320;
[0221] [0221] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[0222] [0222] Ile for the amino acid of position 323;
[0223] [0223] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[0224] [0224] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[0225] [0225] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326;
[0226] [0226] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[0227] [0227] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[0228] [0228] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329;
[0229] [0229] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[0230] [0230] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 331;
[0231] [0231] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[0232] [0232] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the amino acid of position 333;
[0233] [0233] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[0234] [0234] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[0235] [0235] Glu, Lys or Tyr for the amino acid of position 336;
[0236] [0236] Glu, His or Asn for the amino acid at position 337;
[0237] [0237] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[0238] [0238] Ala or Val for the amino acid of position 376;
[0239] [0239] Gly or Lys for the amino acid of position 377;
[0240] [0240] Asp for the amino acid of position 378;
[0241] [0241] Asn for the amino acid of heading 379;
[0242] [0242] Ala, Asn or Ser for the amino acid of position 380;
[0243] [0243] Ala or Ile for the amino acid of position 382;
[0244] [0244] Glu for the amino acid of position 385;
[0245] [0245] Thr for the amino acid of position 392;
[0246] [0246] Read for the amino acid of position 396;
[0247] [0247] Lys for the amino acid of position 421;
[0248] [0248] Asn for the amino acid of heading 427;
[0249] [0249] Phe or Leu for the amino acid of position 428;
[0250] [0250] Met for the amino acid of position 429;
[0251] [0251] Trp for the amino acid of position 434;
[0252] [0252] Ile for the amino acid of position 436; and
[0253] [0253] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[0254] [0254] as indicated by the EU numbering;
[0255] [0255] [20] the use of any of [1] to [16], where the Fc region has a higher binding activity towards an inhibitory Fcγ receptor than towards an activation receptor of Fcγ;
[0256] [0256] [21] the use of [20], in which the inhibitory Fcγ receptor is human FcγRIIb;
[0257] [0257] [22] the use of [20] or [21], wherein the Fcγ activation receptor is human FcγRIa, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) or FcγRIIIa (FcγRIIIa (F) ) human;
[0258] [0258] [23] the use of any of [20] to [22], where the amino acid at position 238 or 328 (EU numbering) in the Fc region is different from the amino acid in the Fc region of Native human IgG;
[0259] [0259] [24] the use of [23], where the amino acid at position 238 of the Fc region is Asp or the amino acid at position 328 of the Fc region is Glu as indicated by the EU number;
[0260] [0260] [25] the use of [23] or [24], in which the amino acid sequence of the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0261] [0261] Asp for the amino acid of position 233;
[0262] [0262] Trp or Tyr for the amino acid of position 234;
[0263] [0263] Ala, Asp, Glu, Leu, Met, Phe, Trp or Tyr for the amino acid of position 237;
[0264] [0264] Asp for the amino acid of position 239;
[0265] [0265] Ala, Gln or Val for the amino acid of position 267;
[0266] [0266] Asn, Asp or Glu for the amino acid of position 268;
[0267] [0267] Gly for the amino acid of position 271;
[0268] [0268] Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser or Thr for the amino acid of position 326;
[0269] [0269] Arg, Lys, or Met for the amino acid of position 330;
[0270] [0270] Ile, Leu, or Met for the amino acid of position 323; and
[0271] [0271] Asp for the amino acid of position 296;
[0272] [0272] as indicated by the EU numbering;
[0273] [0273] [26] a method of tracing an antigen-binding molecule and has the function of eliminating an antigen from the plasma, in which the method comprises:
[0274] [0274] obtaining an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition;
[0275] [0275] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[0276] [0276] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[0277] [0277] culture of a host cell comprising the genes operably linked in (c) above;
[0278] [0278] isolation of an antigen-binding molecule from a culture solution obtained in (d) above;
[0279] [0279] put the antigen-binding molecule obtained in (e) above with an antigen in contact; and
[0280] [0280] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen;
[0281] [0281] [27] a method for producing an antigen-binding molecule and has a function of removing an antigen from plasma, in which the method comprises:
[0282] [0282] bringing an antigen into contact with an antigen-binding molecule comprising an Fc region and two or more antigen-binding domains, in which at least one of the antigen-binding domains has a binding activity to the antigen that varies depending on a condition of ionic concentration;
[0283] [0283] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen;
[0284] [0284] culture of a host cell comprising a vector that carries a gene that encodes an antigen-binding molecule that is confirmed to form an immune complex in (b) above; and
[0285] [0285] isolation of the antigen-binding molecule from a culture solution obtained in (c) above;
[0286] [0286] [28] a method for producing an antigen-binding molecule and has a function of removing an antigen from plasma, wherein the method comprises:
[0287] [0287] obtaining an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition;
[0288] [0288] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[0289] [0289] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[0290] [0290] culture of a host cell comprising the genes operably linked in (c) above;
[0291] [0291] isolation of an antigen-binding molecule from a culture solution obtained in (d) above;
[0292] [0292] put in contact with the antigen binding molecule obtained in (e) above with an antigen;
[0293] [0293] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen;
[0294] [0294] culture of a host cell comprising a vector that carries a gene that encodes an antigen-binding molecule that is confirmed to form an immune complex in (g) above; and
[0295] [0295] isolation of the antigen-binding molecule from a culture solution obtained in (h) above; and
[0296] [0296] [29] a method for producing an antigen-binding molecule and has a function of removing an antigen from plasma, in which the method comprises:
[0297] [0297] obtaining an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition;
[0298] [0298] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[0299] [0299] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[0300] [0300] culture of a host cell comprising the genes operably linked in (c) above; and
[0301] [0301] isolation of an antigen-binding molecule from a culture solution obtained in (d) above; and
[0302] [0302] in which the method also comprises contacting the antigen-binding molecule obtained by the production method with an antigen and assessing the formation of an immune complex comprising the antigen-binding molecule and the antigen.
[0303] [0303] The aforementioned [1] to [25] can be declared again as follows:
[0304] [0304] [1 ’] a pharmaceutical composition for removing an antigen from plasma, wherein the pharmaceutical composition comprises an antigen binding molecule comprising
[0305] [0305] a region of Fc and
[0306] [0306] two or more antigen-binding domains,
[0307] [0307] where at least one of the domains is an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition, and
[0308] [0308] in which the antigen-binding molecule can form an immune complex comprising
[0309] [0309] two or more of the antigen-binding molecules and
[0310] [0310] two or more antigens, in which the antigens comprise two or more antigenic binding units;
[0311] [0311] [2 ’] the pharmaceutical composition of [1’], wherein the ionic concentration condition is a calcium ion concentration condition;
[0312] [0312] [3 '] the pharmaceutical composition of [2'], wherein the antigen-binding domain has an antigen-binding activity under a condition of low calcium ion concentration that is lower than the binding activity the antigen under a condition of high calcium ion concentration;
[0313] [0313] [4 ’] the pharmaceutical composition of any one of [1’] to [3 ’], wherein the ionic concentration condition is a pH condition;
[0314] [0314] [5 '] the pharmaceutical composition of [4'], in which the antigen-binding domain has an antigen-binding activity in an acidic pH range that is lower than the antigen-binding activity antigen in a condition of neutral pH range;
[0315] [0315] [6 '] the pharmaceutical composition of any one of [1'] to [5 '], wherein the antigens comprising two or more antigenic binding units are multimers;
[0316] [0316] [7 '] the pharmaceutical composition of [6'], wherein the antigen is any one of 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), TNF, TNF-alpha, TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL Apo-2 linker, TL2), TNFSF11 (ODANCE TRANCE / RANK linker, OPG linker), TNFSF12 (TWEAK Apo-3 linker, DR3 linker), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 linker gp34, TXGP1), TNFSF5 (CD40 link CD154, gp39, HIGM1, IMD3, TRAP ), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 CD70 ligand), TNFSF8 (CD30 ligand
[0317] [0317] [8 '] the pharmaceutical composition of any one of [1'] to [5 '], wherein the antigens comprising two or more antigenic binding units are monomers;
[0318] [0318] [9 '] the pharmaceutical composition of any one of [1'] to [8 '], wherein the antigen binding molecules are a multispecific or multiparatopic antigen binding molecule or cocktail of antigen binding molecules ;
[0319] [0319] [10 ’] the pharmaceutical composition of any one of [1’] to [9 ’], wherein the Fc region is represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0320] [0320] [11 '] The pharmaceutical composition of any one from [1'] to [9 '], wherein the Fc region is an Fc region with increased FcRn binding activity under a pH range condition acidic compared to that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0321] [0321] [12 '] the pharmaceutical composition of [11'], in which the Fc region is an Fc region with an amino acid substitution of at least one or more amino acids selected from the group consisting of the positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303 , 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385 , 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) at amino acid sequence of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0322] [0322] [13 ’] The pharmaceutical composition of [12’], wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0323] [0323] Read for the amino acid of position 238;
[0324] [0324] Read for the amino acid of position 244;
[0325] [0325] Arg for the amino acid of position 245;
[0326] [0326] Pro for the amino acid of position 249;
[0327] [0327] Gln or Glu for the amino acid of position 250;
[0328] [0328] Arg, Asp, Glu or Leu for the amino acid of position 251;
[0329] [0329] Phe, Ser, Thr or Tyr for the amino acid of position 252;
[0330] [0330] Ser or Thr for the amino acid of position 254;
[0331] [0331] Arg, Gly, Ile or Leu for the amino acid of position 255;
[0332] [0332] Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position 256;
[0333] [0333] Ala, Ile, Met, Asn, Ser or Val for the amino acid of position 257;
[0334] [0334] Asp for the amino acid of position 258;
[0335] [0335] Be for the amino acid of position 260;
[0336] [0336] Read for the amino acid of position 262;
[0337] [0337] Lys for the amino acid of position 270;
[0338] [0338] Leu or Arg for the amino acid of position 272;
[0339] [0339] Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 279;
[0340] [0340] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 283;
[0341] [0341] Asn for the amino acid of heading 285;
[0342] [0342] Phe for the amino acid of position 286;
[0343] [0343] Asn or Pro for the amino acid of position 288;
[0344] [0344] Val for the amino acid of position 293;
[0345] [0345] Ala, Glu, Gln, or Met for the amino acid of position 307;
[0346] [0346] Ala, Glu, Ile, Lys, Leu, Met, Ser, Val or Trp for the amino
[0347] [0347] Pro for the amino acid of position 309;
[0348] [0348] Ala, Asp, or Pro for the amino acid of position 312;
[0349] [0349] Ala or Leu for the amino acid of position 314;
[0350] [0350] Lys for the amino acid of position 316;
[0351] [0351] Pro for the amino acid of position 317;
[0352] [0352] Asn or Thr for the amino acid of position 318;
[0353] [0353] Phe, His, Lys, Leu, Met, Arg, Ser or Trp for the amino acid of position 332;
[0354] [0354] Asn, Thr or Trp for the amino acid of position 339;
[0355] [0355] Pro for the amino acid of position 341;
[0356] [0356] Glu, His, Lys, Gln, Arg, Thr or Tyr for the amino acid of position 343;
[0357] [0357] Arg for the amino acid of position 375;
[0358] [0358] Gly, Ile, Met, Pro, Thr or Val for the amino acid of position 376;
[0359] [0359] Lys for the amino acid of position 377;
[0360] [0360] Asp, Asn or Val for the amino acid of position 378;
[0361] [0361] Ala, Asn, Ser or Thr for the amino acid of position 380;
[0362] [0362] Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 382;
[0363] [0363] Ala, Arg, Asp, Gly, His, Lys, Ser or Thr for the amino acid of position 385;
[0364] [0364] Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr for the amino acid of position 386;
[0365] [0365] Ala, Arg, His, Pro, Ser or Thr for the amino acid of position 387;
[0366] [0366] Asn, Pro, or Ser for the amino acid of heading 389;
[0367] [0367] Asn for the amino acid of position 423;
[0368] [0368] Asn for the amino acid of heading 427;
[0369] [0369] Leu, Met, Phe, Ser or Thr for the amino acid of position 428;
[0370] [0370] Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr for the amino acid of position 430;
[0371] [0371] His or Asn for the amino acid of position 431;
[0372] [0372] Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433;
[0373] [0373] Ala, Gly, His, Phe, Ser, Trp or Tyr for the amino acid of position 434;
[0374] [0374] Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid of position 436;
[0375] [0375] Lys, Leu, Thr or Trp for the amino acid of position 438;
[0376] [0376] Lys for the amino acid of position 440; and
[0377] [0377] Lys for the amino acid of position 442; Ile, Pro, or Thr for the amino acid of position 308;
[0378] [0378] as indicated by the EU numbering following amino acids of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0379] [0379] [14 '] The pharmaceutical composition of any one from [1'] to [9 '], wherein the Fc region is an Fc region with increased FcRn binding activity under a pH range condition neutral compared to that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0380] [0380] [15 '] the pharmaceutical composition of [14'], where the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of positions 237, 248 , 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360 , 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino acid sequence of the Fc region represented
[0381] [0381] [16 ’] the pharmaceutical composition of [15’], wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0382] [0382] Met for the amino acid at position 237;
[0383] [0383] Ile for the amino acid of position 248;
[0384] [0384] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250;
[0385] [0385] Phe, Trp or Tyr for the amino acid of position 252;
[0386] [0386] Thr for the amino acid of position 254;
[0387] [0387] Glu for the amino acid of position 255;
[0388] [0388] Asp, Asn, Glu or Gln for the amino acid of position 256;
[0389] [0389] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257;
[0390] [0390] His for the amino acid of position 258;
[0391] [0391] Wing for the amino acid of position 265;
[0392] [0392] Ala or Glu for the amino acid of position 286;
[0393] [0393] His for the amino acid of position 289;
[0394] [0394] Wing for the amino acid of position 297;
[0395] [0395] Wing for the amino acid of position 303;
[0396] [0396] Wing for the amino acid of position 305;
[0397] [0397] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307;
[0398] [0398] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308;
[0399] [0399] Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309;
[0400] [0400] Ala, His or Ile for the amino acid of position 311;
[0401] [0401] Ala or His for the amino acid of position 312;
[0402] [0402] Lys or Arg for the amino acid of position 314;
[0403] [0403] Ala, Asp or His for the amino acid of position 315;
[0404] [0404] Wing for the amino acid of position 317;
[0405] [0405] Val for the amino acid of position 332;
[0406] [0406] Read for the amino acid of position 334;
[0407] [0407] His for the amino acid of position 360;
[0408] [0408] Wing for the amino acid of position 376;
[0409] [0409] Wing for the amino acid of position 380;
[0410] [0410] Wing for the amino acid of position 382;
[0411] [0411] Wing for the amino acid of position 384;
[0412] [0412] Asp or His for the amino acid of position 385;
[0413] [0413] Pro for the amino acid of position 386;
[0414] [0414] Glu for the amino acid of position 387;
[0415] [0415] Ala or Ser for the amino acid of position 389;
[0416] [0416] Wing for the amino acid of position 424;
[0417] [0417] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 428;
[0418] [0418] Lys for the amino acid of position 433;
[0419] [0419] Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and
[0420] [0420] His, Ile, Leu, Phe, Thr or Val for the amino acid of position 436;
[0421] [0421] as indicated by the EU numbering following amino acids from the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0422] [0422] [17 '] the pharmaceutical composition of any one from [1'] to [13 '], wherein the Fc region includes an Fc region that has a higher Fcγ receptor binding activity than that of the native human IgG Fc region;
[0423] [0423] [18 '] the pharmaceutical composition of [17'], wherein the Fc region comprises in its amino acid sequence at least one or more amino acids that are different from the amino acids of the Fc region of native human IgG selected from of the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246 , 247, 249, 250, 251, 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 (EU numbering);
[0424] [0424] [19 ’] The pharmaceutical composition of [18’], wherein the Fc region comprises in its amino acid sequence at least one or more amino acids selected from the group consisting of:
[0425] [0425] Lys or Tyr for the amino acid of position 221;
[0426] [0426] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[0427] [0427] Phe, Trp, Glu or Lys for the amino acid of position 223;
[0428] [0428] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[0429] [0429] Glu, Lys or Trp for the amino acid of position 225;
[0430] [0430] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[0431] [0431] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[0432] [0432] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[0433] [0433] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[0434] [0434] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[0435] [0435] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[0436] [0436] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[0437] [0437] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,
[0438] [0438] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[0439] [0439] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237;
[0440] [0440] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[0441] [0441] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239;
[0442] [0442] Ala, Ile, Met, or Thr for the amino acid at position 240;
[0443] [0443] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[0444] [0444] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[0445] [0445] His for the amino acid at position 244;
[0446] [0446] Wing for the amino acid of position 245;
[0447] [0447] Asp, Glu, His or Tyr for the amino acid of position 246;
[0448] [0448] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[0449] [0449] Glu, His, Gln or Tyr for the amino acid at position 249;
[0450] [0450] Glu or Gln for the amino acid of position 250;
[0451] [0451] Phe for the amino acid of position 251;
[0452] [0452] Phe, Met, or Tyr for the amino acid of position 254;
[0453] [0453] Glu, Leu or Tyr for the amino acid of position 255;
[0454] [0454] Ala, Met, or Pro for the amino acid of position 256;
[0455] [0455] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[0456] [0456] Asp, Glu, His or Tyr for the amino acid of position 260;
[0457] [0457] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[0458] [0458] Ala, Ile, Met, or Thr for the amino acid of position 263;
[0459] [0459] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[0460] [0460] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[0461] [0461] Ala, Ile, Met, or Thr for the amino acid of position 266;
[0462] [0462] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[0463] [0463] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[0464] [0464] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[0465] [0465] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[0466] [0466] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[0467] [0467] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[0468] [0468] Phe or Ile for the amino acid of position 273;
[0469] [0469] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[0470] [0470] Leu or Trp for the amino acid at position 275;
[0471] [0471] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[0472] [0472] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 278;
[0473] [0473] Wing for the amino acid of position 279;
[0474] [0474] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[0475] [0475] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[0476] [0476] Glu, Gly, Lys, Pro, or Tyr for the amino acid of the position
[0477] [0477] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[0478] [0478] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284;
[0479] [0479] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[0480] [0480] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[0481] [0481] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[0482] [0482] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[0483] [0483] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[0484] [0484] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[0485] [0485] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[0486] [0486] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[0487] [0487] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[0488] [0488] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[0489] [0489] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297;
[0490] [0490] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[0491] [0491] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the 299 amino acid;
[0492] [0492] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
[0493] [0493] Asp, Glu, His or Tyr for the amino acid of position 301;
[0494] [0494] Ile for the amino acid of position 302;
[0495] [0495] Asp, Gly or Tyr for the amino acid of position 303;
[0496] [0496] Asp, His, Leu, Asn or Thr for the amino acid at position 304;
[0497] [0497] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[0498] [0498] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311;
[0499] [0499] Phe for the amino acid of position 313;
[0500] [0500] Read for the amino acid of position 315;
[0501] [0501] Glu or Gln for the amino acid of position 317;
[0502] [0502] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[0503] [0503] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 320;
[0504] [0504] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[0505] [0505] Ile for the amino acid of position 323;
[0506] [0506] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[0507] [0507] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[0508] [0508] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326;
[0509] [0509] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[0510] [0510] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[0511] [0511] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
[0512] [0512] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[0513] [0513] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 331;
[0514] [0514] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[0515] [0515] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the 333 amino acid;
[0516] [0516] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[0517] [0517] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[0518] [0518] Glu, Lys or Tyr for the amino acid of position 336;
[0519] [0519] Glu, His or Asn for the amino acid at position 337;
[0520] [0520] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[0521] [0521] Ala or Val for the amino acid of position 376;
[0522] [0522] Gly or Lys for the amino acid of position 377;
[0523] [0523] Asp for the amino acid of position 378;
[0524] [0524] Asn for the amino acid of heading 379;
[0525] [0525] Ala, Asn or Ser for the amino acid of position 380;
[0526] [0526] Ala or Ile for the amino acid of position 382;
[0527] [0527] Glu for the amino acid of position 385;
[0528] [0528] Thr for the amino acid of position 392;
[0529] [0529] Read for the amino acid of position 396;
[0530] [0530] Lys for the amino acid of position 421;
[0531] [0531] Asn for the amino acid of heading 427;
[0532] [0532] Phe or Leu for the amino acid of position 428;
[0533] [0533] Met for the amino acid of position 429;
[0534] [0534] Trp for the amino acid of position 434;
[0535] [0535] Ile for the amino acid of position 436; and
[0536] [0536] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[0537] [0537] as indicated by the EU numbering;
[0538] [0538] [20 '] the pharmaceutical composition of any one from [1'] to [16 '], in which the Fc region has a higher binding activity towards an inhibitory Fcγ receptor than towards an Fcγ activation receptor;
[0539] [0539] [21 ’] the pharmaceutical composition of [20’], wherein the inhibitory Fcγ receptor is human FcγRIIb;
[0540] [0540] [22 '] the pharmaceutical composition of [20'] or [21 '], wherein the Fcγ activation receptor is human FcγRIa, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) or human FcγRIIIa (F);
[0541] [0541] [23 '] the pharmaceutical composition of any one from [20'] to [22 '], where the amino acid at position 238 or 328 (EU numbering) in the Fc region is different from the amino acid in the Fc region of Native human IgG;
[0542] [0542] [24 '] the pharmaceutical composition of [23'], wherein the amino acid at position 238 of the Fc region is Asp or the amino acid at position 328 of the Fc region is Glu as indicated by the numbering EU ; and
[0543] [0543] [25 ’] the pharmaceutical composition of [23’] or [24 ’], wherein the amino acid sequence of the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0544] [0544] Asp for the amino acid of position 233;
[0545] [0545] Trp or Tyr for the amino acid of position 234;
[0546] [0546] Ala, Asp, Glu, Leu, Met, Phe, Trp or Tyr for the amino acid of position 237;
[0547] [0547] Asp for the amino acid of position 239;
[0548] [0548] Ala, Gln or Val for the amino acid of position 267;
[0549] [0549] Asn, Asp or Glu for the amino acid of position 268;
[0550] [0550] Gly for the amino acid of position 271;
[0551] [0551] Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser or Thr for the amino acid of position 326;
[0552] [0552] Arg, Lys, or Met for the amino acid of position 330;
[0553] [0553] Ile, Leu, or Met for the amino acid of position 323; and
[0554] [0554] Asp for the amino acid of position 296;
[0555] [0555] as indicated by the EU numbering;
[0556] [0556] In addition, the aforementioned [1] to [25] can also be declared again as follows:
[0557] [0557] [1 "] a method for removing an antigen from an individual's plasma, wherein the method comprises administering to the individual an antigen binding molecule comprising
[0558] [0558] a region of Fc and
[0559] [0559] two or more antigen-binding domains,
[0560] [0560] in which at least one of the domains is an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition, and
[0561] [0561] in which the antigen binding molecule can form an immune complex comprising
[0562] [0562] two or more of the antigen-binding molecules and
[0563] [0563] two or more antigens, wherein the antigens comprise two or more antigenic binding units;
[0564] [0564] [2 "] the method of [1"], in which the condition of ionic concentration is a condition of calcium ion concentration;
[0565] [0565] [3 "] the method of [2"], in which the antigen-binding domain has an antigen-binding activity under a condition of low calcium ion concentration that is lower than the activity binding to the antigen under a condition of high ionic calcium concentration;
[0566] [0566] [4 "] the method of any one from [1"] to [3 "], in which the ionic concentration condition is a pH condition;
[0567] [0567] [5 "] the [4"] method, in which the antigen-binding domain has an antigen-binding activity in an acidic pH range that is lower than the antigen-binding activity in a condition of neutral pH range;
[0568] [0568] [6 "] the method of any one from [1"] to [5 "], wherein the antigens comprising two or more antigenic binding units are multimers;
[0569] [0569] [7 "] the [6"] method, where the antigen is any one of 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), TNF, TNF -alpha, TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL Apo-2 linker, TL2), TNFSF11 (ODF TRAN-CE / RANK linker, OPG linker), TNFSF12 (TWEAK Apo-3 linker, DR3 linker ), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM linker, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (AITR linker, TL6), TNFSF1 Conectin TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 linker gp34, TXGP1), TNFSF5 (CD40 CD154 linker, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 CD70 ligand), TNFSF8 (CD30 CD153 ligand), TNFSF9 (4-1BB ligand CD137 ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta Pan Specific and IL-8;
[0570] [0570] [8 "] the method of any one from [1"] to [5 "], wherein the antigens comprising two or more antigenic binding units are monomers;
[0571] [0571] [9 "] the method of any one from [1"] to [9 "], in which the antigen binding molecules are a multispecific or multiparatopic antigen binding molecule or cocktail of molecules binding to the antigen antigen;
[0572] [0572] [10 "] the method of any one from [1"] to [9 "], in which the Fc region is represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0573] [0573] [11 "] the method of any one from [1"] to [9 "], in which the Fc region is an Fc region with increased FcRn binding activity under a bandwidth condition acidic pH compared to that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0574] [0574] [12 "] the method of [11"], in which the Fc region is a region of Fc with a substitution of at least one or more amino acids selected from the group consisting of positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) in the amino acid sequence the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0575] [0575] [13 "] the [12"] method, in which the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0576] [0576] Read for the amino acid at position 238;
[0577] [0577] Read for the amino acid at position 244;
[0578] [0578] Arg for the amino acid of position 245;
[0579] [0579] Pro for the amino acid of position 249;
[0580] [0580] Gln or Glu for the amino acid of position 250;
[0581] [0581] Arg, Asp, Glu or Leu for the amino acid of position 251;
[0582] [0582] Phe, Ser, Thr or Tyr for the amino acid of position 252;
[0583] [0583] Ser or Thr for the amino acid of position 254;
[0584] [0584] Arg, Gly, Ile or Leu for the amino acid of position 255;
[0585] [0585] Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position 256;
[0586] [0586] Ala, Ile, Met, Asn, Ser or Val for the amino acid of position 257;
[0587] [0587] Asp for the amino acid of position 258;
[0588] [0588] Be for the amino acid of position 260;
[0589] [0589] Read for the amino acid of position 262;
[0590] [0590] Lys for the amino acid of position 270;
[0591] [0591] Leu or Arg for the amino acid of position 272;
[0592] [0592] Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 279;
[0593] [0593] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 283;
[0594] [0594] Asn for the amino acid of heading 285;
[0595] [0595] Phe for the amino acid of position 286;
[0596] [0596] Asn or Pro for the amino acid of position 288;
[0597] [0597] Val for the amino acid of position 293;
[0598] [0598] Ala, Glu, Gln, or Met for the amino acid of position 307;
[0599] [0599] Ala, Glu, Ile, Lys, Leu, Met, Ser, Val or Trp for the amino acid of position 311;
[0600] [0600] Pro for the amino acid of position 309;
[0601] [0601] Ala, Asp, or Pro for the amino acid of position 312;
[0602] [0602] Ala or Leu for the amino acid of position 314;
[0603] [0603] Lys for the amino acid of position 316;
[0604] [0604] Pro for the amino acid of position 317;
[0605] [0605] Asn or Thr for the amino acid of position 318;
[0606] [0606] Phe, His, Lys, Leu, Met, Arg, Ser or Trp for the amino acid of position 332;
[0607] [0607] Asn, Thr or Trp for the amino acid of position 339;
[0608] [0608] Pro for the amino acid of position 341;
[0609] [0609] Glu, His, Lys, Gln, Arg, Thr or Tyr for the amino acid of position 343;
[0610] [0610] Arg for the amino acid of position 375;
[0611] [0611] Gly, Ile, Met, Pro, Thr or Val for the amino acid of position 376;
[0612] [0612] Lys for the amino acid of position 377;
[0613] [0613] Asp, Asn or Val for the amino acid of position 378;
[0614] [0614] Ala, Asn, Ser or Thr for the amino acid of position 380;
[0615] [0615] Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 382;
[0616] [0616] Ala, Arg, Asp, Gly, His, Lys, Ser or Thr for the amino acid of position 385;
[0617] [0617] Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr for the amino acid of position 386;
[0618] [0618] Ala, Arg, His, Pro, Ser or Thr for the amino acid of position 387;
[0619] [0619] Asn, Pro, or Ser for the amino acid of heading 389;
[0620] [0620] Asn for the amino acid of heading 423;
[0621] [0621] Asn for the amino acid of heading 427;
[0622] [0622] Leu, Met, Phe, Ser or Thr for the amino acid of position 428;
[0623] [0623] Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr for the amino acid of position 430;
[0624] [0624] His or Asn for the amino acid of position 431;
[0625] [0625] Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433;
[0626] [0626] Ala, Gly, His, Phe, Ser, Trp or Tyr for the amino acid of position 434;
[0627] [0627] Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid of position 436;
[0628] [0628] Lys, Leu, Thr or Trp for the amino acid of position 438;
[0629] [0629] Lys for the amino acid of position 440; and
[0630] [0630] Lys for the amino acid of position 442; Ile, Pro, or Thr for the amino acid of position 308;
[0631] [0631] as indicated by the EU numbering following amino acids from the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0632] [0632] [14 "] the method of any one from [1"] to [9 "], where the Fc region is an Fc region with increased FcRn binding activity under a range condition neutral pH compared to that of the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0633] [0633] [15 "] the method of [14"], in which the Fc region is a region of Fc with a substitution of at least one or more amino acids selected from the group consisting of positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino acid sequence of the Fc region represented by any of SEQ ID NOS: 13 , 14, 15, and 16;
[0634] [0634] [16 "] the [15"] method, in which the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0635] [0635] Met for the amino acid of position 237;
[0636] [0636] Ile for the amino acid of position 248;
[0637] [0637] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250;
[0638] [0638] Phe, Trp or Tyr for the amino acid of position 252;
[0639] [0639] Thr for the amino acid of position 254;
[0640] [0640] Glu for the amino acid of position 255;
[0641] [0641] Asp, Asn, Glu or Gln for the amino acid of position 256;
[0642] [0642] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257;
[0643] [0643] His for the amino acid of position 258;
[0644] [0644] Wing for the amino acid of position 265;
[0645] [0645] Ala or Glu for the amino acid of position 286;
[0646] [0646] His for the amino acid of position 289;
[0647] [0647] Wing for the amino acid of position 297;
[0648] [0648] Wing for the amino acid of position 303;
[0649] [0649] Wing for the amino acid of position 305;
[0650] [0650] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307;
[0651] [0651] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308;
[0652] [0652] Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309;
[0653] [0653] Ala, His or Ile for the amino acid of position 311;
[0654] [0654] Ala or His for the amino acid of position 312;
[0655] [0655] Lys or Arg for the amino acid of position 314;
[0656] [0656] Ala, Asp or His for the amino acid of position 315;
[0657] [0657] Wing for the amino acid of position 317;
[0658] [0658] Val for the amino acid of position 332;
[0659] [0659] Read for the amino acid of position 334;
[0660] [0660] His for the amino acid at position 360;
[0661] [0661] Wing for the amino acid of position 376;
[0662] [0662] Wing for the amino acid of position 380;
[0663] [0663] Wing for the amino acid of position 382;
[0664] [0664] Wing for the amino acid of position 384;
[0665] [0665] Asp or His for the 385 position amino acid;
[0666] [0666] Pro for the amino acid of position 386;
[0667] [0667] Glu for the amino acid of position 387;
[0668] [0668] Ala or Ser for the amino acid of position 389;
[0669] [0669] Wing for the amino acid of position 424;
[0670] [0670] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 428;
[0671] [0671] Lys for the amino acid of position 433;
[0672] [0672] Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and
[0673] [0673] His, Ile, Leu, Phe, Thr or Val for the amino acid of position 436;
[0674] [0674] as indicated by the EU numbering following amino acids from the Fc region represented by any of SEQ ID NOS: 13, 14, 15, and 16;
[0675] [0675] [17 "] the method of any one from [1"] to [13 "], wherein the Fc region includes an Fc region that has a higher Fcγ receptor binding activity than that of the native human IgG Fc region;
[0676] [0676] [18 "] the [17"] method, wherein the Fc region comprises in its amino acid sequence at least one or more amino acids that are different from the amino acids of the Fc region of native human IgG selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 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,
[0677] [0677] [19 "] the [18"] method, in which the Fc region comprises in its amino acid sequence at least one or more amino acids selected from the group consisting of:
[0678] [0678] Lys or Tyr for the amino acid of position 221;
[0679] [0679] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[0680] [0680] Phe, Trp, Glu or Lys for the amino acid of position 223;
[0681] [0681] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[0682] [0682] Glu, Lys or Trp for the amino acid of position 225;
[0683] [0683] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[0684] [0684] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[0685] [0685] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[0686] [0686] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[0687] [0687] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[0688] [0688] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[0689] [0689] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[0690] [0690] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235;
[0691] [0691] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[0692] [0692] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237;
[0693] [0693] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[0694] [0694] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239;
[0695] [0695] Ala, Ile, Met, or Thr for the amino acid at position 240;
[0696] [0696] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[0697] [0697] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[0698] [0698] His for the amino acid at position 244;
[0699] [0699] Wing for the amino acid of position 245;
[0700] [0700] Asp, Glu, His or Tyr for the amino acid of position 246;
[0701] [0701] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[0702] [0702] Glu, His, Gln or Tyr for the amino acid of position 249;
[0703] [0703] Glu or Gln for the amino acid of position 250;
[0704] [0704] Phe for the amino acid of position 251;
[0705] [0705] Phe, Met, or Tyr for the amino acid of position 254;
[0706] [0706] Glu, Leu or Tyr for the amino acid of position 255;
[0707] [0707] Ala, Met, or Pro for the amino acid of position 256;
[0708] [0708] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[0709] [0709] Asp, Glu, His or Tyr for the amino acid of position 260;
[0710] [0710] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[0711] [0711] Ala, Ile, Met, or Thr for the amino acid of position 263;
[0712] [0712] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[0713] [0713] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[0714] [0714] Ala, Ile, Met, or Thr for the amino acid of position 266;
[0715] [0715] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[0716] [0716] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[0717] [0717] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[0718] [0718] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[0719] [0719] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[0720] [0720] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[0721] [0721] Phe or Ile for the amino acid of position 273;
[0722] [0722] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[0723] [0723] Leu or Trp for the amino acid at position 275;
[0724] [0724] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[0725] [0725] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 278;
[0726] [0726] Wing for the amino acid of position 279;
[0727] [0727] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[0728] [0728] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[0729] [0729] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282;
[0730] [0730] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[0731] [0731] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284;
[0732] [0732] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[0733] [0733] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[0734] [0734] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[0735] [0735] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[0736] [0736] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[0737] [0737] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[0738] [0738] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[0739] [0739] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[0740] [0740] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[0741] [0741] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[0742] [0742] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297;
[0743] [0743] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[0744] [0744] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the 299 amino acid;
[0745] [0745] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 300;
[0746] [0746] Asp, Glu, His or Tyr for the amino acid of position 301;
[0747] [0747] Ile for the amino acid of position 302;
[0748] [0748] Asp, Gly or Tyr for the amino acid of position 303;
[0749] [0749] Asp, His, Leu, Asn or Thr for the amino acid of position 304;
[0750] [0750] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[0751] [0751] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311;
[0752] [0752] Phe for the amino acid of position 313;
[0753] [0753] Read for the amino acid of position 315;
[0754] [0754] Glu or Gln for the amino acid of position 317;
[0755] [0755] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[0756] [0756] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 320;
[0757] [0757] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[0758] [0758] Ile for the amino acid of position 323;
[0759] [0759] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[0760] [0760] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[0761] [0761] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326;
[0762] [0762] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[0763] [0763] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[0764] [0764] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329;
[0765] [0765] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[0766] [0766] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid at position 331;
[0767] [0767] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[0768] [0768] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the amino acid of position 333;
[0769] [0769] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[0770] [0770] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[0771] [0771] Glu, Lys or Tyr for the amino acid of position 336;
[0772] [0772] Glu, His or Asn for the amino acid at position 337;
[0773] [0773] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[0774] [0774] Ala or Val for the amino acid of position 376;
[0775] [0775] Gly or Lys for the amino acid of position 377;
[0776] [0776] Asp for the amino acid of position 378;
[0777] [0777] Asn for the amino acid of heading 379;
[0778] [0778] Ala, Asn or Ser for the amino acid of position 380;
[0779] [0779] Ala or Ile for the amino acid of position 382;
[0780] [0780] Glu for the amino acid of position 385;
[0781] [0781] Thr for the amino acid at position 392;
[0782] [0782] Read for the amino acid of position 396;
[0783] [0783] Lys for the amino acid of position 421;
[0784] [0784] Asn for the amino acid of heading 427;
[0785] [0785] Phe or Leu for the amino acid of position 428;
[0786] [0786] Met for the amino acid of position 429;
[0787] [0787] Trp for the amino acid of position 434;
[0788] [0788] Ile for the amino acid of position 436; and
[0789] [0789] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[0790] [0790] as indicated by the EU numbering;
[0791] [0791] [20 "] the method of anyone from [1"] to [16 "], in which the Fc region has a higher binding activity towards an inhibitory Fcγ receptor than towards to an Fcγ activation receptor;
[0792] [0792] [21 "] the [20"] method, wherein the inhibitory Fcγ receptor is human FcγRIIb;
[0793] [0793] [22 "] the [20"] or [21 "] method, in which the Fcγ activation receptor is human FcγRIa, human FcγRIIa (R), human FcγRIIa (H), FcγRIIIa ( V) human or human FcγRIIIa (F);
[0794] [0794] [23 "] the method of any one from [20"] to [22 "], where the amino acid at position 238 or 328 (EU numbering) in the Fc region is different from the amino acid in the IgG Fc region native human;
[0795] [0795] [24 "] the [23"] method, wherein the amino acid at position 238 of the Fc region is Asp or the amino acid at position 328 of the Fc region is Glu as indicated by the EU number; and
[0796] [0796] [25 "] the [23"] or [24 "] method, in which the amino acid sequence of the Fc region comprises at least one or more amino acids selected from the group consisting of:
[0797] [0797] Asp for the amino acid of position 233;
[0798] [0798] Trp or Tyr for the amino acid of position 234;
[0799] [0799] Ala, Asp, Glu, Leu, Met, Phe, Trp or Tyr for the amino acid of position 237;
[0800] [0800] Asp for the amino acid of position 239;
[0801] [0801] Ala, Gln or Val for the amino acid of position 267;
[0802] [0802] Asn, Asp or Glu for the amino acid of position 268;
[0803] [0803] Gly for the amino acid of position 271;
[0804] [0804] Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser or Thr for the amino acid of position 326;
[0805] [0805] Arg, Lys, or Met for the amino acid of position 330;
[0806] [0806] Ile, Leu, or Met for the amino acid of position 323; and
[0807] [0807] Asp for the amino acid of position 296;
[0808] [0808] as indicated by the EU numbering; Brief description of the drawings
[0809] [0809] Fig. 1 is a diagram showing that an antibody with pH-dependent binding repeatedly binds to soluble antigens.
[0810] [0810] Fig. 2 is a diagram showing that increasing FcRn binding under neutral conditions results in improving the effect of a pH-dependent binding antibody to bind antigens repeatedly: (i) an antibody binds to soluble antigens; (ii) the antibody is incorporated into a cell by PEGcytosis via FcRn; (iii) soluble antigens dissociate from the antibody in the endosome; (iv) the soluble antigens are transferred to the lysosome and degraded; (v) after dissociation of the soluble antigens, the antibody is recycled to plasma via FcRn; and (vi) the recycled antibody can bind to soluble antigens again.
[0811] [0811] Fig. 3 shows Biacore sensorgrams showing the interaction of anti-human IgA antibodies with human IgA under conditions of Ca2 + 1.2 mM and Ca2 + 3 M.
[0812] [0812] Fig. 4 is a graph showing the time course changes in plasma antibody concentration in normal mice in a group administered with antibody to human IgA + GA1-IgG1, group administered with antibody to human IgA + GA2-IgG1, groups administered with antibody to human IgA + GA2-FcγR (-) or GA2-N434W.
[0813] [0813] Fig. 5 is a graph showing the time course changes in plasma human IgA concentration in normal mice in the group administered with human IgA only, group administered with antibody to human IgA + GA1-IgG1, group ad -
[0814] [0814] Fig. 6 is a graph showing time course changes in the plasma concentration of unbound human IgA in normal mice in a group administered with antibody to human IgA + GA1-IgG1, a group administered with antibody to human IgA + GA2-IgG1, group administered with antibody to human IgA + GA2-FcγR (-) and group administered with antibody to human IgA + GA2-N434W.
[0815] [0815] Fig. 7 is an illustrative diagram showing the efficiency of antigen elimination by antibody molecule of an antibody dependent on pH / Ca that has the natural IgG1 constant region that forms a large immune complex with a multimeric antigen .
[0816] [0816] Fig. 8 is an illustrative diagram showing the efficiency of antigen elimination by antibody molecule of a pH / Ca dependent multispecific antibody that recognizes two or more epitopes in a monomeric antigen and is suitable for the formation of a large immune complex.
[0817] [0817] Fig. 9 shows the results of chromatographic analyzes with gel filtration that confirmed that human IgE and clone 278, which is a pH-dependent anti-IgE antibody, forms large immune complexes in a pH-dependent manner.
[0818] [0818] Fig. 10 is a graph showing the time course changes in plasma antibody concentration in normal mice from the group administered with clone 278 + human IgE and the group administered with antibody to human IgE + Xolair.
[0819] [0819] Fig. 11 is a graph showing the time course changes in plasma human IgE concentration in normal mice in the group administered with human IgE only, the group administered with antibody to human IgE + clone 278, and the group administered with antibody to human IgE + clone 278.
[0820] [0820] Fig. 12 is a graph showing time course changes in plasma concentrations of GA2-IgG1 and GA2-F1087 antibodies in normal mice.
[0821] [0821] Fig. 13 is a graph showing time course changes in plasma hIgA concentration in normal mice administered with GA2-IgG1 or GA2-F1087.
[0822] [0822] Fig. 14 is a graph showing time course changes in plasma concentrations of 278-IgG1 and 278-F1087 antibodies in C57BL / 6J mice.
[0823] [0823] Fig. 15 is a graph showing time course changes in plasma hIgE (Asp6) concentration in C57BL / 6J mice administered 278-IgG1 or 278-F1087.
[0824] [0824] Fig. 16 is a graph showing the time course changes in the plasma concentration of GA2-F760 or GA2-F1331 in transgenic human FcRn mice administered with GA2-F760 or GA2-F1331.
[0825] [0825] Fig. 17 is a graph showing the time course changes in the plasma concentration of human IgA in transgenic human FcRn mice administered with GA2-F760 or GA2-F1331.
[0826] [0826] Fig. 18 is a graph showing the time course changes in the plasma concentration of 278-F760 or 278-F1331 in transgenic human FcRn mice administered with 278-F760 or 278-F1331.
[0827] [0827] Fig. 19 is a graph showing the time course changes in plasma human IgE concentration in transgenic human FcRn mice administered with 278-F760 or 278-F1331.
[0828] [0828] Fig. 20 presents sensorgrams showing the interaction of PHX-IgG1 with hsIL-6R at pH 7.4 and pH 6.0.
[0829] [0829] Fig. 21 shows the result of evaluating the simultaneous binding of Fv4-IgG1 and PHX-F29 with IL6R by the electrochemiluminescence (ECL) method.
[0830] [0830] Fig. 22 is a graph showing the time course changes in the concentration of anti-IL6R antibody in the plasma of normal mice in the group administered with hsIL6R + Fv4-IgG1- and groups administered with hsIL6R + Fv4-IgG1 + PHX-IgG1.
[0831] [0831] Fig. 23 is a graph showing time course changes in the concentration of human IL6R in the plasma of normal mice in the group administered with hsIL6R + Fv4-IgG1 and groups administered with hsIL6R + Fv4-IgG1 + PHX- IgG1.
[0832] [0832] Fig. 24 shows Biacore sensorgrams obtained when a mixed antibody solution alone or a mixed antibody-antigen solution was applied to FcγR. The dashed line shows the sensorgram obtained when the antibody solution alone was applied, and the solid line shows the sensorgram obtained when the mixed antibody-antigen solution was applied.
[0833] [0833] Fig. 25 presents Biacore sensorgrams obtained when a mixed antibody solution alone or a mixed antibody-antigen solution was applied to human FcRn. The solid line shows the sensorgram obtained when the antibody solution alone was applied, and the dashed line shows the sensorgram obtained when the mixed antibody-antigen solution was applied.
[0834] [0834] Fig. 26 shows Biacore sensorgrams obtained when a mixed antibody solution alone or a mixed antibody-antigen solution was applied to the mouse FcRn. The dashed line shows the sensorgram obtained when the antibody solution alone was applied, and the solid line shows the sensorgram obtained when the mixed antibody-antigen solution was applied.
[0835] [0835] Fig. 27 shows Biacore sensorgrams obtained when a mixed antibody solution alone or a mixed antibody-antigen solution was applied to the mouse FcRn. The dashed line shows the sensorgram obtained when the antibody solution alone was applied, and the solid line shows the sensorgram obtained when the mixed antibody-antigen solution was applied. Mode for Carrying Out the Invention
[0836] [0836] The definitions and detailed description below are provided to aid the understanding of the present invention illustrated in this application. Amino Acids
[0837] [0837] In this application, amino acids are described in one or three letter codes or both, for example, 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 modification
[0838] [0838] For amino acid modification in the amino acid sequence of an antigen-binding molecule, methods known as site-directed mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately adopted. In addition, several known methods can also be adopted as amino acid alteration methods for substitution by unnatural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci USA (2003) 100 (11), 6353-6357). For example, it is appropriate to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA that has an unnatural amino acid linked by an amber suppressor tRNA complementary to the UAG codon (amber codon) which is a stop codons.
[0839] [0839] In this specification, the meaning of the word "and / or" describing the site of the amino acid change includes each combination where "e" and "or" are appropriately combined. Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following variation of amino acid modifications:
[0840] [0840] amino acid (s) at position (a) 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96 and positions (g) 33, 55, and 96. Antigenic binding units
[0841] [0841] In this application, "antigen binding units" refers to the "number of epitopes present in a single antigen molecule in a form normally present in plasma in the absence of an antigen binding molecule, in which the molecule antigen comprises an epitope that binds to a monovalent binding unit of an antigen binding domain contained in an antigen binding molecule of the present invention ". Examples of an antigen that has two antigenic binding units are antigens including multimers usually present in plasma in the form of homodimers such as GDF, PDGF or VEGF. For example, in homodimerized GDF molecules, there are two epitope units each linked by a monovalent variable region binding unit contained in an anti-GDF antibody molecule that comprises two variable regions having the same sequence ( that is, not the bispecific antibody to be described later). Immunoglobulin molecules such as IgE are also included as examples of antigens with two antigenic binding units. IgE normally exists in the plasma as a tetramer comprising a heavy chain dimer and a light chain dimer; and in the tetramer, there are two epitope units each linked by a unique valence of a variable region contained in an anti-IgE antibody molecule comprising two variable regions that have the same sequence (i.e., not the bispecific antibody described below) . IgA normally exists in plasma in two forms: a tetramer comprising a heavy chain dimer and a light chain dimer and an octamer produced when the tetramers still form a complex via a J chain; and the tetramer and octamer respectively have two units and four epitope units each linked by a unique valence of a variable region contained in an anti-IgA antibody molecule comprising two variable regions that have the same sequence (i.e., not the bispecific antibody described below). Examples of antigens with three antigenic binding units are antigens including multimers usually present in plasma in the form of homotrimers such as TNFα, RANKL or CD154 from the TNF superfamily.
[0842] [0842] Examples of antigen molecules with an antigen binding unit are molecules generally present in plasma in the form of monomers as a soluble IL-6 receptor (hereinafter also referred to as sIL-6R), IL-6, HMGB -1 and CTGF. Heterodimers such as IL-12 comprising IL-12p40 and IL-12p35, IL-23 comprising IL-12p40 and IL-23p19 (also referred to as IL-30B), IL-23 comprising EBI3 and IL27p28 and IL-35 comprising IL-12p35 and EBI3 contain two structurally different subunit molecules. In an anti-subunit antibody molecule comprising two variable regions that bind to one of these subunits (that is, not the bispecific antibody described below), the epitope bound by a single valence of the variable regions is one unit; therefore, the antigenic binding unit of these heterodimers is that. The antigenic binding unit of heterotrimers such as the multi-subunit complex TNFα-TNFβ-hCG is similarly a unit.
[0843] [0843] Since then "the antigenic binding unit" means the number of epitopes attached by a monovalent unit of binding to an antigen-binding domain, when there is one type of paratope or multiple types of paratopes present in a molecule of binding tion to the antigen, the antigen binding unit of the antigen will be different even if the antigen bound by these antigen binding molecules is the same antigen. For example, in the case of the aforementioned heterodimers, when the monovalent binding unit in the antigen binding domain contained in the antigen binding molecule binds to a type of subunit in the heterodimer, the antigen binding unit of this antigen is one, whereas when the antigen binding molecule is a biparatopic or a bispecific antigen binding molecule and comprises two monovalent binding units that bind to each of the subunits that form the heterodimer , the antigen binding unit of this antigen is two. Multimers
[0844] [0844] In this application, when the phrase "two or more multimers" is simply quoted, the meaning of the word "multimer" includes both homomultimer and heteromultimer. The bonding modes between the subunits contained in a multimer are covalent bonds like peptide bonds or disulfide bonds and stable non-covalent bonds like ionic bonds, van der Waals bonds and hydrogen bonds, but are not limited to these. Homomultimers include a plurality of identical subunits, whereas hetero-multimers contain a plurality of different subunits. For example, the meaning of the word "dimer" includes both homodimers and heterodimers; and while a homodimer contains two identical subunits, a heterodimer contains two different subunits. In addition, in the case of a polypeptide, the term "monomer" that expresses each of the units that form a multimer refers to each of the continuous structural units linked by peptide bonds. Antigens
[0845] [0845] In this application, "antigens" are not particularly limited in structure, as long as they comprise epitopes to which antigen-binding domains are bound.
[0846] [0846] Other antigens include, for example, the molecules below: 17-1A, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor , A33, ACE, Ace-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, adressin, 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, artemine, anti-Id, ASPARTIC, atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7- H, lymphocyte stimulation factor B (BlyS), BACE, BACE-1, Bad, BAFF, BAFF- R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP -7 (OP-1), MBP-8 (BMP-8a, OP-2), BMPR, BMPR-1A (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR -II (BRK-3), BM P, b-NGF, BOK, bom-besine, bone-derived neutrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin V, cathepsin V X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, C-
[0847] [0847] "Epitope" means an antigenic determinant in an antigen and refers to an antigenic site to which the antigen-binding domain of an antigen-binding molecule presented here binds. In this way, for example, the epitope can be defined according to its structure. Alternatively, the epitope can 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 can be specified by the amino acid residues that make up the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be specified by its specific sugar chain structure. A linear epitope is an epitope that contains an epitope whose primary amino acid sequence has been recognized. Such a linear epitope typically contains
[0848] [0848] As described later, when an antigen binding molecule binds to a plurality of epitopes on an antigen molecule as with a bispecific antibody and such, the antigen that can form a complex with the antigen binding molecule to be any of the antigens exemplified above, or combinations thereof, that is, monomers or heteromultimers. Non-limiting examples of heteromultimers include heterodimers such as IL-12 comprising IL-12p40 and IL-12p35, IL-23 comprising IL-12p40 and IL-23p19 (also referred to as IL-30B), IL-23 comprising EBI-
[0849] [0849] While the receptors are cited as examples of the aforementioned antigens, when these receptors exist in forms soluble in biological fluids such as plasma, they can form complexes with the antigen-binding molecules of the present invention. Therefore, while the aforementioned receptors exist in their soluble forms in biological fluids such as plasma, they can be used as antigens that can form complexes of the present invention by binding to an antigen-binding molecule of the present invention. An example of a non-limiting embodiment of such a soluble receptor is soluble IL-6R, which is a protein consisting of amino acids at positions 1 to 357 in the IL-6R polypeptide sequence of SEQ ID NO: 1 as described in Mullberg et al . (J. Immunol. (1994) 152 (10), 4958-4968).
[0850] [0850] Soluble antigens are cited as examples of the aforementioned antigens, and the solutions in which the antigens exist are not limited. Soluble antigens can exist in biological fluids, or more specifically in all fluids that fill the space between tissues and cells or vessels in organisms. In a non-limiting mode, the antigens to which the antigen-binding molecules of the present invention bind may be present in extracellular fluids. In vertebrates, extracellular fluid is a general term for plasma, interstitial fluid, lymph, compact connective tissue, cerebrospinal fluid, spinal fluid, puncture fluid, synovial or such components in bone and cartilage, alveolar fluid (bronchoalveolar lavage fluid), fluid peritoneal, pleural fluid, pericardial fluid, cyst fluid, aqueous humor (hydatoid) or such transcellular fluids (various fluids in the glandular cavities and fluids in the digestive tract cavity and other body cavity fluids produced in consequence active transport / cell secretory activities).
[0851] [0851] When the epitope to which the antigen-binding domain contained in an antigen-binding molecule binds is a unique epitope, a non-limiting modality of an antigen that can form a complex of the present invention to bind with antigen-binding molecule includes the molecules exemplified below, which include homodimers, homotrimers, or the like, as an antigenic binding unit: 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) , TNF, TNF-alpha, TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL Apo-2 linker, TL2), TNFSF11 (ODANCE TRANCE / RANK linker, OPG linker), TNFSF12 (TWEAK Apo-3 linker, DR3), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM linker, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR linker AITR link, TL6) , TNFSF1A (Conectin TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb T NFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (ligand CD27 CD70), TNFSF8 (CD30 CD153 ligand), TNFSF9 (CD137 ligand 4-1BB ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta Pan Spec and IL-8. Epitopes
[0852] [0852] "Epitope" means an antigenic determinant in an antigen and refers to an antigen site to which the antigen binding domain of an antigen binding molecule described in this application binds. In this way, for example, the epitope can be defined according to its structure. Alternatively, the epitope can 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 can be specified by the amino acid residues that make up the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be specified by its specific sugar chain structure.
[0853] [0853] A linear epitope is an epitope that contains an epitope whose primary amino acid sequence has been recognized. Such a linear epitope typically contains at least three and even more commonly at least five, for example, approximately 8 to 10 or 6 to 20 amino acids in a specific sequence.
[0854] [0854] In contrast to the linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence containing the epitope is not the only determinant of the recognized epitope (for example, the primary amino acid sequence of a conformational epitope is not necessarily recognized by an antibody that defines the epitope). Conformational epitopes can contain a greater number of amino acids compared to linear epitopes. A conformational antibody that recognizes the epitope recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds up and forms a three-dimensional structure, the main chains of amino acids and / or polypeptides that form a conformational epitope are aligned, and the epitope is made recognizable by the antibody. Methods for determining epitope formations include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin marking and electron paramagnetic resonance, but are not limited to these. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).
[0855] [0855] The structure of the antigen-binding domain that binds to an epitope is called a paratope. An epitope and a paratope bond with stability through the action of hydrogen bonds,
[0856] [0856] Examples of a method for assessing epitope binding by a test antigen-binding molecule that contains an IgA-directed antigen-binding domain are described below. According to the examples below, methods for assessing the binding of the epitope by a test antigen binding molecule that contains an antigen binding domain of an IgA antigen except can also be properly conducted.
[0857] [0857] For example, if a test antigen binding molecule containing an IgA antigen binding domain recognizes a linear epitope on the IgA molecule can be confirmed, for example, as mentioned below. For example, a linear peptide comprising an amino acid sequence that forms the IgA constant region is synthesized for the purpose above. The peptide can be synthesized chemically or obtained by genetic engineering techniques using a region that encodes the sequence of amino acids that corresponds to the constant region in an IgA cDNA. Then, a test antigen binding molecule containing an antigen binding domain towards IgA is evaluated for its binding activity towards a linear peptide comprising the amino acid sequence that forms the constant region. For example, an ELISA using a linear peptide immobilized as an antigen can be performed to assess the binding activity of the antigen-binding molecule towards the peptide. Alternatively, the binding activity towards a linear peptide can be assessed based on the level of inhibition by the linear peptide of the binding of the antigen binding molecule towards cells with IgA. These tests can demonstrate the binding activity of the antigen-binding molecule towards the linear peptide.
[0858] [0858] The recognition of a conformational epitope by a test antigen binding molecule comprising an antigen binding domain and targeting the IgA protein can be confirmed as stated below. For the aforementioned objective, as described in this application, a general genetic recombination technique is used to transfer a recombinant gene that encodes IgA to host cells (for example, animal cells, insect cells or yeast cells) that allow formation of the native conformational epitope on the IgA protein. IgA containing the conformational epitope is prepared from the culture of recombinant cells produced in this way. The recognition of a conformational epitope by a test antigen binding molecule that comprises a targeting of the IgA antigen binding domain is, for example, when the test antigen binding molecule binds strongly to the IgA molecule when it is brought into contact with immobilized IgA that contains the conformational epitope, while the antigen binding molecule does not substantially bind to a linear peptide comprising an amino acid sequence that constitutes the immobilized IgA amino acid sequence. Alternatively, it is also possible to use, instead of the aforementioned linear peptide, the IgA-binding antigen test molecule that has been denatured by a reducing agent that cleaves disulfide bonds, such as dithiothreitol, dithioerythritol, β-mercaptoethanol,
[0859] [0859] Methods for analyzing IgA-binding activity of a test antigen-binding molecule that contains an IgA-binding antigen domain include, for example, the methods described in Antibodies: A Laboratory Manual (Ed Harlow , David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the evaluation can be carried out based on the principle of ELISA or EIA using IgA as antigen.
[0860] [0860] In ELISA format, the binding activity of a test antigen binding molecule that contains an IgA antigen binding domain towards IgA can be quantitatively assessed by comparing the levels of the signal generated by the reaction enzymatic. Specifically, a test antigen-binding molecule is added to an ELISA plate to which IgA has been immobilized. Then, the test antigen binding molecule that bound to the immobilized IgA on the plate is detected using an antibody labeled with the enzyme that recognizes the test antigen binding molecule. In ELISA, a serial dilution of the test antigen-binding molecule can be prepared and the antibody-binding titer towards IgA to be determined to compare the binding activity of the test-antigen binding molecule towards IgA.
[0861] [0861] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in buffer or the like, can be detected using a flow cytometer.
[0862] [0862] FACSCantoTM II
[0863] [0863] FACSAriaTM
[0864] [0864] FACSArrayTM
[0865] [0865] FACSVantageTM SE
[0866] [0866] FACSCaliburTM (all are trade names of BD Biosciences)
[0867] [0867] EPICS ALTRA HyPerSort
[0868] [0868] Cytomics FC 500
[0869] [0869] EPICS XL-MCL ADC EPICS XL ADC
[0870] [0870] Cell Lab Quanta / Cell Lab Quanta SC (all are trade names of Beckman Coulter).
[0871] [0871] Preferred methods for assessing binding activity against an antigen from an antigen binding molecule test containing an antigen binding domain against IgE include, for example, the following method. First, cells that express IgE are reacted with a test antigen-binding molecule, and then they are stained with a secondary FITC-labeled antibody that recognizes the antigen-binding molecule. The test antigen binding molecule is appropriately diluted with an appropriate buffer to prepare the molecule to a desired concentration. For example, the molecule can be used at a concentration within the range of 10 µg / ml to 10 ng / ml. Then, the fluorescence intensity and cell count are determined using FACSCalibur (BD). The fluorescence intensity is obtained by analysis using the CELL QUEST (BD) program, that is, the average geometric value reflects the amount of antibody bound to the cells. That is, the binding activity of a molecule binding to the test antigen, which is represented by the amount of the molecule binding to the test antigen bound, can be determined by measuring the mean geometric value.
[0872] [0872] It can be assessed whether a test antigen binding molecule containing an IgA antigen binding domain shares a common epitope with another antigen binding molecule based on competition between the two molecules for the same epitope. The competition between the antigen-binding molecules can be detected by a cross-block assay or the like. For example, the competitive ELISA assay is a preferred cross-block assay.
[0873] [0873] Specifically, in the cross-blocking assay, IgA protein immobilized to the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen binding molecule and then a molecule binding to test antigen is added to that. The amount of the test antigen binding molecule bound to the IgA protein in the wells is indirectly correlated with the binding capacity of a candidate competing antigen binding molecule that competes for binding to the same epitope. That is, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test-antigen-binding molecule to the wells coated with the IgA protein.
[0874] [0874] The amount of the test antigen-binding molecule linked to the wells via the IgA protein can be easily determined by marking the antigen-binding molecule in advance. For example, a biotin-labeled antigen-binding molecule is measured using an avidin / peroxidase conjugate and a suitable substrate. In particular, the cross-block assay that uses enzyme markers such as peroxidase is called a "competitive ELISA assay". The antigen-binding molecule can also be labeled with other labeling substances that allow detection or measurement. Specifically, radiomarkers, fluorescent labels, and the like, are known.
[0875] [0875] When the candidate competing antigen binding molecule can block binding by a test antigen binding molecule containing an IgA antigen binding domain by at least 20%, preferably at least 20 to 50%, and more preferred by at least 50% compared to binding activity in a control experiment conducted in the absence of the competing antigen binding molecule, it is determined that the test antigen binding molecule binds substantially to the same epitope li - cattle for the competing antigen binding molecule, or competing for binding to the same epitope.
[0876] [0876] When the structure of an epitope linked by a test antigen binding molecule containing an IgA antigen binding domain has already been identified, it can be assessed whether the test and control antigen binding molecules share a common epitope by comparing the binding activities of the two antigen-binding molecules to a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.
[0877] [0877] To measure the binding activities above, for example, the binding activities of the molecules binding to the test and control antigen to a linear peptide into which a mutation is introduced are compared in the ELISA format above. In addition to ELISA methods, the binding activity to the mutant peptide bound to a column can be determined by making the test and control antigen binding molecules flow in the column, and then quantifying the antigen binding molecule eluted in the column. elution solution. Methods for adsorbing a mutant peptide to a column, for example, in the form of a GST fusion peptide, are known.
[0878] [0878] Alternatively, when the epitope identified in an
[0879] [0879] In the above method, one can evaluate whether an antigen binding molecule that "does not substantially bind to cells that express mutant IgE", for example, using the following method. First, test antigen-binding and control molecules bound to cells that express mutant IgE are labeled with a labeled antibody. Then, the fluorescence intensity of the cells is determined. When FACSCalibur is used to detect fluorescence by flow cytometry, the determined fluorescence intensity can be analyzed using the CELL QUEST program. From the geometric mean values in the presence and absence of the polypeptide complex, the comparison value (∆mean-Geo) can be calculated according to the following formula to determine the proportion of increase in fluorescence intensity as a result of binding by antigen binding molecule.
[0880] [0880] ∆medium-Geo = medium-Geo (in the presence of the polypeptide complex) / medium-Geo (in the absence of the polypeptide complex)
[0881] [0881] The geometric mean comparison value (da-Geo-mean value for the mutant IgE molecule) determined by the above analysis, which reflects the amount of a test antigen-binding molecule bound to cells expressing IgE mutant, is compared with the comparison value of the ∆mean-Geo that reflects the amount of the test antigen-binding molecule bound to cells that express IgE. In this case, the concentrations of the test antigen-binding molecule used to determine the ∆mean-Geo comparison values for cells that express IgE and cells that express mutant IgE are particularly adjusted, preferably, to be the same or substantially the same. . An antigen binding molecule that has been confirmed to recognize an epitope in IgE is used as a control antigen binding molecule.
[0882] [0882] If the valormean-Geo comparison value of a test antigen-binding molecule for cells expressing the mutant IgE is less than the damean-Geo comparison value of the binding molecule to the test antigen for cells that express IgE in at least 80%, preferably 50%, more preferably, 30%, and particularly preferably 15% so the test antigen binding molecule "does not bind substantially to cells that express
[0883] [0883] In the present invention, an "antigen-binding domain" can have any structure as long as it binds to an antigen of interest. Such domains preferably include, for example:
[0884] [0884] variable regions of heavy chain and light chain of the antibody;
[0885] [0885] a module of about 35 amino acids called domain A that is contained in the cell membrane protein in vivo Avímero (International Publication No. WO 2004/044011, International Publication No.WO 2005/040229);
[0886] [0886] Adnectin containing the 10Fn3 domain that binds to the protein portion of fibronectin, a glycoprotein expressed on the cell membrane (International Publication No.WO 2002/032925);
[0887] [0887] A body that consists of a bundle of three helices with 58 amino acids based on the structure of the IgG-binding domain of protein A (International Publication No.WO 1995/001937);
[0888] [0888] Projected ankyrine repeat proteins (DARPins) which are an exposed region on the molecular surface of ankyrine (AR) repeats that have a structure in which a subunit consisting of a loop comprising 33 amino acid residues, two antiparallel helices, and a loop is repeatedly stacked (International Publication No.WO 2002/020565);
[0889] [0889] Anticalins and the like, which are domains that consist of four loops that support one side of a cylinder structure composed of eight antiparallel strips arranged in a circular fashion that are highly conserved among the lipocalin molecules as lipocalin associated with neutrophil gelatinase ( NGAL) (International Publication No.WO 2003/029462); and
[0890] [0890] The concave region formed by the parallel leaf structure within the horseshoe-shaped structure consisting of stacked repetitions of the leucine-rich repetition module (LRR) of the variable lymphocyte receptor (VLR) that lacks the immune structure - globulin and is used in the immunity system acquired in vertebrates without jaws such as lampery and conger fish (WO 2008/016854). Preferred antigen-binding domains of the present invention include, for example, those having variable regions of the heavy and light chain of the antibody. Preferred examples of antigen binding domains include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab", and " F (ab ') 2 ".
[0891] [0891] The antigen binding domains of the antigen binding molecules of the present invention can bind to an identical epitope. Such an identical epitope may be present, for example, in a protein comprising the amino acid sequence of SEQ ID NO: 2. Alternatively, each of the antigen-binding domains of the antigen-binding molecules of the present invention may attach to a different epitope. In the present invention, the different epitope may be present, for example, in a protein that comprises the amino acid sequence of SEQ ID NO: 2. Immune complex
[0892] [0892] The immune complex refers to a relatively stable structure produced when at least one antigen and at least one antigen-binding molecule bind together to form a higher molecular weight complex. A non-limiting modality of the immune complex is, for example, an antigen-antibody aggregate. A method for assessing the formation of immune complexes comprising two or more antigen binding molecules and two or more antigen binding units will be described later in this specification. Specificity
[0893] [0893] "Specific" means that one of the molecules that specifically binds does not show any significant binding to molecules other than the single or plurality of molecules that bind to it. In addition, "specific" is also used when an antigen-binding domain is specific to a particular epitope among multiple epitopes on an antigen. When an epitope linked by an antigen-binding domain is contained in multiple different antigens, antigen-binding molecules containing the antigen-binding domain can bind to several antigens that have the epitope. In this application, "does not show that any significant link" means to show no more than 50%, generally not more than 30%, preferably not more than 15%, particularly preferably not more than 10%, and even more preferably not more than 5% of the binding activity to a partner molecule towards molecules other than the partner molecule. Antibody
[0894] [0894] In the present invention, "antibody" refers to a natural immunoglobulin or an immunoglobulin produced by partial or complete synthesis. Antibodies can be isolated from natural sources such as naturally occurring plasma and serum, or culture supernatants from antibody-producing hybridomas. Alternatively, antibodies can be synthesized partially or completely using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of an immunoglobulin isotype or subclass that belongs to it. Known human immunoglobulins include antibodies from 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. Various sequences of allotypes of human IgG1, human IgG2, human IgG3 and human IgG4 constant regions due to gene polymorphisms are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242. Any of such sequences can be used in the present invention. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356 to 358 as indicated by the EU numbering can be DEL or EEM. Various sequences of allotypes due to genetic polymorphisms have been described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242 for the human Igκ (Kappa) constant region and Igλ (Lambda) constant region and any of the sequences can be used in the present invention.
[0895] [0895] Methods for producing an antibody with the desired binding activity are known to those skilled in the art. Below is an example that describes a method for producing an antibody that binds to IgA (anti-IgA antibody). Antibodies that bind to antigens other than IgA can also be produced according to the example described below.
[0896] [0896] Anti-IgA antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. The anti-IgA antibodies produced are preferably monoclonal antibodies derived from mammals. Such mammalian-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector that carries an antibody gene by genetic engineering techniques. In turn, "humanized antibodies" or "chimeric antibodies" are included
[0897] [0897] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as described below. Specifically, mammals are immunized by conventional immunization methods using an IgA protein as a sensitizing antigen. The resulting immune cells are fused with parental cells known by conventional cell fusion methods. Then, hybridomas that produce an anti-IgA antibody can be selected by screening the monoclonal antibody-producing cells using conventional screening methods.
[0898] [0898] Specifically, monoclonal antibody production is carried out, for example, as shown below. For example, a purified native IgA protein can be used as a sensitizing antigen. In addition, a recombinant protein comprising an IgA polypeptide sequence of SEQ ID NO: 2, which is registered as L00022 | IGHE * 02 in IMGT / GENE-DB, is purified to obtain soluble IgA from the culture supernatant. To express the recombinant protein, firstly, the IgA heavy chain constant region gene of which the nucleotide sequence is described in SEQ ID NO: 3 can be expressed to obtain the IgA protein shown in SEQ ID NO: 2, which is used as a sensitizing antigen to obtain antibodies. That is, suitable host cells are transformed by inserting a gene sequence that encodes IgA into a known expression vector. The desired IgA protein is purified by methods known to the host cells or their culture supernatant. For expression of the IgA heavy chain constant region gene, the polynucleotide sequence of SEQ ID NO: 3 can be operably linked to the 3 'end of the signal sequence. In another non-limiting modality, the polynucleotide sequence of SEQ ID NO: 3 can be operationally linked to the ex-
[0899] [0899] The purified IgA protein can be used as a sensitizing antigen for immunization of mammals. A partial IgA peptide can also be used as a sensitizing antigen. In this case, a partial peptide can be prepared by chemical synthesis based on the amino acid sequence of human IgA, or by inserting a partial IgA gene into an expression vector for expression. Alternatively, a partial peptide can be produced by degrading an IgA protein with a protease. The length and region of the partial IgA peptide are not limited to specific modalities. A preferred region can be selected arbitrarily from the amino acid sequence of SEQ ID NO: 2. The number of amino acids that form a peptide to be used as a sensitizing antigen is preferably at least five or more, six or more, or seven or more. More specifically, a peptide with 8 to 50 residues, more preferably, 10 to 30 residues can be used as a sensitizing antigen.
[0900] [0900] For the sensitizing antigen, alternatively, it is possible
[0901] [0901] There is no specific limitation on mammals to be immunized with the sensitizing antigen. However, it is preferable to select mammals because they consider their compatibility with the progenitor cells to be used for cell fusion. In general, rodents such as mice, rats, and hamsters, rabbits, and monkeys are preferably used.
[0902] [0902] The above animals are immunized with a sensitizing antigen by known methods. In general, the immunization methods performed include, for example, intraperitoneal or subcutaneous injection of a sensitizing antigen in mammals. Specifically, a sensitizing antigen is appropriately 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. Then, the sensitizing antigen is administered to a mammal several times at intervals of 4 to 21 hours.
[0903] [0903] Alternatively, hybridomas that produce a desired antibody can be prepared using immunization with DNA, as mentioned below. DNA immunization is an immunization method that provides immunological stimulation by expressing a sensitizing antigen in an immunized animal as a result of administering a DNA vector constructed to allow the expression of a gene encoding the protein antigen in the animal. In comparison to conventional immunization methods in which a protein antigen is administered to the animals to be immunized, DNA immunization is expected to be superior where:
[0904] [0904] - in the case where the antigen is a membrane protein, immunological stimulation can be provided while maintaining the structure of the member protein like IgA; and
[0905] [0905] - there is no need to purify the antigen for immunization.
[0906] [0906] In order to prepare a monoclonal antibody of the present invention using DNA immunization, first, a DNA that expresses an IgA protein is administered to an animal to be immunized. The DNA encoding IgA can be synthesized by methods known as PCR. The obtained DNA is inserted into a suitable expression vector, and then it is administered to an animal to be immunized. Preferably, the expression vectors used include, for example, commercially available expression vectors such as pcDNA3.1. Vectors can be administered to an organism using conventional methods. For example, immunization with DNA is done using a gene gun to introduce gold particles coated with an expression vector into cells in the body of an animal to be immunized. The antibodies that recognize IgA can also be produced by the methods described in PCT / JP2011 / 077619.
[0907] [0907] After immunizing a mammal, as described above, the increase in the titer of an IgA binding antibody is confirmed in the serum. Then, immune cells are collected from the mammal, and then subjected to cell fusion. In particular, splenocytes are preferably used as immune cells.
[0908] [0908] A mammalian myeloma cell is used as a cell to be fused with the aforementioned immune cells. Myeloma cells preferably comprise a selection marker suitable for screening. A selection marker gives cells characteristics for their survival (or death) under a specific culture condition. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereafter in this document, abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereafter in this document, abbreviated as TK deficiency) are known as selection markers. Cells with HGPRT or TK deficiency are sensitive to hypoxanthine-aminopterin-thymidine (hereafter in this document, abbreviated as sensitivity to HAT). HAT-sensitive cells do not synthesize DNA in a HAT selection medium and are then killed. However, when cells are fused with normal cells, they can continue DNA synthesis using the normal cell salvage pathway, and therefore they can grow even in the HAT selection medium.
[0909] [0909] HGPRT-deficient and TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-
[0910] [0910] For example, myeloma cells that include the following cells can preferably be used:
[0911] [0911] P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);
[0912] [0912] P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7);
[0913] [0913] NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519);
[0914] [0914] MPC-11 (Cell (1976) 8 (3), 405-415);
[0915] [0915] SP2 / 0 (Nature (1978) 276 (5685), 269-270);
[0916] [0916] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);
[0917] [0917] S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);
[0918] [0918] R210 (Nature (1979) 277 (5692), 131-133), etc.
[0919] [0919] Cellular fusions between immunocytes and myeloma cells are essentially performed using known methods, for example, a method by Kohler and Milstein and others (Methods Enzymol. (1981) 73: 3-46).
[0920] [0920] More specifically, cell fusion can be performed, for example, in a conventional culture medium in the presence of a cell fusion promoting agent. Fusion promoters include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, an auxiliary substance such as dimethyl sulfoxide is also added to improve the fusion efficiency.
[0921] [0921] The ratio of immune cells to myeloma cells can be determined as desired, preferably, for example, one myeloma cell for each one to ten immunocytes. Culture media to be used for cell fusions include, for example, media that are suitable for the cultivation of myeloma cell lines, such as RPMI1640 medium and MEM medium, and another conventional culture medium used for this type of culture cell phone. In addition, serum supplement such as fetal bovine serum (FSB) can be added, preferably, to the culture medium.
[0922] [0922] For cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed well in the above culture medium. Next, a PEG solution (for example, the average molecular weight is about 1,000 to 6,000) preheated to about 37 ° C is added to this at a concentration of generally 30% to 60% (w / v). This is gently mixed to produce the desired fusion cells (hybridomas). Then, a suitable culture medium mentioned above is gradually added to the cells, and it is centrifuged repeatedly to remove the supernatant. In this way, cell fusion agents and the like that are unfavorable for hybridoma growth can be removed.
[0923] [0923] Hybridomas thus obtained can be selected by culture using a conventional selective medium, for example, HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridomas (unfused cells) can be killed by continuous culture in the above HAT medium for a sufficient period of time. Typically, the period is several days to several weeks. Then, the hybridomas that produce the desired antibody are selected and cloned in a unique way by conventional methods.
[0924] [0924] The hybridomas thus obtained can be selected using a selection medium based on the selection marker possessed by the myeloma used for cell fusion. For example, cells deficient in HGPRT or TK can be selected by culture using the HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively proliferate in the HAT medium. Cells other than the desired hybridomas (unfused cells) can be killed by continuous culture in the above HAT medium for a sufficient period of time. Specifically, the desired hybrids can be selected by culture generally for several days to several weeks. Next, hybridomas that produce the desired antibody are selected and cloned uniquely by conventional methods of limiting dilution.
[0925] [0925] The desired antibodies can preferably be selected and cloned in isolation by screening methods based on the known antigen / antibody reaction. For example, an antibody's activity of binding to IgA can be assessed based on the ELISA principle. For example, IgA is immobilized to the wells of an ELISA plate. Hybridoma culture supernatants are placed in contact with immobilized IgA in the wells, and antibodies that bind to the immobilized cells are detected. When monoclonal antibodies are derived from mice, antibodies bound to cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas that produce a desired antibody that has the ability to bind to the antigen are selected by the above screening, and they can be cloned by a limiting dilution method or the like.
[0926] [0926] The monoclonal antibody-producing hybridomas thus prepared can be subcultured in a conventional culture medium, and stored in liquid nitrogen for a long period.
[0927] [0927] The above hybridomas are cultured by a conventional method, and the desired monoclonal antibodies can be prepared from the culture supernatants. Alternatively, hybridomas are administered and cultured in compatible mammals, and monoclonal antibodies are prepared from ascites. The previous method is suitable for preparing antibodies with high purity.
[0928] [0928] Antibodies encoded by antibody genes that are cloned from cells that produce antibodies such as the above hybrids - can also be preferably used. A cloned antibody gene is inserted into a suitable vector, and it is introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting genes into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.
[0929] [0929] For example, a cDNA that encodes the variable region (V region) of an anti-IgA antibody is prepared from hybridoma cells that produce the anti-IgA antibody. For this purpose, the total RNA is first extracted from the hybridomas. Methods used to extract mRNAs from cells include, for example:
[0930] [0930] - the guanidine ultracentrifugation method (Biochemis-try (1979) 18 (24), 5294-5299), and
[0931] [0931] - the AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)
[0932] [0932] The extracted mRNAs can be purified using the mRNA purification kit (GE Healthcare Bioscience) or similar. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience) are also commercially available. MRNAs can be prepared from hybridomas that use such kits. The cDNAs encoding the V region of the antibody can be synthesized from the mRNAs prepared using a reverse transcriptase. CDNAs can be synthesized using the first AMV reverse transcriptase strand cDNA synthesis kit (Seikagaku Co.) or similar. In addition, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002; Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be appropriately used to synthesize and amplify cDNAs. In such a cDNA synthesis process, the appropriate restriction enzyme sites described below can be introduced at both ends of a cDNA.
[0933] [0933] The cDNA fragment of interest is purified from the resulting PCR product, and then it is linked to a vector DNA. The recombinant vector is then constructed, and introduced into E. coli or similar. After colony selection, the desired recombinant vector can be prepared from colony-forming E. coli. Next, it is tested whether the recombinant vector has the cDNA nucleotide sequence of interest by a method known as the didesoxy nucleotide chain termination method.
[0934] [0934] The 5'-RACE method that uses primers to amplify the variable region gene is conveniently used to isolate the gene that encodes the variable region. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNAs extracted from hybridoma cells as a template. A kit available for commercialization such as the SMART RACE cDNA amplification kit is used appropriately to synthesize the 5'-RACE cDNA library.
[0935] [0935] The antibody gene is amplified by PCR using the 5'-RACE cDNA library prepared as a template. Primers to amplify the mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Therefore, it is preferable that the subclass is determined in advance using a kit available for commercialization such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).
[0936] [0936] Specifically, for example, primers that allow the amplification of genes encoding the γ1, γ2a, γ2b, and γ3 heavy chains and the κ and γ light chains are used to isolate genes that encode mouse IgG. In general, a primer that targets a constant region site near the variable region is used as a 3 'side primer to amplify an IgG variable region gene. However, a primer attached to a 5 'RACE cDNA library construction kit is used as a 5' side primer.
[0937] [0937] PCR products thus amplified are used to reform immunoglobulins composed of a combination of heavy and light chains. A desired antibody can be selected using the IgA binding activity of a reformed immunoglobulin as an indicator. For example, when the objective is to isolate an antibody against IgA, it is more preferable that the binding of the antibody to IgA is specific. An IgA-binding antibody can be selected, for example, by one of the following steps:
[0938] [0938] (1) placing an IgA in contact with an antibody comprising the V region encoded by a cDNA isolated from a hybridoma;
[0939] [0939] (2) detecting binding of the antibody to IgA; and
[0940] [0940] (3) select an antibody that binds to IgA.
[0941] [0941] Methods for detecting the binding of an antibody to IgA are known. Specifically, the binding of an antibody to IgA can be detected by the techniques described above as ELISA.
[0942] [0942] Preferred antibody screening methods that use binding activity as an indicator also include separation methods using phage vectors. Methods of screening using phage vectors are advantageous when antibody genes are isolated from libraries of the heavy and light chain subclasses of a population of cells that express a polyclonal antibody. The genes encoding the heavy and light chain variable regions can be linked by a suitable linker sequence to form a single chain Fv (scFv). Phages that have scFv on their surface can be produced by inserting a gene that encodes scFv into a phage vector. The phages are placed in contact with an antigen of interest. Then, a scFv DNA encoding that has the binding activity of interest can be isolated by collecting the phage bound to the antigen. This process can be repeated as needed to enrich the scFv that has the binding activity of interest.
[0943] [0943] After isolation of the cDNA encoding the V region of the anti-IgA antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites introduced at both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs in the nucleotide sequence of the antibody gene at a low frequency. In addition, a restriction site for an enzyme that produces an adhesive end is preferably introduced into a vector to insert a single copy digested fragment in the correct orientation
[0944] [0944] To produce an anti-IgA monoclonal antibody, the antibody's genes are inserted into an expression vector so that genes are expressed under the control of an expression regulatory region. The regulatory region of expression for antibody expression includes, for example, enhancers and promoters. In addition, a suitable signal sequence can be linked to the amino termination so that the expressed antibody is secreted outside the cells. In the examples described below, a peptide that has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 5) can be used as a signal sequence. However, other suitable signal sequences can be linked. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the resulting polypeptide is secreted outside the cells as a mature polypeptide. Then, the appropriate host cells are transformed with the expression vector, and recombinant cells that express the DNA encoding the anti-IgA antibody are obtained.
[0945] [0945] The DNAs encoding the antibody heavy chain (H chain) and the light chain (L chain) are inserted separately into different expression vectors to express the antibody gene. An antibody molecule that has the H and L chains can be expressed by co-transfecting the same host cell with vectors into which the H chain and L chain genes are inserted, respectively. Alternatively, host cells can be transformed with a single expression vector into which the DNAs encoding the H and L chains are inserted (see International Publication No. WO 1994/011523).
[0946] [0946] There are several host cell / expression vector combinations known for the preparation of antibodies by introducing isolated antibody genes into suitable hosts. All of these expression systems are applicable to the isolation of the antibodies of the present invention. Suitable eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, animal cells include, for example, the following cells.
[0947] [0947] mammalian cells: CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myelin (Sp2 / O, NS0, etc.), BHK (monkey kidney cell line) baby hamster), HEK293 (human embryonic kidney cell line with cut adenovirus DNA (Ad) 5), PER.C6 cell (human embryonic retinal cell line transformed with E1A and E1B genes from
[0948] [0948] amphibian cells: Xenopus oocytes, or similar; and
[0949] [0949] insect cells: sf9, sf21, Tn5, or the like.
[0950] [0950] In addition, as a plant cell, an antibody gene expression system that uses cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Cultured callus cells can be used appropriately to transform plant cells.
[0951] [0951] In addition, the following cells can be used as fungal cells:
[0952] [0952] yeasts: the genus Saccharomyces, as Saccharomyces serevisiae, and the genus Pichia, as Pichia pastoris; and
[0953] [0953] filamentous fungi: the genus Aspergillus, as Aspergillus niger.
[0954] [0954] In addition, antibody gene expression systems using prokaryotic cells are also known. For example, when using bacterial cells, E. coli cells, Bacillus subtilis cells, and the like, can be suitably used in the present invention. Expression vectors carrying the antibody genes of interest are introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be prepared from the transformed cell culture.
[0955] [0955] In addition to the host cells described above, transgenic animals can also be used to produce a recombinant antibody. That is, the antibody can be obtained from an animal in which the gene encoding the antibody of interest is introduced.
[0956] [0956] When an antigen-binding molecule described herein is administered to a human, an antigen-binding domain derived from a genetically recombinant antibody that has been artificially modified to reduce heterologous antigenicity against a human and the like, can be used appropriately as the antigen binding domain of the antigen binding molecule. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are produced appropriately by known methods.
[0957] [0957] An antibody variable region used to produce the antigen binding domain of an antigen binding molecule described here is generally formed by three complementarity determining regions (CDRs) that are separated by four regions structural (FRs). CDR is a region that substantially determines the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, FR-forming amino acid sequences often have a high identity even among antibodies with different binding specificities. Therefore, in general, the binding specificity of a particular antibody can be introduced into another antibody by CDR grafting.
[0958] [0958] A humanized antibody is also called a reformed human antibody. Specifically, the humanized antibodies prepared by applying the CDR grafting technology, which grafts the CDRs of a non-human animal antibody, such as a mouse antibody to a human antibody and the like, are known. Common genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting a mouse antibody CDR into a human RF. In overlap extension PCR, a nucleotide sequence encoding a mouse antibody CDR to be grafted is added to primers to synthesize a human antibody FR. Primers are prepared for each of the four FRs. It is generally considered that when grafting a mouse CDR into a human RF, selecting a human RF that has high identity to a mouse RF is advantageous for maintaining the function of the CDR. That is, it is generally preferable to use a human RF that comprises an amino acid sequence that has high identity to the FR amino acid sequence adjacent to the mouse CDR to be grafted.
[0959] [0959] The nucleotide sequences to be linked are designed so that they are connected to each other in the structure. Human FRs are individually synthesized using the respective primers. As a result, products are obtained in which the DNA encoding the mouse CDR is linked to the DNAs encoding the individual FR. Nucleotide sequences that encode the mouse CDR for each product are designed to overlap with each other. Next, a complementary strand synthesis reaction is conducted to annex the overlapping CDR regions of the synthesized products using a human antibody gene as a template. Human FRs are linked via mouse CDR sequences by this reaction.
[0960] [0960] The full-length V region gene, in which three CDRs and four FRs are essentially linked, is amplified using primers that ring with their 5 'or 3' end, which are added with enzyme recognition sequences appropriate restriction An expression vector for a humanized antibody can be produced by inserting the obtained DNA as described above and a DNA encoding a human antibody C region into an expression vector so that they bind to the structure. After the recombinant vector is transfected into a host to establish the recombinant cells, the recombinant cells are cultured, and the DNA encoding the humanized antibody is expressed to produce the humanized antibody in the cell culture (see European Patent publication No. EP 239400 and International Patent Publication No. WO 1996/002576).
[0961] [0961] By measuring qualitatively or quantitatively and evaluating the antigen-binding activity of the humanized antibody produced as described above, one can adequately select human antibody FRs that allow CDRs to form a favorable antigen-binding site when bound through the CDRs. Amino acid residues in FRs can be replaced as needed, so that the CDRs of a reformed human antibody form a suitable antigen binding site. For example, mutations in the amino acid sequence can be introduced into FRs by applying the PCR method used to engraft a mouse CDR into a human RF. More specifically, partial mutations in the nucleotide sequence can be introduced in RF-priming primers. Mutations in the nucleotide sequence are introduced in the FRs synthesized by the use of such primers. The mutant FR sequences that have the desired characteristics can be selected by measuring and evaluating the activity of the mutant antibody with substituted amino acid to bind to the antigen by the method mentioned above (Cancer Res. (1993) 53: 851-856) .
[0962] [0962] Alternatively, the desired human antibodies can be obtained by immunizing transgenic animals that have the entire human antibody gene repertoire (see International Publications Nos. WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO 1996/033735) by immunization with DNA.
[0963] [0963] In addition, techniques for preparing human antibodies by separation 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 phage surface by the phage display method. Phages that express a scFv that binds to the antigen can be selected. The DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined by analyzing the genes of the selected phages. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by merging the sequence of region V in the structure with the sequence of region C of a desired human antibody, and inserting this into a suitable expression vector. The expression vector is introduced into cells suitable for expression, such as those described above. The human antibody can be produced by expression of the gene that encodes the human antibody in cells. These methods are already known (see International Publications Nos. WO 1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO 1995/015388).
[0964] [0964] In addition to the techniques described above, B cell cloning techniques (identification of each antibody coding sequence, cloning and its isolation; use in the construction of the expression vector in order to prepare each antibody ( IgG1, IgG2, IgG3, or IgG4, in particular), and the like) as described in Bernasconi et al. (Science (2002) 298: 2199-2202) or in International Publication No. WO 2008/081008 can be appropriately used to isolate the antibody genes. EU Numbering and Kabat Numbering
[0965] [0965] According to the methods used in the present invention, the amino acid positions assigned to the CDR and FR of the antibody are specified according to the numbering of Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethlehem) , Md., 1987 and 1991)). In the present invention, when an antigen-binding molecule is an antibody or antigen-binding fragment, the amino acids of the variable region are indicated according to the Kabat numbering system (Kabat numbering), while the amino acids of the constant region are indicated according to the EU numbering system based on Kabat's amino acid positions. Ion concentration conditions Metal ion concentration conditions
[0966] [0966] In one embodiment of the present invention, the ion concentration refers to a concentration of metal ion. "Metal ions" refers to ions of group I elements except hydrogen such as alkali metals and copper group elements, group II elements such as alkaline earth metals and zinc group elements, group III elements except boron, elements of group IV except carbon and silicon, elements of group VIII such as iron group and elements of platinum group, 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 to become cations. This is referred to as a tendency to ionization. Metals with a tendency to strong ionization are considered to be chemically attractive.
[0967] [0967] In the present invention, preferred metal ions include, for example, calcium ion. Calcium ion is involved in the modulation of many biological phenomena, including contraction of muscles such as skeletal, smooth and cardiac muscles; activation of movement, phagocytosis and similar leukocytes; activation of change in shape, secretion and the like of platelets; lymphocyte activation; mast cell activation including histamine secretion; cellular responses measured by catecholamine α receptor or acetylcholine receptor; exocytosis; release of transmitting substances from neuron terminals; and axoplastic flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin and myosin light chain, which have several calcium ion binding sites and are believed to be derived from a common origin in terms of molecular evolution. There are also many known calcium binding reasons. Such well-known motifs include, for example, cadherin domains, calmodulin EF-hand, C2 domain of Protein kinase C, Gla domain of blood clotting protein Factor IX, type C lectins of an akoglycoprotein receptor and mannose binding factor. An LDL receptor domain, annexin, thrombospondin type 3 domain and EGF type domains.
[0968] [0968] In the present invention, when the metal ion is a calcium ion
[0969] [0969] Here, the high calcium ion concentration is not particularly limited to a specific value; however, the concentration can preferably be selected between 100 M and 10 mM. In another mode, the concentration can be selected between 200 M and 5 mM. In an alternative mode, the concentration can be selected between 400 M and 3 mM. In yet another modality, the concentration can be selected between 200 M and 2 mM. In addition, the concentration can be selected between 400 M and 1 mM. In particular, a selected concentration between 500 M and 2.5 mM, which is close to the concentration of calcium ion in plasma (blood) in vivo, is preferred.
[0970] [0970] Here, the low calcium ion concentration is not particularly limited to a specific value; however, the concentration can preferably be selected between 0.1 M and 30 M. In another embodiment, the concentration can be selected between 0.2 M and 20 M. In yet another embodiment, the concentration can be selected between 0.5 M and 10 M. In an alternative mode, the concentration can be selected between 1 M and 5 M. Also, the concentration can be selected between 2 M and 4 M. In particular, a selected concentration between 1 M and 5 M, which it is close to the concentration of ionized calcium in initial endosomes in vivo, it is preferred.
[0971] [0971] Here, "antigen-binding activity is less 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 more weaker at a selected calcium ion concentration between 0.1 M and 30 M than at a selected calcium ion concentration between 100 M and 10 mM. Preferably, it means that the antigen-binding activity of an antigen-binding molecule is weaker at a selected calcium ion concentration between 0.5 M and 10 M than at a calcium ion concentration selected between 200 M and 5 mM. It particularly preferably means that the antigen-binding activity in the calcium ion concentration in the initial endosome in vivo is weaker than that in the calcium ion concentration in the plasma in vivo; and specifically, it means that the antigen-binding activity of an antigen-binding molecule is weaker at a selected calcium ion concentration between 1 M and 5 M than at a selected calcium ion concentration between 500 M and 2 , 5 mM.
[0972] [0972] Whether the antigen-binding activity of an antigen-binding molecule is changed depending on metal ion concentrations can be determined, for example, through the use of known measurement methods such as those described in the section "Activations". Connection "above. For example, in order to confirm that the antigen-binding activity of an antigen-binding molecule becomes greater at a high calcium ion concentration than at a low calcium ion concentration, the activity of binding to antigen of the antigen-binding molecule at low and high calcium ion concentrations is compared.
[0973] [0973] In the present invention, the expression "antigen-binding activity is less at a low calcium ion concentration than at a high calcium ion concentration" can also be expressed as "the activity of binding to antigen of an antigen-binding molecule is higher at a high calcium ion concentration than at a low calcium ion concentration ". In the present invention, "antigen-binding activity is less at a low calcium ion concentration than at a high calcium ion concentration" is sometimes written as "the antigen-binding ability is more weak at a low calcium ion concentration than at a high calcium ion concentration ". Also, "antigen-binding activity at a low calcium ion concentration is reduced to be less than that at a high calcium ion concentration" can be written as "the antigen-binding ability at a concentration low calcium ion is made weaker than at a high calcium ion concentration ".
[0974] [0974] When determining antigen-binding activity, conditions other than calcium ion concentration can be appropriately selected by those skilled in the art and are not particularly limited. For example, activity can be determined at 37 ° C in HEPES buffer. For example, Bi acore (GE Healthcare) or similar can be used for the determination. When the antigen is a soluble antigen, the antigen-binding activity of an antigen-binding molecule can be assessed by flowing the antigen as an analyte on a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of an antigen binding molecule to the membrane antigen can be assessed by flowing the antigen binding molecule as an analyte on a chip on which the antigen is immobilized.
[0975] [0975] Since the antigen-binding activity of an antigen-binding molecule of the present invention is weaker at a low calcium ion concentration than at a high calcium ion concentration, the ratio antigen-binding activity between low and high calcium ion concentrations is not particularly limited. However, the ratio of the KD (dissociation constant) of the antigen-binding molecule to an antigen at a low calcium ion concentration compared to KD at a high calcium ion concentration, that is, the value of KD (3 M Ca) / KD (2 mM Ca), is preferably 2 or more, more preferably 10 or more and even more preferably 40 or more. The upper limit of the KD (3 M Ca) / KD (2 mM Ca) value is not particularly limited, and can be any value such as 400, 1000 or 10000 as long as the molecule can be produced using techniques known to those speci - technical experts.
[0976] [0976] When the antigen is a soluble antigen, KD (dissociation constant) can be used to represent antigen-binding activity. However, when the antigen is a membrane antigen, apparent KD (apparent dissociation constant) can be used to represent the activity. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be determined using methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard graph or flow cytometry.
[0977] [0977] Alternatively, for example, the dissociation rate constant (kd) can also preferably be used as an index to represent the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention between concentrations. low and high calcium concentrations. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index to represent the ratio of binding activity, the ratio of the dissociation rate constant (kd) between calcium concentrations low and high, that is, the value of kd (low calcium concentration) / kd (high calcium concentration), is preferably 2 or more, more preferably 5 or more, more preferably still 10 or more, and even more preferably 30 or more. The upper limit of the Kd (low calcium concentration) / kd (high calcium concentration) value is not particularly limited and can be any value such as 50, 100 or 200 as long as the molecule can be produced using techniques known to those skilled in the art. in the technique.
[0978] [0978] When the antigen is a soluble antigen, kd (constant in the dissociation rate) can be used to represent antigen-binding activity. However, when the antigen is a membrane antigen, the apparent kd (constant in the apparent dissociation rate) can be used to represent antigen-binding activity. The kd (constant of the dissociation rate) and the apparent kd (constant of the apparent dissociation rate) can be determined using methods known to those skilled in the art, for example, using Biacore (GE healthcare) or cytometry flow. In the present invention, when the antigen-binding activity of an antigen-binding molecule is determined at different calcium ion concentrations, it is preferable to use the same conditions except for calcium concentrations.
[0979] [0979] For example, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, it can be obtained through the evaluation of antigen or antibody binding domains including the steps of:
[0980] [0980] determination of antigen-binding activity of an antigen or antibody-binding domain at a low calcium concentration;
[0981] [0981] determination of antigen-binding activity of an antigen-binding domain or antigen-binding molecule at a high calcium concentration; and
[0982] [0982] selection of an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.
[0983] [0983] In addition, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is less at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, can be obtained through the evaluation of antigen-binding domains or antigen-binding molecule, or a library thereof, including the steps of:
[0984] [0984] contact of an antigen with an antigen-binding domain or antigen-binding molecule or a library of it at a high calcium concentration;
[0985] [0985] incubation at a low calcium concentration of an antigen binding domain or antigen binding molecule that bound to the antigen in step (a); and
[0986] [0986] isolation of an antigen-binding domain or antigen-binding molecule dissociated in step (b).
[0987] [0987] Also, the antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, can be obtained by evaluating domains or molecules binding to the antigen binding antigen, or a library thereof, including the steps of:
[0988] [0988] contact of an antigen with a library of antigen-binding domains or antigen-binding molecules at a low calcium concentration;
[0989] [0989] selection of an antigen-binding domain or antigen-binding molecule that does not bind to the antigen in step (a);
[0990] [0990] allow the antigen-binding domain or antigen-binding molecule selected in step (c) to bind to the antigen at a high calcium concentration; and
[0991] [0991] isolation of an antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).
[0992] [0992] Also, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, can be obtained through an evaluation method comprising the steps of:
[0993] [0993] contact at a high calcium concentration from a library of antigen binding domains or antigen binding molecules with a column on which an antigen is immobilized;
[0994] [0994] elution of an antigen-binding domain or antigen-binding molecule that bound to the column in step (a) of the column at a low calcium concentration; and
[0995] [0995] isolation of the antigen-binding domain or antigen-binding molecule eluted in step (b).
[0996] [0996] Also, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, can be obtained through a comprehensive evaluation method
[0997] [0997] allow at a low calcium concentration that an antigen binding domain library or antigen binding molecules pass through a column in which an antigen is immobilized;
[0998] [0998] collection of an antigen binding domain or antigen binding molecule that was eluted without binding to the column in step (a);
[0999] [0999] allow the antigen-binding domain or antigen-binding molecule collected in step (b) to bind to the antigen at a high calcium concentration; and
[01000] [01000] isolation of an antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).
[01001] [01001] In addition, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is a modality of the present invention, can be obtained through an evaluation method comprising the steps of:
[01002] [01002] contact of an antigen-binding molecule with a library of antigen-binding domains or antigen-binding molecule at a high calcium concentration;
[01003] [01003] obtaining an antigen-binding domain or antigen-binding molecule that bound to the antigen in step (a);
[01004] [01004] incubation at a low calcium concentration of the antigen-binding domain or antigen-binding molecule obtained in step (b); and
[01005] [01005] isolation of an antigen-binding domain or antigen-binding molecule whose antigen-binding activity in step (c) is weaker than the criterion for selecting step (b).
[01006] [01006] The steps described above can be repeated two or more times. Thus, the present invention provides antigen-binding domains or antigen-binding molecules whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which are obtained through of assessment methods that further comprise the repetition step two or more times from steps (a) to (c) or (a) to (d) in the assessment methods described above. The number of cycles in steps (a) to (c) or (a) to (d) is not particularly limited, but is generally 10 or less.
[01007] [01007] In the evaluation methods of the present invention, the antigen binding activity of an antigen binding domain or antigen binding molecule at a low calcium concentration is not particularly limited, as long as it is a antigen-binding activity at an ionized calcium concentration between 0.1 M and 30 M, but preferably it is antigen-binding activity at an ionized calcium concentration between 0.5 M and 10 M. More preferably, it is an activity antigen-binding activity in the concentration of ionized calcium in the initial endosome in vivo, specifically between 1 M and 5 M. However, the antigen-binding activity of an antigen-binding domain or antigen-binding molecule at a concentration High calcium content is not particularly limited, as long as it is an antigen binding activity at an ionized calcium concentration between 100 M and 10 mM, but preferably it is antigen binding activity in a con ionized calcium concentration between 200 M and 5 mM. Most preferably, it is an antigen-binding activity in the ionized calcium concentration in plasma in vivo, specifically between 0.5 mM and 2.5 mM.
[01008] [01008] The antigen-binding activity of an antigen-binding domain or antigen-binding molecule can be measured using methods known to those skilled in the art. Conditions other than ionized calcium concentration can be determined
[01009] [01009] In the present invention, the step of selecting an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is greater at a high calcium concentration than at a low calcium concentration is synonymous the step of selecting an antigen-binding domain or antigen-binding molecule whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.
[01010] [01010] Although antigen-binding activity is greater at a high calcium concentration than at a low calcium concentration, the difference in antigen-binding activity between high and low calcium concentrations is not particularly limited ; however, antigen-binding activity at a high calcium concentration is preferably twice or more, more preferably 10 times or more, and even more preferably 40 times or more than at a low calcium concentration.
[01011] [01011] The antigen binding domains or antigen binding molecules of the present invention to be evaluated using the evaluation methods described above can be any antigen binding domains and antigen binding molecules. For example, it is possible to evaluate the antigen binding domains and antigen binding molecules described above. For example, antigen-binding domains or antigen-binding molecules having natural or substituted amino acid sequences can be evaluated. Libraries
[01012] [01012] In one embodiment, an antigen binding domain or antigen binding molecule of the present invention can be obtained from a library that is mainly composed of a plurality of antigen binding molecules whose sequences are different from each other and whose antigen-binding domains have at least one amino acid residue that alters the antigen-binding activity of antigen-binding molecules depending on ion concentrations. Ion concentrations preferably include, for example, metal ion concentration and proton concentration.
[01013] [01013] Here, 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 a plurality of antigen binding molecules or a plurality of fusion polypeptides containing antigen binding molecules in a library are not identical, but are different from each other.
[01014] [01014] Here, the expression "sequences are different from each other" in the expression "a plurality of antigen-binding molecules whose sequences are different from each other" means that the sequences of antigen-binding molecules in a library are different from each other. Specifically, in a library, the number of different sequences from each other reflects the number of independent clones with different sequences and can also be referred to as "library size". The library size of a conventional phage display library ranges from 106 to 1012.
[01015] [01015] Here, the expression "a plurality of" in the expression "a library composed mainly of a plurality of antigen-binding molecules" generally refers to, in the case of, for example, antigen-binding molecules, polypeptides fusion molecules, polynucleotide molecules, vectors or viruses of the present invention, a group of two or more types of the substance. For example, when two or more substances are different from each other in a particular characteristic, it means that there are two or more types of the substance. Such examples may include, for example, mutant amino acids observed at specific amino acid positions in an 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 flexible residues or except for particular mutant amino acids in positions
[01016] [01016] Still, here, the expression "composed mainly of" in the expression "a library composed mainly of a plurality of antigen binding molecules" reflects the number of antigen binding molecules whose antigen binding activity varies from - depending on ion concentrations, among independent clones with different sequences in a library. Specifically, it is preferred that there are at least 104 antigen-binding molecules having such binding activity in a library. More preferably, the antigen binding domains of the present invention can be obtained from a library containing at least 105 antigen binding molecules having such binding activity. Most preferably, the antigen binding domains of the present invention can be obtained from a library containing at least 106 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 107 antigen binding molecules having such a binding activity. Even more preferably, the antigen binding domains of the present invention can be obtained from a library containing at least 108 antigen binding molecules having such binding activity. Alternatively, this can also preferably be expressed as the ratio of the number of antigen-binding molecules whose antigen-binding activity varies depending on ion concentrations to the number of independent clones having different sequences in a library. Specifically, the antigen binding domains of the present invention can be obtained from a library in which antigen binding molecules having such binding activity are responsible for 0.1% to 80%, preferably 50% to 60% , more preferably 1% to 40%, even more preferably 2% to 20% and particularly preferably 4% to 10% of independent clones with different sequences in the library. In the case of fusion polypeptides, polynucleotide molecules or vectors, similar expressions may be possible using the number of molecules or the ratio of the total number of molecules. In the case of viruses, similar expression may also be possible using the number of virions or the ratio for the total number of virions. Amino acids that alter the antigen-binding activity of antigen-binding domains depending on ion concentrations.
[01017] [01017] Antigen or antibody binding domains of the present invention to be evaluated using the evaluation methods described above can be prepared in any way. For example, when the metal ion is calcium ion, it is possible to use pre-existing antigen-binding molecules, pre-existing libraries (phage library, etc.), antigen-binding molecules, or libraries prepared from hybridomas obtained through immunization of animals or from D cells of immunized animals, antigen-binding molecules or libraries obtained by introducing amino acids capable of calcium chelation (eg aspartic acid and glutamic acid) or unnatural amino acid mutations in the antibodies or libraries described above (calcium-chelable amino acids (such as aspartic acid and glutamic acid), libraries with a high content of unnatural amino acids, libraries prepared by introducing calcium-chelable amino acids (such as aspartic acid and glutamic acid) or unnatural amino acid mutations at particular or similar positions.
[01018] [01018] Examples of amino acids that alter the antigen-binding activity of antigen-binding molecules depending on ion concentrations as described above can be any types of amino acids as long as the amino acids form a calcium-binding motif. Reasons for calcium binding are well known to those of skill in the art and have been described in detail (eg, 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)). Specifically, any known calcium binding motifs, including type C lecithins such as ASGPR, CD23, MBR and DC-SIGN, can be included in antigen binding molecules of the present invention. Preferred examples of such preferred calcium binding motifs also include, in addition to those described above, for example, the calcium binding motif in the antigen binding domain of SEQ ID NO: 6.
[01019] [01019] Also, as amino acids that alter the antigen-binding activity of antigen-binding molecules depending on calcium ion concentrations, for example, amino acids having metal chelation activity can also preferably be used . Examples of such metal chelation amino acids include, for example, serine (Ser (S)), threonine (Thr (R)), asparagine (Asn (N)), glutamine (Gln (Q)), aspartic acid ( Asp (D)) and glutamic acid (Glu (E)).
[01020] [01020] The positions in the antigen-binding domains in which the amino acids described above are contained are not particularly limited to particular positions and can be any positions within the heavy chain variable region or light chain variable region that forms. an antigen binding domain, as long as they alter the antigen binding activity of antigen binding molecules depending on calcium ion concentrations. Specifically, the antigen binding domains of the present invention can be obtained from a library composed mainly of antigen binding molecules whose sequences are different from each other 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 embodiment, the antigen-binding domains of the present invention can be obtained from a library composed primarily of antigen-binding molecules whose sequences are different from each other and whose heavy chain CDR3 domains contain the amino acids mentioned above. In yet another embodiment, the antigen binding domains of the present invention can be obtained from a library composed mainly of antigen binding molecules whose sequences are different from each other and whose heavy chain CDR3 domains contain the mentioned amino acids above in positions 95, 96, 100a and / or 101 as indicated according to the Kabat numbering system.
[01021] [01021] However, in one embodiment of the present invention, the antigen-binding domains of the present invention can be obtained from a library composed mainly of antigen-binding molecules whose sequences are different from each other and whose light chain antigen-binding domains contain amino acids that alter the antigen-binding activity of antigen-binding molecules depending on calcium ion concentrations. In another embodiment, the antigen binding domains of the present invention can be obtained from a library composed primarily of antigen binding molecules whose sequences are different from each other and whose light chain CDR1 domains contain the amino acids mentioned above. In yet another embodiment, the antigen-binding domains of the present invention can be obtained from a library composed primarily of antigen-binding molecules whose sequences are different from each other and whose light chain CDR1 domains contain the amino acids mentioned above in positions 30, 31 and / or 32 as indicated according to the Kabat numbering system.
[01022] [01022] In another embodiment, the antigen-binding domains of the present invention can be obtained from a library composed mainly of antigen-binding molecules whose sequences are different from each other and whose light chain CDR2 domains contain the amino acid residues mentioned above. In yet another embodiment, the present invention provides libraries composed mainly of antigen binding molecules whose sequences are different from each other and whose light chain CDR2 domains contain the amino acid residues mentioned above at position 50 as indicated according to the Kabat numbering system.
[01023] [01023] In yet another embodiment of the present invention, the antigen binding domains of the present invention can be obtained from a library composed mainly of antigen binding molecules whose sequences are different from each other and whose sequences are different. The light chain CDR3 domains contain the amino acid residues mentioned above. In an alternative embodiment, antigen-binding domains of the present invention can be obtained from a library composed mainly of antigen-binding molecules.
[01024] [01024] Still, in a different embodiment of the present invention, the antigen binding domains of the present invention can be obtained from a library composed mainly of antigen binding molecules whose sequences are different from each other and in which two or three CDRs selected from CDR1, CDR2 and light chain CDR3 described above contain the amino acid residues mentioned above. In addition, the antigen binding domains of the present invention can be obtained from a library composed primarily of antigen binding molecules whose sequences are different from each other and whose light chains contain the amino acid residues mentioned above in either or more than positions 30, 31, 32, 50 and / or 92 as indicated according to the Kabat numbering system.
[01025] [01025] In a particularly preferred embodiment, the main structure sequences of the light chain and / or heavy chain variable region of an antigen binding molecule preferably contain main structure sequences of human germline. Thus, in one embodiment of the present invention, when the main frame sequences are fully human sequences, it is expected that when such an antigen-binding molecule of the present invention is administered to humans (for example, to treat diseases), it induces little or no immunogenic response. In the above sense, the term "containing a germline sequence" in the present invention means that a part of the main structure sequences of the present invention is identical to a part of any human germline main structure 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 main germline backbone sequences, that molecule is also a antigen of the present invention "containing a germline sequence".
[01026] [01026] Preferred examples of the main structures include, for example, currently known fully human main structure region sequences, which are included on the V-Base website (http://vbase.mrc-cpe.cam.ac.uk) the others. Such backbone region sequences can be appropriately used as a germline sequence contained in an 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)). Suitable germline sequences can be selected from Vk, which is grouped into seven subgroups: Vλ, which is grouped into ten subgroups; and VH, which is grouped into seven subgroups.
[01027] [01027] Fully human VH sequences preferably include, but are not limited to, for example, VH sequences of:
[01028] [01028] subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1- 18, VH1-24, VH1-45, VH1-46, VH1-58 and VH1-69);
[01029] [01029] VH2 subgroup (for example, VH2-5, VH2-26 and VH2-70);
[01030] [01030] subgroup VH3 (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);
[01031] [01031] subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-39, VH4-59 and VH4-61);
[01032] [01032] subgroup VH5 (VH5-51);
[01033] [01033] subgroup VH6 (VH6-1); and
[01034] [01034] subgroup VH7 (VH7-4 and VH7-81).
[01035] [01035] These are also described in well-known documents (Matsuda et al., (J. Exp. Med. (1998) 188, 1973-1975) and the like and so, people skilled in the art can appropriately design antigen-binding molecules of the present based on information from these sequences, it is also preferable to use other main structures or subregions of main structure that are entirely human.
[01036] [01036] Fully human Vκ sequences preferably include, but are not limited to, for example:
[01037] [01037] A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14 and O18 grouped in subgroup Vk1 ;
[01038] [01038] A1, A2, A3, A5, A7, A17, A18, A19, A23, O1 AND O11 grouped in subgroup Vk2;
[01039] [01039] A11, A27, L2, L6, L10, L16, L20 and L25 grouped in sub-group Vk3;
[01040] [01040] B3 grouped in subgroup Vk4;
[01041] [01041] B2 (here also referred to as Vk5-2) grouped in a sub-group Vk5; and
[01042] [01042] A10, A14 and A26 grouped in subgroup Vk6
[01043] [01043] (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001- 1022); Bresing-Kuppers and others ( Gene (1997) 191, 173-181).
[01044] [01044] Fully human Vλ sequences preferably include, but are not limited to, for example:
[01045] [01045] 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 grouped in subgroup VL1;
[01046] [01046] V2-1, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17 and V2-19 grouped in the VL1 subgroup;
[01047] [01047] V3-2, V3-3 and V3-4 grouped in subgroup VL3;
[01048] [01048] V4-1, V4-2, V4-3, V4-4 and V4-6 grouped in the VL4 subgroup; and
[01049] [01049] V5-1, V5-2, V5-4 and V5-6 grouped in the VL5 subgroup (Ka-wasaki et al. (Genome Res. (1997) 7, 250-261)).
[01050] [01050] Typically, these backbone sequences are different from one another in one or more amino acid residues. These backbone 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 the ion concentrations "of the present invention. Other examples of the all-human main structures 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, for example , KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (for example, Kabat et al. (1991) supra; Wu and others (J. Exp. Med. (1970) 132, 211-250)) .
[01051] [01051] Without wishing to be limited to any particular theory, a reason for the expectation that the use of germline sequences precludes adverse immune responses in most individuals is believed to be as follows. As a result of the affinity maturation process during normal immune responses, somatic mutation often occurs in the variable regions of immunoglobulin. Such mutations occur mainly around CDRs whose sequences are hypervariable, but also affect
[01052] [01052] Common methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed to produce the antigen binding molecules of the present invention in which the variable region sequences, heavy and light variable region sequences, CDR sequences, or main structure sequences described above contain amino acids that alter the antigen binding activity of the molecules of antigen binding depending on calcium ion concentrations.
[01053] [01053] For example, a library that contains a plurality of antigen-binding molecules of the present invention whose sequences are different from each other can be constructed by combining variable regions of heavy chain prepared as a sequence library. random variable region with a light chain variable region selected as a main structure sequence originally containing at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentrations . As a non-limiting example, when the ion concentration is calcium ion concentration, such preferred libraries include, for example, those constructed by combining the light chain variable region sequence of SEQ ID NO: 6 ( Vk5-2) and the heavy chain variable region produced as a randomized variable region sequence library.
[01054] [01054] Alternatively, a light chain variable region sequence selected as a main structure region originally containing at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule as mentioned above can be designed to contain several amino acid residues other than the above amino acid residues. Here, such wastes are referred to as flexible wastes. The number and position of flexible residues are not particularly limited, as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on ion concentrations. Specifically, the heavy chain and / or light chain CDR sequences and / or FR sequences can contain one or more flexible residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced in the light chain variable region sequence of SEQ ID NO: 6 (Vk5-2) include the amino acid residues listed in Table 1 or 2.
[01056] [01056] (Position indicates Kabat numbering)
[01058] [01058] (Position indicates Kabat numbering)
[01059] [01059] Here, flexible residues refer to variations of amino acid residue present in hypervariable positions in which several different amino acids are present in the variable regions of light chain and heavy chain when the amino acid sequences of known and / or native antibodies or antigen-binding domains are compared. Hypervariable positions are generally located in the CDR regions. In one embodiment, the data provided by Ka- bat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda, Md.) (1987 and 1991) are useful to determine
[01060] [01060] 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 produced as a variable region sequence library. randomized with variable regions of light chain in which at least one amino acid residue that alters the antigen-binding activity of antigen-binding molecules depending on ion concentrations as mentioned above is introduced. When the ion concentration is calcium ion concentration, non-limiting examples of such libraries include, for example, libraries in which heavy chain variable regions are produced as a random variable region sequence library.
[01061] [01061] Non-limiting examples of such amino acid residues contained in light chain CDR1 include those at positions 30, 31 and / or 32 in the light chain variable region CDR1 as indicated by EU numbering. In addition, non-limiting examples of such amino acid residues contained in the light chain CDR2 include an amino acid residue at position 50 in the light chain variable region CDR2 as indicated by Kabat numbering. In addition, non-limiting examples of such amino acid residues contained in the light chain CDR3 include an amino acid residue at position 92 in the light chain variable region CDR3 as indicated by Kabat numbering. Amino acid residues may be contained alone or in combination as long as they form a calcium-binding motif and / or as long as the antigen-binding activity of an antigen-binding molecule varies depending on concentrations of calcium ion. However, as troponin C, calmodulin, parvalbumin and myosin light chain, which have several calcium ion binding sites and are believed to be derived from a common origin in terms of molecular evolution, CDR1 is known,
[01062] [01062] When heavy chain variable regions produced as a random variable region sequence library and light chain variable regions in which at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on the ion concentrations that were introduced are combined as described above, the sequences of the light chain variable regions can be designed to contain flexible residues in the same manner as described above. The number and position of such flexible residues are not particularly limited to the particular embodiments as long as the antigen-binding activity of antigen-binding molecules of the present invention varies depending on ion concentrations. Specifically, the CDR sequences and / or heavy chain and / or light chain FR sequences may contain one or more flexible residues. When the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced in the light chain variable region sequence include the amino acid residues listed in Tables 1 and 2.
[01063] [01063] Preferred heavy chain variable regions to be combined include, for example, randomized variable region libraries. Known methods are combined as appropriate to produce a randomized variable region library. In a non-limiting embodiment of the present invention, an immune library built on the basis of antibody genes derived from lymphocytes from animals immunized with a specific antigen, patients with infections, people with an elevated antibody titer in the blood as a result of vaccination , cancer patients or patients with autoimmune disease, can preferably be used as a randomized variable region library.
[01064] [01064] In another non-limiting embodiment of the present invention, a synthetic library produced by replacing the CDR sequences of V genes in genomic DNA or functional remodeled V genes with a set of synthetic oligonucleotides containing sequences encoding codon sets of an appropriate length can be also preferably used as a randomized variable region library. In this case, since sequence diversity is observed in the heavy chain CDR3 sequence, it is also possible to replace the CDR3 sequence only. A criterion for granting origin to the diversity of amino acids in the variable region of an antigen binding molecule is that this diversity is given to amino acid residues in exposed surface positions in the antigen binding molecule. The exposed surface position refers to a position that is considered to be capable of being exposed on the surface and / or contacted with an antigen, based on the structure, set of structures and / or modeled structure of an antigen binding molecule . In general, such positions are CDRs. Preferably, exposed surface positions are determined using coordinates of a three-dimensional model of an antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Exposed surface positions can be determined using algorithms known in the art (for example, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16 ,
[01065] [01065] In another non-limiting embodiment of the present invention, a pure library, which is constructed from antibody genes derived from lymphocytes from healthy people and whose repertoire consists of pure sequences, which are antibody sequences with no predisposition, can be also particularly preferably used as a randomized variable region library (Gejima and others (Human Antibodies (2002) 11, 121-129); Cardoso et al. (Scand, J. Immunol. (2000) 51, 337-344 Here, an amino acid sequence comprising a native sequence refers to an amino acid sequence obtained from such a pure library.
[01066] [01066] In one embodiment of the present invention, an antigen binding domain of the present invention can be obtained from a library containing a plurality of antigen binding molecules of the present invention whose sequences are different from each other, prepared by combination of light chain variable regions constructed as a random variable region sequence library with a heavy chain variable region selected as a backbone sequence containing origi-
[01067] [01067] Alternatively, the sequence of a heavy chain variable region selected as a backbone sequence that originally contains "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule" as mentioned above it can be designed to contain flexible waste. The number and position of the flexible residues are not particularly limited as long as the antigen-binding activity of an antigen-binding molecule of the present invention varies depending on ion concentrations. Specifically, heavy chain and / or light chain CDR and / or FR sequences may contain one or more flexible residues. When the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced in the heavy chain variable region sequence of SEQ ID NO: 11 (6RL # 9-IgG1) include all residues of heavy chain CDR1 and CDR2 amino acids and the heavy chain CDR3 amino acid residues except those in positions 95, 96 and / or 100a. Alternatively, non-limiting examples of flexible residues to be introduced into the heavy chain variable region sequence of SEQ ID NO: 12 (6KC4-1 # 85-IgG1) include all amino acid residues of heavy chain CDR1 and CDR2 and the amino acid residues of heavy chain CDR3 except those at amino acid positions 95 and / or 101.
[01068] [01068] Alternatively, a library containing a plurality of antigen-binding molecules whose sequences are different from each other can be constructed by combining light chain variable regions constructed as a random variable region sequence library or light chain variable regions having - germline sequences with variable regions of heavy chain in which "at least one amino acid residue responsible for the ion-dependent change in the antigen-binding activity of an antigen-binding molecule" was introduced as mentioned above. When the ion concentration is calcium ion concentration, non-limiting examples of such libraries preferably include those in which light chain variable regions constructed as a randomized variable region sequence library or light chain variable regions having lineage sequences germline are combined with the sequence of a heavy chain variable region in which a particular residue (s) has been replaced with at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on of calcium ion concentrations. Non-limiting examples of such amino acid residues include amino acid residues from the heavy chain CDR1. Additional non-limiting examples of such amino acid residues include amino acid residues of the heavy chain CDR2. 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 C-DR3 amino acid residues include the amino acids at positions 95, 96, 100a and / or 101 in the heavy chain variable region CDR3 as indicated by the Kabat numbering. In addition, these amino acid residues may be contained alone or in combination as long as they form a calcium binding motif and / or the antigen binding activity of an antigen binding molecule varies depending on calcium concentrations.
[01069] [01069] When light chain variable regions constructed as a random variable region sequence library or light chain variable regions having germline sequence are combined with a heavy chain variable region in which at least one amino acid residue that alters the antigen-binding activity of an ion concentration-dependent antigen-binding molecule as mentioned above was introduced, the sequence of the heavy chain variable region can also be designed to contain flexible residues in the same manner as described above. The number and position of flexible residues are not particularly limited, as long as the antigen-binding activity of an antigen-binding molecule of the present invention varies depending on ion concentrations. Specifically, the heavy chain and / or FR CDR sequences can contain one or more flexible residues. In addition, randomized variable region libraries can preferably be used as amino acid sequences of CDR1, CDR2 and / or CDR3 of the heavy chain variable region instead of the amino acid residues that alter the antigen-binding activity of an antigen-binding molecule . When germline sequences are used as variable light chain regions, non-limiting examples of such sequences include those of SEQ ID NO: 7 (Vk1), SEQ ID NO: 8 (Vk2), SEQ ID NO: 9 (Vk3 ) and SEQ ID NO: 10 (Vk4).
[01070] [01070] Any of the amino acids described above that alter the antigen-binding activity of a calcium ion concentration-dependent antigen-binding molecule can preferably be used, as long as they form a calcium-binding motif. Specifically, 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. Condition of proton concentrations
[01071] [01071] In one embodiment of the present invention, the condition of ion concentrations refers to the condition of proton concentrations or pH condition. In the present invention, the proton concentration, that is, the nucleus of hydrogen atoms, is treated as a synonym with hydrogen index (pH). When the hydrogen ion activity in an aqueous solution is represented as aH +, pH is defined as -log10aH +. When the ionic resistance of the aqueous solution is low (for example, less than 10-3), aH + is almost equal to the proton resistance. For example, the ionic product of water at 25 ° C and 1 atmosphere is Kw = aH + aOH = 10-14 and then in pure water, aH + = aOH = 10-
[01072] [01072] In the present invention, when the pH condition is used as the ion concentration condition, pH conditions include high proton concentrations or low pHs, that is, an acidic pH range, and low proton concentrations or pHs high, that is, a
[01073] [01073] In this report, the neutral pH range is not limited to a specific value and is preferably selected between pH 6.7 and pH 10.0. In another embodiment, the pH can be selected between pH 6.7 and pH 9.5. In yet another embodiment, the pH can be selected between pH 7.0 and pH 9.0. In yet another modality, the pH can be selected between pH7.0 and pH8.0. In particular, the preferred pH includes pH 7.4, which is close to the plasma (blood) pH in vivo.
[01074] [01074] In this report, an acidic pH range is not limited to a specific value and is preferably selected between pH4.0 and pH6.5. In another embodiment, the pH can be selected between pH4.5 and pH6.5. In yet another embodiment, the pH can be selected between pH5.0 and pH6.5. In yet another modality, the pH can be selected between pH5.5 and pH6.5. In particular, the preferred pH includes pH 5.8, which is close to the proton concentration in the initial endosome in vivo.
[01075] [01075] In the present invention, “the antigen-binding activity of an antigen-binding molecule at a high proton concentration or low pH (an acid pH range) is less than that at a low proton concentration or pH high (a neutral pH range) ”means that the antigen-binding activity of an antigen-binding molecule at a pH selected between pH 4.0 and pH6.5 is weaker than that at a pH selected from pH6.7 and pH10.0; preferably it means that the antigen-binding activity of an antigen-binding molecule at a pH selected between pH 4.5 and pH6.5 is weaker than that at a pH selected between pH6.7 and pH9.5; more preferably, it means that the antigen-binding activity of an antigen-binding molecule at a pH selected between pH5.0 and pH6.5 is weaker than that at a pH selected between pH7.0 and pH9.0; even more preferential means that the antigen-binding activity of an antigen-binding molecule at a pH selected between pH5.5 and pH6.5 is weaker than that at a pH selected between pH7.0 and pH8.0; particularly preferably it means that the antigen-binding activity at the pH in the initial endosome in vivo is weaker than the antigen-binding activity at the plasma pH in vivo; and specifically it means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.
[01076] [01076] Whether the antigen-binding activity of an antigen-binding molecule has changed by the pH condition can be determined, for example, through the use of known measurement methods such as those described in the "Binding Activity" section above. Specifically, the binding activity is measured under different pH conditions using the measurement methods described above. For example, the antigen-binding activity of an antigen-binding molecule is compared under the conditions of the acid pH range and the neutral pH range to confirm that the antigen-binding activity of the antigen-binding molecule changes to be larger under the neutral pH range condition than that under the acid pH range condition.
[01077] [01077] Furthermore, in the present invention, the expression "antigen-binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is lower than that at a low concentration of protons or high pH, that is, in a neutral pH range "can also be expressed as" the antigen-binding activity of an antigen-binding molecule at a low proton concentration or high pH, that is , in a neutral pH range, is higher than that in a high concentration of protons or low pH, that is, in an acidic pH range ". In the present invention, "the antigen-binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is lower than that at a low proton concentration or high pH, that is that is, in a neutral pH range "can be described as" antigen-binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is weaker than the ability to bind to antigen in a low proton concentration or high pH, that is, in a neutral pH range ". Alternatively, "antigen-binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is reduced because it is lower than that at a low or high proton concentration pH, that is, in a neutral pH range "can be described as" the antigen-binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is reduced because it is weaker than that the ability to bind to the antigen at a low proton concentration or high pH, that is, in a neutral pH range ".
[01078] [01078] Conditions other than proton concentration or pH for measuring antigen-binding activity can be suitably selected by those skilled in the art and are not particularly limited. Measurements can be performed, for example, at 37 ° C using HEPES buffer. Measurements can be performed, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the antigen-binding activity of an antigen-binding molecule can be determined by evaluating the binding activity for the soluble antigen by dumping the antigen as an analyte on a chip immobilized with the molecule of antigen binding. When the antigen is a membrane antigen, the activity of binding to the membrane antigen can be assessed by dumping the antigen binding molecule as an analyte on a chip immobilized with the antigen.
[01079] [01079] Although the antigen-binding activity of an antigen-binding molecule of the present invention at a high proton concentration or low pH, that is, in a low pH range, is weaker than that at a concentration of low protons or high pH, that is, in a neutral pH range, the ratio of antigen-binding activity to that in a high proton concentration or low pH, that is, an acid pH range, and in a low proton concentration or high pH, that is, a neutral pH range, is not particularly limited, and the KD (pH5.8) / KD (pH7.4) value, which is the ratio of the constant dissociation (KD) for an antigen at a high proton concentration or low pH, that is, in an acid pH range for KD at a low proton concentration or high pH, that is, in a pH range neutral, it is preferably 2 or more; more preferably, the KD (pH5.8) / KD (pH7.4) value is 10 or more; and most preferably the KD (pH5.8) / KD (pH7.4) value is 40 or more. The upper limit of the KD (pH5.8) / KD (pH7.4) value is not particularly limited, and can be any value such as 400, 1000 or 10000, as long as the molecule can be produced through the techniques of those skilled in the art.
[01080] [01080] When the antigen is a soluble antigen, the dissociation constant (KD) can be used as the value for binding activity
[01081] [01081] Alternatively, for example, the dissociation rate constant (kd) can be appropriately used as an index to indicate the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention to that at a high proton concentration and low pH, that is, an acid pH range and a low proton concentration or high pH, that is, a neutral pH range. When kd (dissociation rate constant) is used as an index to indicate the ratio of binding activity instead of KD (dissociation constant), the value of kd (in an acidic pH range) / kd (in a neutral pH range), which is the ratio of kd (dissociation rate constant) to the antigen in a high proton concentration or low pH, that is, in an acid pH to kd range (constant dissociation) at a high proton concentration or high pH, that is, in a neutral pH range, it is preferably 2 or more, more preferably 5 or more, more preferably and still 10 or more, and especially preferably 30 or more. The upper limit value (in an acidic pH range) / kd (in a neutral pH range) is not particularly limited and can be any value such as 50, 100 or 200, as long as the molecule can be produced using techniques those experts in the art.
[01082] [01082] When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the value for antigen binding activity and when the antigen is a membrane antigen, the dissociation rate constant apparent (kd) can be used.
[01083] [01083] For example, an antigen or antibody binding domain whose antigen binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is less than that in a low proton concentration or high pH, that is, in a neutral pH range, which is a modality provided by the present invention, can be obtained through the evaluation of antigen binding domains or antigen binding molecules comprising following steps (a) to (c) that follow:
[01084] [01084] obtaining the antigen binding activity of an antigen or antibody binding domain in an acidic pH range;
[01085] [01085] obtaining the antigen-binding activity of an antigen or antibody-binding domain in a neutral pH range; and
[01086] [01086] selection of an antigen or antibody binding domain whose antigen binding activity in the acidic pH range is less than that in the neutral pH range.
[01087] [01087] Alternatively, an antigen or antibody-binding domain whose antigen-binding activity at a high proton concentration or low pH, that is, in an acid pH range, is less than that at a concentration of low protons or high pH, that is, in a neutral pH range, which is a modality provided by the present invention, can be obtained through the evaluation of antigen or antibody binding domains, or its library, comprising the steps ( a) to (c) that follow:
[01088] [01088] contact of an antigen or antibody binding domain, or its library, in a neutral pH range with an antigen;
[01089] [01089] placing in an acid pH range of the antigen binding domain or antigen binding molecule linked to the antigen in step (a); and
[01090] [01090] isolation of the antigen or antibody-binding domain dissociated in step (b).
[01091] [01091] An antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, ie, in an acidic pH range, is less than that at a low proton concentration or high pH, that is, in a neutral pH range, which is another modality provided by the present invention, can be obtained through the evaluation of antigen or antibody binding domains, or a library thereof, comprising the steps ( a) to (d) that follow:
[01092] [01092] contact in an acidic pH range of an antigen with an antigen or antibody binding domain library;
[01093] [01093] selection of the antigen-binding domain or antigen-binding molecule that does not bind to the antigen in step (a);
[01094] [01094] allow the antigen or antibody binding domain selected in step (b) to bind with the antigen in a neutral pH range; and
[01095] [01095] isolation of the antigen-binding domain or antigen-binding molecule linked to the antigen in step (c).
[01096] [01096] An antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, ie, in an acidic pH range, is less than that at a low proton concentration or high pH, that is, in a neutral pH range, which is yet another modality provided by the present
[01097] [01097] contact in a neutral pH range of a library of antigen binding domains or antigen binding molecules with a column immobilized with an antigen;
[01098] [01098] elution in an acidic pH range from the column of the antigen binding domain or antigen binding molecule attached to the column in step (a); and
[01099] [01099] isolation of the antigen or antibody binding domain eluted in step (b).
[01100] [01100] An antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, that is, in an acid pH range, is less than that at low or low proton concentration High pH, that is, in a neutral pH range, which is yet another modality provided by the present invention, can be obtained through an evaluation method comprising the steps (a) to (d) that follow:
[01101] [01101] allow, in an acid pH range, that a library of antigen-binding domains or antigen-binding molecules pass through a column immobilized with an antigen;
[01102] [01102] collection of the binding domain to antigen or antibody eluted without binding to the column in step (a);
[01103] [01103] allow the antigen or antibody binding domain collected in step (b) to bind with the antigen in a neutral pH range; and
[01104] [01104] isolation of the antigen-binding domain or antigen-binding molecule linked to the antigen in step (c).
[01105] [01105] An antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, ie, in an acidic pH range, is less than that at a low proton concentration or high pH, that is, in a neutral pH range, which is yet another modality provided by the present invention, can be obtained through an evaluation method comprising the steps (a) to (d) that follow:
[01106] [01106] contact of an antigen with a library of antigen-binding domains or antigen-binding molecules in a neutral pH range;
[01107] [01107] obtaining the antigen-binding domain or antigen-binding molecule linked to the antigen in step (a);
[01108] [01108] placement in an acid pH range of the antigen binding domain or antigen binding molecule obtained in step (b); and
[01109] [01109] isolation of the antigen or antibody binding domain whose antigen binding activity in step (c) is weaker than the standard selected in step (b).
[01110] [01110] The steps described above can be repeated two or more times. In this way, the present invention provides antigen binding domains and antigen binding molecules whose antigen binding activity in an acidic pH range is less than that in a neutral pH range, which are obtained through a evaluation method that also includes the stages of repetition of steps (a) to (c) or (a) to (d) in the evaluation methods described above. The number of times that steps (a) to (c) or (a) to (d) are repeated is not particularly limited; however, the number is 10 or less in general.
[01111] [01111] In the evaluation methods of the present invention, the antigen binding activity of an antigen or antibody binding domain at a high proton concentration or low pH, that is, in an acid pH range, is not particularly limited, as long as it is the antigen binding activity at a pH between 4.0 and 6.5 and includes the antigen binding activity at a pH between 4.5 and 6.6 co-
[01112] [01112] The antigen-binding activity of an antigen or antibody-binding domain can be measured using methods known to those skilled in the art. Those skilled in the art can properly determine conditions other than ionized calcium concentration. The antigen-binding activity of an antigen or antibody-binding domain can be assessed based on the dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), rate constant apparent dissociation (kd) and similar. These can be determined using methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard chart or FACS.
[01113] [01113] Here, the step of selecting an antigen or antibody binding domain whose antigen binding activity in a low proton concentration or high pH, that is, in a neutral pH range, is greater than that at a high proton concentration or low pH, that is, in an acid pH range, is synonymous with the step with selecting an antigen or antibody binding domain whose antigen binding activity at a high proton concentration or low pH, that is, in an acidic pH range, is less than that in low proton concentration or high pH, that is, in a neutral pH range.
[01114] [01114] As long as the antigen-binding activity at a low proton concentration or high pH, that is, in a neutral pH range, is greater than that at a high proton concentration or low pH, that is, in an acid pH range, the difference between antigen binding activity at a low or high proton concentration, that is, a neutral pH range, and that at a high or low pH proton concentration, that is, an acidic pH range, is not particularly limited; however, antigen-binding activity at a low proton concentration or high pH, i.e., in a neutral pH range, is preferably twice or more, more preferably 10 times or more, and even more preferably 40 times. or more than that at a high proton concentration or low pH, that is, in an acid pH range.
[01115] [01115] The antigen-binding domain or antigen-binding molecule of the present invention assessed using the evaluation methods described above can be any antigen-binding domain or antigen-binding molecule, and for example, a binding to the antigen or antibody mentioned above can be assessed. For example, antigen binding domain or antigen binding molecule with the native sequence can be assessed, and antigen or antibody binding domain in which its amino acid sequences have been replaced can be assessed.
[01116] [01116] The antigen binding domain or antigen binding molecule of the present invention to be evaluated using the evaluation methods described above can be prepared in any way. For example, conventional antigen-binding molecules, conventional libraries (phage library, etc.), antigen-binding molecules or libraries prepared by B cells from immunized animals or hybridomas obtained through immunizing animals, molecules antigen-binding or libraries (libraries with high amino acid content with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or unnatural amino acids, libraries introduced with amino acids with a chain pKa side of 4.0-8.0 (eg histidine and glutamic acid) or mutations of unnatural amino acid at specific positions, etc.) obtained through the introduction of amino acids with a side chain pKa of 4.0 -8.0 (for example, histidine and glutamic acid) or mutations of unnatural amino acid in the antibodies or libraries described above can be used.
[01117] [01117] Methods for obtaining an antigen or antibody binding domain whose antigen binding activity in a low proton concentration or high pH, that is, in a neutral pH range, is greater than that in a high proton concentration or low pH, that is, in an acid pH range, from antigen binding domains or antigen binding molecules prepared from hybridomas obtained through immunization of animals or B cells of immunized animals preferably include, for example, the antigen binding molecule or antigen binding molecule in which at least one of the amino acids in the antigen binding domain or antigen binding molecule is replaced with a amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or an unnatural amino acid mutation, or the antigen or antibody-binding domain inserted with an amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acid, such as those described in International publication No. WO 2009/125825.
[01118] [01118] The sites for introducing amino acid mutations with a side chain pKa of 4.0-8.0 (eg histidine or glutamic acid) or unnatural amino acids are not particularly limited, and can be any position as long as antigen binding activity in an acidic pH range becomes weaker than that in a neutral pH range (the KD value (in an acidic pH range) / KD (in a pH range neutral) or kd (in an acidic pH range) / kd (in a neutral pH range) is increased) compared to before replacement or insertion. For example, when the antigen-binding molecule is an antibody, the variable region of antibody and CDRs are suitable. 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. It is possible to replace a single amino acid having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or a single unnatural amino acid; it is possible to insert a single amino acid having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or single unnatural amino acid; it is possible to replace two or more amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or two or more unnatural amino acids; and it is possible to insert two or more amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or two or more unnatural amino acids. Alternatively, other amino acids can be deleted, added, inserted and / or replaced concomitantly, with the exception of substitution in amino acids having a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or amino acids
[01119] [01119] Preferred examples of antigen-binding molecules containing the mutation in amino acids with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or unnatural amino acids as described above and whose activity of antigen binding in an acidic pH range is less than that in a neutral pH range include antigen binding molecules whose antigen binding activity in the neutral pH range after mutation in amino acids with a pKa of side chain of 4, -8.0 (eg histidine and glutamic acid) or unnatural amino acids is comparable to that before mutation in amino acids with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or unnatural amino acids. Here, "an antigen-binding molecule after mutation with amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids tends to
[01120] [01120] However, when an antigen binding molecule is a substance containing an antibody constant region, preferred modalities of antigen binding molecules whose antigen binding activity in an acidic pH range is less than that in a neutral pH range includes methods in which the antibody constant regions contained in the antigen binding molecules have been modified. Specific examples of modified antibody constant regions preferably include the constant regions of SEQ ID NOs: 11, 12, 13 and 14.
[01121] [01121] Amino acids that alter the antigen binding activity of the antigen binding domain depending on the proton concentration conditions
[01122] [01122] The antigen binding domains or antigen binding molecules of the present invention to be evaluated using the evaluation methods described above can be prepared in any way. For example, when an ion concentration condition is a proton concentration condition or a pH condition, pre-existing antigen-binding molecules, pre-existing libraries (phage library, etc.), antigen-binding molecules or prepared libraries from B cells of immunized animals or from hybrids but obtained by immunizing animals, antigen-binding molecules or libraries (libraries with a high amino acid content with a side chain pKa of 4.0-8.0 (for histidine and glutamic acid) or unnatural amino acids, libraries introduced with amino acid mutations with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or non-natural amino acids in specific positions, etc.) obtained by introducing mutations of amino acids with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or unnatural amino acids in the antibodies or libraries described above can be used.
[01123] [01123] In one embodiment of the present invention, a library containing multiple antigen-binding molecules of the present invention whose sequences are different from one another can also be constructed by combining variable regions of heavy chain, produced as a region sequence library. randomized variable, with variable light chain regions introduced with "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on the condition of proton concentration".
[01124] [01124] Such amino acid residues include, but are not limited to, for example, 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 light chain CDR2. Amino acid residues also include, but are not limited to, for example, amino acid residues contained in light chain CDR3.
[01125] [01125] The amino acid residues described above contained in the light chain CDR1 include, but are not limited to, for example, amino acid residues of positions 24, 27, 28, 31, 32 and / or 34 according to numbering Kabat in the light chain variable region CDR1. However, the amino acid residues contained in the light chain CDR2 include, but are not limited to, for example, amino acid residues of positions 50.5 1, 52, 53, 54, 55 and / or 56 according to Kabat numbering in Light chain variable region CDR2. However, 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 numbering
[01126] [01126] Even when the heavy chain variable region produced as a random variable region sequence library is combined with the light chain variable region described above introduced with "at least one amino acid residue that changes the activity. antigen-binding activity of an antigen-binding molecule depending on the condition of proton concentration ", it is possible to design so that the flexible 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 the flexible residues are not particularly limited to a specific modality, as long as the antigen binding activity of an antigen binding molecule of the present invention changes depending on the condition of proton concentration. Specifically, the heavy chain and / or light chain CDR and / or FR sequences may contain one or more flexible residues. For example, flexible residues to be introduced into the sequences of the light chain variable regions include, but are not limited to, for example, the amino acid residues listed in Tables 3 and 4. However, amino acid sequences from varying regions - light chain levels other than flexible residues and amino acid residues that change the antigen-binding activity of an antigen-binding molecule depending on the condition of proton concentration suitably include, but are not limited to, sequence - germline copies such as Vk1 (SEQ ID NO: 7), Vk2 (SEQ ID NO: 8), Vk3 (SEQ ID NO: 9) and Vk4 (SEQ ID NO: 10).
[01127] [01127] (Position indicates Kabat numbering)
[01129] [01129] (Position indicates Kabat numbering)
[01130] [01130] Any amino acid residue can be used appropriately as the amino acid residues described above that change the antigen-binding activity of an antigen-binding molecule depending on the condition of the proton concentration. Specifically, such amino acid residues include amino acids with a side chain pKa of 4.0-8.0. Such electron-releasing amino acids preferably include, for example, naturally occurring amino acids such as histidine and glutamic acid, as well as unnatural amino acids such as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3.5-Br2-Tyr (pKa 7.21) and 3.5-12-Tyr (pKa 7.38) (Bioorg.
[01131] [01131] Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap Extension PCR can be appropriately employed to modify the amino acids from antigen-binding domains. In addition, several known methods can also be used as an amino acid modification method to replace amino acids with those other than natural amino acids (Annu. Rev. Biophyys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which amber suppressor tRNA, which is complementary to the UAG codon (amber codon) which is a stop codon, is linked with an amino acid. - the unnatural can be used properly.
[01132] [01132] The preferred heavy chain variable region that is used in combination includes, for example, randomized variable region libraries. Known methods are appropriately combined as a method for producing a randomized variable region library. In a non-limiting embodiment of the present invention, an immune library constructed on the basis of antibody genes derived from animals immunized with specific antigens, patients with infection or people with an elevated blood antibody titer as a result of vaccination, cancer patients or lymphocytes from autoimmune diseases can be appropriately used as a randomized variable region library.
[01133] [01133] In another non-limiting embodiment of the present invention, in the same manner as described above, a synthetic library in which the CDR sequences of genomic DNA V genes or functional reconstructed V genes are replaced with a set of synthetic oligonucleotides containing sequences encoding codon sets of an appropriate length can also be suitably used as a randomized variable region library.
[01134] [01134] In yet another non-limiting embodiment of the present invention, a pure library constructed from antibody genes derived from lymphocytes from healthy people and consisting of pure sequences, which are an impartial repertoire of antibody sequences, it can also be particularly suitably used as a randomized variable region library (Gejima et al. (Human Antibodies (2002) 11, 121-129); and Cardoso et al. (Scand. J. Immunol. (2000) 51, 337- 344)). Region of Fc
[01135] [01135] An Fc region contains the amino acid sequence derived from the constant region of an antibody heavy chain. An Fc region is a portion of the antibody heavy chain constant region, which starts at the N-terminal end of the hinge region, which corresponds to the papain cleavage site in an amino acid around position 216 according to with the EU numbering system and contains the hinge, CH2 and CH3 domains. Although the Fc region can be obtained from human IgG1, it is not limited to a particular IgG subclass. As described below, a favorable example of the Fc region is an Fc region that has an FcRn binding activity in an acidic pH range. In addition, a favorable example of the Fc region is an Fc region that has Fcγ receptor binding activity as described later. A non-limiting embodiment of such an Fc region is, for example, the Fc region of IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16) human. FcRn
[01136] [01136] Unlike the Fcγ receptor that belongs to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, exhibiting sequence identity of 22% to 29% to the molecule. - MHC class I la (Ghetie el al., Immunol. Today (1997) 18 (12): 592- 598). FcRn is expressed as a heterodimer consisting of soluble β or light chain (β2 microglobulin) complexed with the transmembrane α or heavy chain. Like MHC, the α chain of FcRn comprises 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 Fc region of the antigen (Raghavan et al., Immunity (1994) 1: 303-315).
[01137] [01137] FcRn is expressed in the maternal placenta and in the mammalian yolk sac, and transfer of IgG from the mother to the fetus is involved. In addition, in the neonatal small intestine of rodents, where FcRn is expressed, FcRn is involved in the transfer of maternal IgG through the brush border epithelium of ingested colostrum or milk. FcRn is expressed in a variety of other tissues and in the endothelial cell systems of the various species. FcRn is also expressed in adult human endotheliums, muscular blood vessels and hepatic sine capillary tubes. It is believed that FcRn plays a role in maintaining the plasma IgG concentration by mediating the recycling of IgG to the serum by binding to IgG. Typically, the binding of FcRn to IgG molecules is strictly pH dependent. Optimal binding is observed in an acidic pH range below 7.0.
[01138] [01138] Human FcRn whose precursor is a polypeptide having the signal sequence of SEQ ID NO: 17 (the polypeptide with the signal sequence is shown in SEQ ID NO: 18) forms a complex with human β2-microglobulin in vivo. Soluble human fcRn complexed with β2-microglobulin is produced using conventional recombinant expression techniques. The FcRn regions of the present invention can be evaluated for their binding activity to soluble human FcRn complexed with β2-microglobulin. In this application, unless otherwise specified, human FcRn refers to a suitable form of binding to an FcRn region of the present invention. Examples include a complex between human FcRn and human β2-microglobulin. A non-limiting modality is exemplified by a complex formed between a mouse FcRn (SEQ ID NO: 73) and a mouse β2-microglobulin (SEQ ID NO: 74). Binding activity of the Fc region to FcRn, especially human FcRn
[01139] [01139] The binding activity of an Fc region of the present invention to FcRn, especially 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 appropriately determine conditions beyond pH. The antigen binding activity and human FcRn binding activity of an antigen binding molecule can be assessed based on the dissociation constant (KD), apparent dissociation constant (KD), dissociation rate (kd), apparent dissociation (kd) and the like. These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard graph or flow cytometer can be used.
[01140] [01140] When the human FcRn binding activity of an Fc region of the present invention is measured, conditions beyond pH are not particularly limited and can be appropriately selected by those skilled in the art. Measurements can be performed, for example, at 37 ° C using MES buffer, as described in International Publication WO No. 2009125825. Alternatively, the human FcRn binding activity of an Fc region of the present invention it can be measured by methods known to those skilled in the art and can be measured using, for example, Biacore (GE Healthcare) or the like. The binding activity of an Fc region of the present invention to human FcRn can be assessed by pouring, as an analyte, human FcRn, from an Fc region or an antigen binding molecule of the present invention containing the Fc region on a chip immobilized with an Fc region, an antigen-binding molecule of the present invention containing the human Fc or FcRn region.
[01141] [01141] A neutral pH range as the condition where the Fc region contained in an antigen binding molecule of the present invention has the FcRn binding activity generally means pH 6.7 at pH 10.0 . Preferably, the neutral pH range is a range indicated with arbitrary pH values between pH 7.0 and 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 it is particularly preferably pH 7.4 which is close to the pH of the plasma ( blood) in vivo. When the binding affinity between the human FcRn and human FcRn binding domain at pH 7.4 is too low to assess, pH 7.0 can be used instead of pH 7.4. In this application, an acidic pH range as the condition where the Fc region contained in an antigen binding molecule of the present invention has FcRn binding activity generally means pH 4.0 at pH 6.5. Preferably, the acidic pH range means pH 5.5 to pH 6.5, particularly preferably pH 5.8 to pH 6.0 which is close to the pH in the first endosome in vivo. As for the temperature used as the measurement condition, the binding affinity between the human FcRn and human FcRn binding domain can be evaluated at any temperature between 10 ° C and 50 ° C. Preferably, the binding affinity between the human FcRn binding domain and human FcRn can be determined at 15 ° C to 40 ° C. More preferably, the binding affinity between the human FcRn and human FcRn binding domain can be determined in the same way at an arbitrary temperature between 20 ° C and 35 ° C, as with any temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 ° C. In an embodiment of the present invention, the temperature includes, but is not limited to, for example, 25 ° C.
[01142] [01142] According to the Journal of Immunology (2009) 182, 7663-7671, the human FcRn binding activity of native human IgG1 in an acidic pH range (pH 6.0) is 1.7 M (KD) , and the activity is barely detectable in a neutral pH range. Thus, in a preferred embodiment, antigen-binding molecules that comprise an Fc region from which the human FcRn binding activity in an acidic pH range is 20 M (KD) or stronger can be traced. In a more preferred embodiment, antigen binding molecules that comprise an Fc region in which the human FcRn binding activity in an acidic pH range is 2.0 M (KD) or stronger can be tracked. In an even more preferred mode, antigen binding molecules that comprise an Fc region from which the human FcRn binding activity in an acidic pH range is 0.5 M (KD) or stronger can be traced. . The aforementioned KD values are determined by the method described in the Journal of Immunology (2009) 182: 7663-7671 (immobilizing the antigen binding molecule on a chip and loading human FcRn as an analyte).
[01143] [01143] In the present invention, the preferred Fc regions have an FcRn binding activity in an acidic pH range condition. When an Fc region originally has FcRn binding activity under an acidic pH range condition, the domain can be used as is. When the domain has little or no FcRn binding activity under an acidic pH range condition, an Fc region that has a desired FcRn binding activity can be obtained by modifying amino acids of a molecule binding to the antigen. Fc regions that have a desired or increased FcRn binding activity under an acidic pH range condition can also be appropriately obtained by modifying the amino acids in an Fc region. Amino acid modifications of an Fc region that result in such a desired binding activity can be found by comparing the FcRn binding activity under an acidic pH range condition before and after amino acid modification. Those skilled in the art can appropriately modify amino acids using known techniques similar to the above mentioned techniques used to modify Fcγ receptor binding activity.
[01144] [01144] The Fc regions comprised in the antigen binding molecules of the present invention, which have an FcRn binding activity under an acidic pH range condition, can be obtained by any method. Specifically, FcRn binding domains that have increased FcRn binding activity or FcRn binding activity under an acidic pH range condition can be obtained by modifying the human IgG-type immunoglobulin amino acids used as an initial Fc region. The preferred Fc regions of an IgG-like immunoglobulin for modification include, for example, those of human IgGs (IgG1, IgG2, IgG3, and IgG4 and variants thereof). Although the Fc region has an FcRn binding activity under an acidic pH range condition or can increase human FcRn binding activity under an acidic pH range condition, amino acids at any position can be modified into other amino acids . When the antigen-binding molecule contains the human IgG1 Fc region as the Fc region, it is preferable that the resulting Fc region contains a modification that results in the effect of increasing FcRn binding under a condition of acidic pH range comparing with the binding activity of the initial human IgG1 Fc region. Amino acids that allow such modification include, for example, amino acids from positions 252, 254, 256, 309, 311, 315, 433, and / or 434 according to the EU numbering and their combination amino acids at positions 253, 310 , 435, and / or 426 as described in WO 1997/034631. Favorable examples include amino acids of 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 as indicated by the EU numbering as described in WO 2000/042072. Similarly favorable examples of amino acids that allow such modification include, the amino acids of positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and / or 436 according to EU numbering as described in WO 2002/060919. In addition, the amino acids that allow such modification include, for example, amino acids at positions 250, 314, and 428 according to the EU numbering as described in WO2004 / 092219. In addition, favorable examples of amino acids that allow such modification include amino acids of 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. In addition, favorable examples of amino acids that allow such modification include amino acids of positions 251, 252, 307, 308, 378, 428, 430, 434, and / or 436 according to the EU numbering as described in WO 2010 / 045193. The modification of these amino acids increases the FcRn binding of the Fc region of an IgG-like immunoglobulin under an acidic pH range condition.
[01145] [01145] When the human IgG1 Fc region is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region includes at least one or more amino acid changes selected from the group consisting of:
[01146] [01146] Arg or Leu for the amino acid of position 251;
[01147] [01147] Phe, Ser, Thr or Tyr for the amino acid of position 252;
[01148] [01148] Ser or Thr for the amino acid of position 254;
[01149] [01149] Arg, Gly, Ile or Leu for the amino acid of position 255;
[01150] [01150] Ala, Arg, Asn, Asp, Gln, Glu or Thr for the amino acid at position 256;
[01151] [01151] Ile or Thr for the amino acid of position 308;
[01152] [01152] Pro for the amino acid of position 309;
[01153] [01153] Glu, Leu or Ser for the amino acid of position 311;
[01154] [01154] Ala or Asp for the amino acid of position 312;
[01155] [01155] Ala or Leu for the amino acid of position 314;
[01156] [01156] Ala, Arg, Asp, Gly, His, Lys, Ser or Thr for the amino acid of position 385;
[01157] [01157] Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr for the amino acid of position 386;
[01158] [01158] Ala, Arg, His, Pro, Ser or Thr for the amino acid of position 387;
[01159] [01159] Asn, Pro, or Ser for the amino acid of heading 389;
[01160] [01160] Leu, Met, Phe, Ser or Thr for the amino acid of position 428;
[01161] [01161] Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433;
[01162] [01162] His, Phe or Tyr for the amino acid of position 434; and
[01163] [01163] Arg, Asn, His, Lys, Met, or Thr for the amino acid at the
[01164] [01164] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding in an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including Ile for the amino acid of position 308, Pro for the amino acid of position 309, and / or Glu for the amino acid of position 311 according to the EU number. Another non-limiting embodiment of this modification may include Thr for the amino acid of position 308, Pro for the amino acid of position 309, Leu for the amino acid of position 311, Ala for the amino acid of position 312 and / or Ala for the amino acid of position 314. In addition, another non-limiting embodiment of this modification may include Ile or Thr for the amino acid of position 308, Pro for the amino acid of position 309, Glu, Leu or Ser for the amino acid of position 311, Ala for the amino acid of position 312, and / or Ala or Leu for the amino acid of position 314. Another non-limiting modality of this modification may include Thr for the amino acid of position 308, Pro for the amino acid of position 309, Ser for the amino acid of position 311, Asp for the amino acid at position 312, and / or Leu for the amino acid at position 314.
[01165] [01165] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - human IgG1 initial Fc region can be modifications including Leu for the amino acid at position 251, Tyr for the amino acid at
[01166] [01166] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - human IgG1 initial Fc region can be modifications including Leu, Met, Phe, Ser, or Thr for the amino acid of position 428, Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433, His, Phe, or Tyr for the amino acid of position 434, and / or Arg, Asn, His, Lys, Met, or Thr for the amino acid of position 436 according to the EU numbering. Another non-limiting modality of this modification may include His or Met for the amino acid at position 428, and / or his or Met for the amino acid at position 434.
[01167] [01167] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including Arg for the amino acid of position 385, Thr for the amino acid of position 386, Arg for the amino acid of position 387, and / or Pro for the amino acid of position 389 according to with the EU numbering. Another non-limiting embodiment of this modification may include Asp for the amino acid at position 385, Pro for the amino acid at position 386, and / or Ser for the amino acid at position 389.
[01168] [01168] Furthermore, when the human IgG1 Fc region is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the activity of binding of the initial Fc region of human IgG1 includes at least one or more amino acid modifications selected from the group consisting of:
[01169] [01169] Gln or Glu for the amino acid of position 250; and
[01170] [01170] Leu or Phe for the amino acid of position 428 according to EU numbering. The number of amino acids to be modified is not particularly limited; and the amino acid can be modified at only one site or the amino acids can be modified at two sites.
[01171] [01171] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including Gln for the amino acid at position 250, and / or Leu or Phe for the amino acid at position 428 according to the EU numbering. Another non-limiting embodiment of this modification may include Glu for the amino acid at position 250, and / or Leu or Phe for the amino acid at position 428.
[01172] [01172] When the human IgG1 Fc region is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region includes at least two or more amino acid modifications selected from the group consisting of:
[01173] [01173] Asp or Glu for the amino acid of position 251;
[01174] [01174] Tyr for the amino acid of position 252;
[01175] [01175] Gln for the amino acid of position 307;
[01176] [01176] Pro for the amino acid of position 308;
[01177] [01177] Val for the amino acid of position 378;
[01178] [01178] Wing for the amino acid of position 380;
[01179] [01179] Read for the amino acid of position 428;
[01180] [01180] Ala or Lys for the amino acid of position 430;
[01181] [01181] Ala, His, Ser or Tyr for the amino acid of position 434; and
[01182] [01182] Ile for the amino acid of position 436, as indicated by the EU numbering. The number of amino acids to be modified is not particularly limited; and the amino acid can be modified at only two sites or the amino acids can be modified at three or more sites.
[01183] [01183] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including Gln for the amino acid at position 307, and Ala or Ser for the amino acid at position 434 according to EU numbering. Another non-limiting modality of this modification can include Pro for the amino acid of position 308, and Ala for the amino acid of position 434. In addition, another non-limiting modality of this modification can include Tyr for the amino acid of position 252, and Ala for the amino acid at position 434. A non-limiting modality other than this modification may include Val for the amino acid at position 378, and Ala for the amino acid at position 434. Another non-limiting modality other than this modification may include Leu for the amino acid at position 428, and Ala for the amino acid of position 434. Another non-limiting modality other than this modification may include Ala for the amino acid of position 434, and Ile for the amino acid of position 436. In addition, another non-limiting modality of this modification can include Pro for the amino acid at position 308, and Tyr for the amino acid at position 434. In addition, another non-limiting modality of this modification may include Gln for the amino acid of position 307, and Ile for the amino acid of position 436.
[01184] [01184] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including any of Gln for the amino acid at position 307, Ala for the amino acid at position 380, and Ser for the amino acid at position 434 according to the EU numbering. Another non-limiting embodiment of this modification may include Gln for the amino acid at position 307, Ala for the amino acid at position 380 and Ala for the amino acid at position
[01185] [01185] When the Fc region of human IgG1 is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region includes the modification of at least two or more amino acids selected from the group consisting of:
[01186] [01186] Read for the amino acid of position 238;
[01187] [01187] Read for the amino acid of position 244;
[01188] [01188] Arg for the amino acid of position 245;
[01189] [01189] Pro for the amino acid of position 249;
[01190] [01190] Tyr for the amino acid of position 252;
[01191] [01191] Pro for the amino acid of position 256;
[01192] [01192] Ala, Ile, Met, Asn, Ser or Val for the amino acid of position 257;
[01193] [01193] Asp for the amino acid of position 258;
[01194] [01194] Be for the amino acid of position 260;
[01195] [01195] Read for the amino acid of position 262;
[01196] [01196] Lys for the amino acid of position 270;
[01197] [01197] Leu or Arg for the amino acid of position 272;
[01198] [01198] Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 279;
[01199] [01199] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 283;
[01200] [01200] Asn for the amino acid of heading 285;
[01201] [01201] Phe for the amino acid of position 286;
[01202] [01202] Asn or Pro for the amino acid of position 288;
[01203] [01203] Val for the amino acid of position 293;
[01204] [01204] Ala, Glu, or Met for the amino acid of position 307;
[01205] [01205] Ala, Ile, Lys, Leu, Met, Val or Trp for the amino acid of position 311;
[01206] [01206] Pro for the amino acid of position 312;
[01207] [01207] Lys for the amino acid of position 316;
[01208] [01208] Pro for the amino acid of position 317;
[01209] [01209] Asn or Thr for the amino acid of position 318;
[01210] [01210] Phe, His, Lys, Leu, Met, Arg, Ser or Trp for the amino acid of position 332;
[01211] [01211] Asn, Thr or Trp for the amino acid of position 339;
[01212] [01212] Pro for the amino acid of position 341;
[01213] [01213] Glu, His, Lys, Gln, Arg, Thr or Tyr for the amino acid of position 343;
[01214] [01214] Arg for the amino acid of position 375;
[01215] [01215] Gly, Ile, Met, Pro, Thr or Val for the amino acid of the position
[01216] [01216] Lys for the amino acid of position 377;
[01217] [01217] Asp or Asn for the amino acid of position 378;
[01218] [01218] Asn, Ser or Thr for the amino acid of position 380;
[01219] [01219] Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 382;
[01220] [01220] Asn for the amino acid of position 423;
[01221] [01221] Asn for the amino acid of heading 427;
[01222] [01222] Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr for the amino acid of position 430;
[01223] [01223] His or Asn for the amino acid of position 431;
[01224] [01224] Phe, Gly, His, Trp or Tyr for the amino acid of position 434;
[01225] [01225] Ile, Leu or Thr for the amino acid of position 436;
[01226] [01226] Lys, Leu, Thr or Trp for the amino acid of position 438;
[01227] [01227] Lys for the amino acid of position 440; and
[01228] [01228] Lys for the amino acid of position 442 according to EU numbering. The number of amino acids to be modified is not particularly limited and the amino acid can only be modified at two sites and the amino acids at three or more sites can be modified.
[01229] [01229] When the human IgG1 Fc region is understood as the Fc region, a non-limiting modification modification that results in the effect of increasing FcRn binding under an acidic pH range condition compared to the binding activity of the re - Human IgG1 initial Fc region can be modifications including Ile for the amino acid at position 257 and Ile for the amino acid at position 311 according to EU numbering. Another non-limiting embodiment of this modification may include Ile for the amino acid at position 257, and His for the amino acid at position 434. Another non-limiting embodiment of this modification may include Val for the amino acid at position 376,
[01230] [01230] In addition, in another non-limiting modality, one can trace by antigen binding molecules that comprise an Fc region with the characteristic of having human FcRn binding activity in the neutral pH range instead of the characteristic described to have a human FcRn binding activity in the acidic pH range. In a preferred embodiment, it can be traced by antigen-binding molecules that comprise an Fc region whose human FcRn binding activity in the neutral pH range is 40 M (KD) or stronger. In a more preferred embodiment, it can be screened for antigen-binding molecules that comprise an Fc region whose human FcRn binding activity in the neutral pH range is 15 M (KD) or stronger.
[01231] [01231] In addition, in another non-limiting modality, one can trace by antigen-binding molecules that comprise an Fc region with the characteristic of having a human FcRn binding activity in the neutral pH range in addition to the characteristic described above to have a human FcRn binding activity in the acidic pH range. In a preferred embodiment, it can be screened for antigen-binding molecules that comprise an Fc region whose human FcRn-binding activity in the neutral pH range is 40 M (KD) or stronger. In a more preferred embodiment, it can be screened for antigen-binding molecules that comprise an Fc region whose human FcRn binding activity in the neutral pH range is 15 M (KD) or stronger.
[01232] [01232] In the present invention, the preferred Fc regions have a human FcRn binding activity in the acidic pH range and / or neutral pH range. When an Fc region originally has human FcRn binding activity in the acidic pH range and / or neutral pH range, it can be used as is. When an Fc region has poor or no human FcRn binding activity in the acidic pH range and / or neutral pH range, the antigen binding molecules that comprise an Fc region that has a binding activity for The desired human FcRn can be obtained by modifying amino acids in the Fc region comprised in the antigen binding molecules. Fc regions that have a desired human FcRn binding activity in the acidic pH range and / or neutral pH range can also be appropriately obtained by modifying amino acids in a human Fc region. Alternatively, antigen binding molecules that comprise an Fc region that has a desired human FcRn binding activity can be obtained by modifying amino acids from an Fc region that originally has human FcRn binding activity in the range acidic pH and / or neutral pH range. Amino acid modifications of a human Fc region that result in such a desired binding activity can be found by comparing the human FcRn binding activity in the acidic pH range and / or neutral pH range before and after amino acid modification. Those skilled in the art can appropriately modify amino acids using known methods.
[01233] [01233] In addition, a modified altered Fc region from an initial modified Fc region can also be used preferably as an altered Fc region of the present invention. The "initial Fc region" can refer to the polypeptide itself, a composition comprising the initial Fc region or amino acid sequence that encodes the initial Fc region. Initial Fc regions may comprise a known IgG antibody Fc region produced via recombination described briefly in the "Antibodies" section. The origin of early Fc regions is not limited, and can be obtained from humans or any non-human organism. Such organizations
[01234] [01234] Examples of changes include those with one or more mutations, for example, mutations by substituting amino acid residues other than amino acids from initial Fc regions, by inserting one or more amino acid residues in initial Fc regions, or by deletion of one or more amino acids from the initial Fc region. Preferably, the amino acid sequences of altered Fc regions comprise at least a part of the amino acid sequence of a non-native Fc region. Such variants necessarily have less than 100% sequence identity or similarity to their initial Fc region. In a preferred embodiment, the variants have the identity or similarity of amino acid sequence approximately 75% to less than 100%, more preferably approximately 80% to less than 100%, even more preferably approximately 85% to less than 100%, even more preferably approximately 90% to less than 100%, and even more preferably approximately 95% to less than 100% to the amino acid sequence of your initial Fc region. In a non-limiting embodiment of the present invention, at least one amino acid is different between a modified Fc region of the present invention and its initial Fc region. The amino acid difference between a modified Fc region of the present invention and its initial Fc region can also be preferably specified based on amino acid differences in the particular amino acid positions described above according to the EU numbering system.
[01235] [01235] Methods known as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed to modify the amino acids from Fc regions. In addition, several known methods can also be used as an amino acid modification method to replace amino acids with those in addition to natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cellless translation system (Clover Direct (Protein Express)) containing tRNAs in which the amber suppressor tRNA, which is complementary to the UAG codon (amber codon) which is a stop codon, is linked with an unnatural amino acid can be used appropriately.
[01236] [01236] Fc regions comprised in the antigen binding molecules of the present invention that have human FcRn binding activity in the acidic pH range can be obtained by any method. Specifically, it can be traced by antigen binding molecules that comprise an Fc region of which the human FcRn binding activity in the acidic pH range is 20 M (KD) or stronger; in a more favorable modality, an Fc region in which the human FcRn binding activity in the acidic pH range is 2.0 M (KD) or stronger; and in an even more favorable modality, an Fc region in which the human FcRn binding activity in the acidic pH range is 0.5 M (KD) or stronger as a result of the modification of human immunoglobulin IgG-type amino acids used as an initial Fc region. Preferred Fc regions of IgG-like immunoglobulins for modification include, for example, those of human IgGs such as IgG1, IgG2, IgG3 and IgG4 shown in SEQ ID NOS: 13, 14, 15, and 15, respectively, and variants - tas.
[01237] [01237] When an antigen-binding molecule comprises the human IgG1 Fc region as the Fc region, suitable examples of amino acids that can be modified to achieve the aforementioned desired effects on FcRn binding under a band condition acidic pH by modification of human IgG-type immunoglobulin amino acids as an initial Fc region, include amino acids of 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 according to EU numbering as described in WO 2000/042072. Similar favorable examples of amino acids that allow such modification include amino acids of positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and / or 436 of according to EU numbering as described in WO 2002/060919. In addition, the amino acids that allow such modification include, for example, amino acids at positions 250, 314, and 428 according to the EU numbering as described in WO2004 / 092219. In addition, favorable examples of amino acids that allow such modification include amino acids of positions 251, 252, 307, 308, 378, 428, 430, 434, and / or 436 according to EU numbering as described in WO 2010/045193. The modification of these amino acids increases the FcRn binding of the Fc region of an IgG-type immunoglobulin under an acidic pH range condition.
[01238] [01238] Fc regions that have human FcRn binding activity in the neutral pH range can also be obtained by modifying the human IgG-like immunoglobulin amino acids used as the initial Fc region. Fc regions of IgG-like immunoglobulins suitable for modification include, for example, those of human IgGs such as IgG1, IgG2, IgG3 and IgG4 respectively represented by SEQ ID NOs: 13, 14, 15, and 16, and modified forms of these. Amino acids from any position can be modified to other amino acids, although Fc regions have human FcRn binding activity in the neutral pH range or can increase human FcRn binding activity in the neutral range. When the antigen-binding molecule contains the human IgG1 Fc region as the human Fc region, it is preferred that the resulting Fc region contains a modification that results in the effect of increasing human FcRn binding in the neutral pH range compared to the binding activity of the initial human IgG1 Fc region. Amino acids that allow such modification include, for example, one or more amino acids selected from the group of positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442 according to EU numbering. The modification of these amino acids increases the binding of human FcRn of the IgG type immunoglobulin Fc region in the neutral pH range.
[01239] [01239] From those described above, modifications that increase the binding of human FcRn in the neutral pH range are appropriately selected for use in the present invention. Particularly preferred amino acids from the modified Fc regions include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305 , 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 of according to the EU numbering system. The binding activity of human FcRn in the neutral pH range of the Fc region contained in an antigen binding molecule can be increased by replacing at least one amino acid selected from the above amino acids with a different amino acid.
[01240] [01240] Particularly preferred modifications include, for example:
[01241] [01241] Met for the amino acid of position 237;
[01242] [01242] Ile for the amino acid of position 248;
[01243] [01243] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250;
[01244] [01244] Phe, Trp or Tyr for the amino acid of position 252;
[01245] [01245] Thr for the amino acid of position 254;
[01246] [01246] Glu for the amino acid of position 255;
[01247] [01247] Asp, Asn, Glu or Gln for the amino acid of position 256;
[01248] [01248] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257;
[01249] [01249] His for the amino acid of position 258:
[01250] [01250] Wing for the amino acid of position 265;
[01251] [01251] Ala or Glu for the amino acid of position 286;
[01252] [01252] His for the amino acid of position 289;
[01253] [01253] Wing for the amino acid of position 297;
[01254] [01254] Wing for the amino acid of position 303;
[01255] [01255] Wing for the amino acid of position 305;
[01256] [01256] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307;
[01257] [01257] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308;
[01258] [01258] Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309;
[01259] [01259] Ala, His or Ile for the amino acid of position 311;
[01260] [01260] Ala or His for the amino acid of position 312;
[01261] [01261] Lys or Arg for the amino acid of position 314;
[01262] [01262] Ala, Asp or His for the amino acid of position 315;
[01263] [01263] Wing for the amino acid of position 317;
[01264] [01264] Val for the amino acid of position 332;
[01265] [01265] Read for the amino acid of position 334;
[01266] [01266] His for the amino acid of position 360;
[01267] [01267] Wing for the amino acid of position 376;
[01268] [01268] Wing for the amino acid of position 380;
[01269] [01269] Ala for the amino acid of position 382;
[01270] [01270] Ala for the amino acid of position 384;
[01271] [01271] Asp or His for the amino acid of position 385;
[01272] [01272] Pro for the amino acid of position 386;
[01273] [01273] Glu for the amino acid of position 387;
[01274] [01274] Ala or Ser for the amino acid of position 389;
[01275] [01275] Ala for the amino acid of position 424;
[01276] [01276] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 428;
[01277] [01277] Lys for the amino acid of position 433;
[01278] [01278] Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and
[01279] [01279] His, Ile, Leu, Phe, Thr or Val for the amino acid at position 436 of the Fc region according to EU numbering.
[01281] [01281] Table 5-2 is a continuation of Table 5-1.
[01283] [01283] Table 5-3 is a continuation of Table 5-2.
[01285] [01285] Table 5-4 is a continuation of Table 5-3.
[01287] [01287] Table 5-5 is a continuation of Table 5-4.
[01289] [01289] Table 5-6 is a continuation of Table 5-5.
[01291] [01291] Table 5-7 is a continuation of Table 5-6.
[01293] [01293] Table 5-8 is a continuation of Table 5-7.
[01295] [01295] Table 5-9 is a continuation of Table 5-8.
[01297] [01297] Table 5-10 is a continuation of Table 5-9.
[01299] [01299] Table 5-11 is a continuation of Table 5-10.
[01301] [01301] Table 5-12 is a continuation of Table 5-11.
[01303] [01303] Table 5-13 is a continuation of Table 5-12.
[01305] [01305] Table 5-14 is a continuation of Table 5-13.
[01307] [01307] Table 5-15 is a continuation of Table 5-14.
[01309] [01309] Table 5-16 is a continuation of Table 5-15.
[01311] [01311] Table 5-17 is a continuation of Table 5-16.
[01313] [01313] Table 5-18 is a continuation of Table 5-17.
[01315] [01315] Table 5-19 is a continuation of Table 5-18.
[01317] [01317] Table 5-20 is a continuation of Table 5-19.
[01319] [01319] Table 5-21 is a continuation of Table 5-20.
[01321] [01321] Table 5-22 is a continuation of Table 5-21.
[01323] [01323] Table 5-23 is a continuation of Table 5-22.
[01325] [01325] Table 5-24 is a continuation of Table 5-23.
[01327] [01327] Table 5-25 is a continuation of Table 5-24.
[01329] [01329] Table 5-26 is a continuation of Table 5-25.
[01331] [01331] Table 5-27 is a continuation of Table 5-26.
[01333] [01333] Table 5-28 is a continuation of Table 5-27.
[01335] [01335] Table 5-29 is a continuation of Table 5-28.
[01337] [01337] Table 5-30 is a continuation of Table 5-29.
[01339] [01339] Table 5-31 is a continuation of Table 5-30.
[01341] [01341] Table 5-32 is a continuation of Table 5-31.
[01343] [01343] Table 5-33 is a continuation of Table 5-32.
[01345] [01345] Fcγ receptor
[01346] [01346] The Fcγ receptor (also described as FcγR) refers to a receptor capable of binding to the Fc region of monoclonal antibodies IgG1, IgG2, IgG3 or IgG4, and includes all members that belong to the family of proteins substantially encoded by an Fcγ receptor gene. In humans, the family includes FcγRI (CD64) including FcγRIa, FcγRIb and FcγRIc isoforms; FcγRII (CD32) including FcγRIIa isoforms (including allotypes H131 and R131, i.e., FcγRIIa (H) and FcγRIIa (R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRI- Ic; and FcγRIII (CD16) including FcγRIIIa isoforms (including allotypes V158 and F158, i.e., FcγRIIIa (V) and FcγRIIIa (F)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all unidentified human FcγRs, FcγR isoforms and allotypes thereof. However, the Fcγ receptor is not limited to these examples. Without being limited to these, FcγR includes those derived from humans, mice, rats, rabbits and monkeys. FcγR can be derived from any organism. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as all unidentified mouse FcγRs, isoforms of FcγR and allotypes thereof. Such Fcγ receptors preferentially
[01347] [01347] In FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc and FcγRIII (CD16) including FcγRIIIa isoforms (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRII- α that binds to the Fc portion of IgG is associated with the common γ chain with ITAM responsible for the transduction of the intracellular activation signal. However, the cytoplasmic domain of FcγRII (CD32) including FcγRIIa isoforms (including the H131 and R131 allotypes) and FcγRIIc contains ITAM. These receptors are expressed in many immune cells such as macrophages, mast cells and
[01348] [01348] However, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM responsible for the transduction of inhibitory signals. Crosslinking between FcγRIIb and B cell receptor (BCR) in B cells suppresses the BCR activation signal, which results in suppression of antibody production via BCR. Crosslinking of Fcγ- RIII and FcγRIIb in macrophages suppresses phagocytic activity and inflammatory cytokine production. Fcγ receptors that have the ability to transduce the inhibitory signal as described above are also referred to as the inhibitory Fcγ receptor. FcγR binding activity from the Fc region
[01349] [01349] As mentioned above, the Fc regions having an Fcγ receptor binding activity are examples of the Fc regions comprised in the antigen binding molecules of the present invention. A non-limiting embodiment of such an Fc region includes the Fc region of IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16) ) human. If an Fcγ receptor has activity to bind to the Fc region of a monoclonal antibody for IgG1, IgG2, IgG3, or IgG4, it can be evaluated by ALPHA screening (Amplified Homogeneous Lumenescent Proximity Assay), the BIACORE method based on surface plasmon resonance (SPR), and the others (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010), in addition to the ELISA and FACS formats described above.
[01350] [01350] ALPHA tracking is performed by ALPHA technology based on the principle described below using two types of accounts: donor and acceptor accounts. A luminescent signal is only detected when the molecules linked to the donor beads interact biologically with molecules linked to the acceptor beads and when the two beads are located in close proximity. Excited by the laser beam, the photosensitizers in a donor convert the oxygen around the bead into the excited oxygen in singlet. When singlet oxygen diffuses around the donor beads and reaches the acceptor beads located in the immediate vicinity, a chemiluminescent reaction within the acceptor beads is induced. This reaction ultimately results in the emission of light. If the molecules linked to the donor accounts do not interact with molecules linked to the acceptor beads, the singlet oxygen produced by donor beads does not reach the acceptor beads and the chemiluminescent reaction does not occur.
[01351] [01351] For example, a molecule labeled with biotin binding to the antigen and comprising the Fc region is immobilized to the donor beads and Fcγ receptor labeled with glutathione S-transferase (GST) is immobilized to the acceptor beads. The absence of an antigen binding molecule comprising a competitive Fc region variant, the Fcγ receptor interacts with a polypeptide complex comprising a wild Fc region, inducing a signal from 520 to 620 nm as a result. The antigen-binding molecule having an unlabeled Fc region variant competes with the antigen-binding molecule and comprises a native Fc region for interaction with the Fcγ receptor. The relative binding affinity can be determined by quantifying the fluorescence reduction as a result of competition. Methods for biotinylating antigen-binding molecules as antigen-binding molecules using Sulfo-NHS-biotin or the like are known. Appropriate methods for adding the GST marker to an Fcγ receptor include methods involving fusion polypeptides that encode Fcγ and GST in the structure, expressing the fused gene using cells introduced with a vector to which the gene is operationally linked, and then purifying using a glutathione column. The induced signal can preferably be analyzed, for example, by adjusting to a competition model of a site based on nonlinear regression analysis using a GRAPHPAD PRISM program (GraphPad; San Diego).
[01352] [01352] One of the substances to observe their interaction is immobilized as a binder for the thin gold layer of a sensor chip. When light is emitted on the rear surface of the sensor chip so that the total reflection occurs at the interface between the thin layer of gold and the glass, the intensity of the reflected light is partially reduced in a certain place (SPR signal). Another substance to observe their interaction is injected as an analyte into the surface of the sensor chip. The mass of the immobilized ligand molecule increases when the analyte binds to the ligand. This changes the refractive index of the solvent on the surface of the sensor chip. The change in the refractive index causes a positional displacement of the SPR signal (conversely, the dissociation moves the signal back to its original position). In the Biacore system, the amount of displacement described above (that is, the change in mass on the surface of the sensor chip) is plotted on the vertical axis, and thus the change in mass over time is shown as measured data (sensorgram ). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and affinity (KD) is determined from the ratio between these two constants. The inhibition assay is preferably used in the BIACORE methods. Examples of such an inhibition test are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
[01353] [01353] In addition to the Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16), a Fc region with modified FcγR binding, which has a higher Fcγ receptor binding activity than a native human IgG Fc region can be appropriately used as an Fc region included in the present invention. In this application, "native human IgG Fc region" refers to an Fc region in which the sugar chain linked to position 297 (EU numbering) of the human IgG1, IgG2, IgG3 or IgG4 Fc region shown in SEQ ID NOS: 13, 14, 15, or 16 is a sugar chain containing fucose. Such Fc regions with the modified FcγR binding can be produced by modifying amino acids from the Fc region of native human IgG. Whether the FcγR binding activity of an Fc region with the modified FcγR binding is higher than that of a native human IgG Fc region can be determined appropriately using methods described in the section above in the binding activity.
[01354] [01354] In the present invention, "amino acid modification" or "amino acid modification" of an Fc region includes modification in an amino acid sequence that is different from that of the initial Fc region. The initial Fc region can be any Fc region, while a modified variant of the initial Fc region can bind to the human Fcγ receptor in a neutral pH range. In addition, a modified Fc region from an initial Fc region that had already been modified can also be used preferably as an Fc region of the present invention. The "initial Fc region" can refer to the polypeptide itself, a composition comprising the initial Fc region or amino acid sequence that encodes the initial Fc region. The initial Fc regions may comprise known Fc regions produced via recombination described briefly in the "Antibodies" section. The origin of early Fc regions is not limited, and can be obtained from humans or any non-human organism. Such organisms preferably include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels and organisms selected from non-human primates. In another embodiment, initial Fc regions can also be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The initial Fc regions can preferably be obtained 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 used appropriately as an initial Fc region, and in this application it also means that an Fc region of an arbitrated IgG class or subclass derived from any The organism described above can preferably be used as an initial Fc region. Examples of naturally occurring IgG variants or modified forms are described in published documents (Curr. Opin. Biotechnol. (2009) (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460- 470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202; International Publication WO No. 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338); however, they are not limited to examples.
[01355] [01355] Examples of changes include those with one or more mutations, for example, mutations by replacing amino acid residues other than amino acids from initial Fc regions, by inserting one or more amino acid residues in initial Fc regions, or by the deletion of one or more amino acids from the initial Fc region. Preferably, the amino acid sequences of altered Fc regions comprise at least a part of the amino acid sequence of a non-native Fc region. Such variants necessarily have less than 100% sequence identity or similarity to their initial Fc region. In a preferred embodiment, the variants have the identity or similarity of amino acid sequence from approximately 75% to less than 100%, more preferably approximately 80% to less than 100%, even more preferably approximately 85% to less than 100%, even more preferably approximately 90% to less than 100%, and even more preferably approximately 95% to less than 100% at the amino acid sequence of your initial Fc region. In a non-limiting mode of the present invention, at least one amino acid is different between the modified Fc region of an FcγR-binding region of the present invention and its initial Fc region. The amino acid difference between a modified FcγR-binding Fc region of the present invention and its initial Fc region can also be preferably specified based on the specific amino acid differences at the specific amino acid positions described above. according to the EU numbering system.
[01356] [01356] Methods known as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed to modify the amino acids from Fc regions. In addition, several known methods can also be used as an amino acid modification method to replace amino acids with those in addition to natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which the amber suppressor tRNA, which is complementary to the UAG codon (amber codon) which is a stop codon, is linked to an unnatural amino acid can be properly used.
[01357] [01357] Included in the antigen binding molecules of the present invention, an Fc region with modified FcγR binding, which has a higher Fcγ receptor binding activity than that of a human IgG Fc region native, (a modified FcγR binding Fc region) can be obtained by any method. Specifically, the Fc region with the modified FcγR binding can be obtained by modifying human IgG-like immunoglobulin amino acids used as an initial Fc region. Preferred Fc regions of IgG-like immunoglobulins for modification include, for example, those of human IgGs shown in SEQ ID NOS: 13, 14, 15, or 16 (IgG1, IgG2, IgG3, or IgG4, respectively, and variants of these).
[01358] [01358] Amino acids from any position can be modified to other amino acids, while the binding activity towards the Fcγ receptor is higher than that of the native human IgG Fc region. When the antigen-binding molecule contains a human IgG1 Fc region as the human Fc region, it preferably contains a modification that produces the effect of a higher Fcγ receptor binding activity than that of the Fc region of native human IgG, in which the sugar chain linked at position 297 (EU numbering) is a sugar chain containing fucose. Such amino acid modifications have been reported, for example, in international publications such as WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO2006 / 020114, WO2006 / 116260 and WO2006 / 023403.
[01359] [01359] Examples of such amino acids that can be modified include at least one or more amino acids selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232 , 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263,
[01360] [01360] Examples of particularly preferred modifications for use in the present invention include at least one or more amino acid changes selected from the group consisting of:
[01361] [01361] Lys or Tyr for the amino acid of position 221;
[01362] [01362] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[01363] [01363] Phe, Trp, Glu or Lys for the amino acid of position 223;
[01364] [01364] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[01365] [01365] Glu, Lys or Trp for the amino acid of position 225;
[01366] [01366] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[01367] [01367] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[01368] [01368] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[01369] [01369] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[01370] [01370] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[01371] [01371] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[01372] [01372] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[01373] [01373] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,
[01374] [01374] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[01375] [01375] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237;
[01376] [01376] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[01377] [01377] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239;
[01378] [01378] Ala, Ile, Met, or Thr for the amino acid at position 240;
[01379] [01379] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[01380] [01380] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[01381] [01381] His for the amino acid of position 244;
[01382] [01382] Ala for the amino acid of position 245;
[01383] [01383] Asp, Glu, His or Tyr for the amino acid of position 246;
[01384] [01384] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[01385] [01385] Glu, His, Gln or Tyr for the amino acid of position 249;
[01386] [01386] Glu or Gln for the amino acid of position 250;
[01387] [01387] Phe for the amino acid of position 251;
[01388] [01388] Phe, Met, or Tyr for the amino acid of position 254;
[01389] [01389] Glu, Leu or Tyr for the amino acid of position 255;
[01390] [01390] Ala, Met, or Pro for the amino acid of position 256;
[01391] [01391] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[01392] [01392] Asp, Glu, His or Tyr for the amino acid of position 260;
[01393] [01393] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[01394] [01394] Ala, Ile, Met, or Thr for the amino acid of position 263;
[01395] [01395] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[01396] [01396] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[01397] [01397] Ala, Ile, Met, or Thr for the amino acid of position 266;
[01398] [01398] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[01399] [01399] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[01400] [01400] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[01401] [01401] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[01402] [01402] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[01403] [01403] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[01404] [01404] Phe or Ile for the amino acid of position 273;
[01405] [01405] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[01406] [01406] Leu or Trp for the amino acid of position 275;
[01407] [01407] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[01408] [01408] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 278;
[01409] [01409] Wing for the amino acid of position 279;
[01410] [01410] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[01411] [01411] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[01412] [01412] Glu, Gly, Lys, Pro, or Tyr for the amino acid of the position
[01413] [01413] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[01414] [01414] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284;
[01415] [01415] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[01416] [01416] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[01417] [01417] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[01418] [01418] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[01419] [01419] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[01420] [01420] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[01421] [01421] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[01422] [01422] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[01423] [01423] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[01424] [01424] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[01425] [01425] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297;
[01426] [01426] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[01427] [01427] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 299;
[01428] [01428] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
[01429] [01429] Asp, Glu, His or Tyr for the amino acid of position 301;
[01430] [01430] Ile for the amino acid of position 302;
[01431] [01431] Asp, Gly or Tyr for the amino acid of position 303;
[01432] [01432] Asp, His, Leu, Asn or Thr for the amino acid of position 304;
[01433] [01433] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[01434] [01434] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311;
[01435] [01435] Phe for the amino acid of position 313;
[01436] [01436] Read for the amino acid of position 315;
[01437] [01437] Glu or Gln for the amino acid of position 317;
[01438] [01438] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[01439] [01439] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 320;
[01440] [01440] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[01441] [01441] Ile for the amino acid of position 323;
[01442] [01442] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[01443] [01443] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[01444] [01444] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326;
[01445] [01445] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[01446] [01446] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[01447] [01447] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
[01448] [01448] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[01449] [01449] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 331;
[01450] [01450] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[01451] [01451] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the amino acid of position 333;
[01452] [01452] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[01453] [01453] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[01454] [01454] Glu, Lys or Tyr for the amino acid of position 336;
[01455] [01455] Glu, His or Asn for the amino acid of position 337;
[01456] [01456] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[01457] [01457] Ala or Val for the amino acid of position 376;
[01458] [01458] Gly or Lys for the amino acid of position 377;
[01459] [01459] Asp for the amino acid of position 378;
[01460] [01460] Asn for the amino acid of heading 379;
[01461] [01461] Ala, Asn or Ser for the amino acid of position 380;
[01462] [01462] Ala or Ile for the amino acid of position 382;
[01463] [01463] Glu for the amino acid of position 385;
[01464] [01464] Thr for the amino acid of position 392;
[01465] [01465] Read for the amino acid of position 396;
[01466] [01466] Lys for the amino acid of position 421;
[01467] [01467] Asn for the amino acid of position 427;
[01468] [01468] Phe or Leu for the amino acid of position 428;
[01469] [01469] Met for the amino acid of position 429;
[01470] [01470] Trp for the amino acid of position 434;
[01471] [01471] Ile for the amino acid of position 436; and
[01472] [01472] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[01473] [01473] as indicated by the EU numbering. The number of amino acids to be modified is not particularly limited; and the amino acid can be modified at only one site or the amino acids can be modified at two or more sites. Examples of combinations of amino acid modifications at two or more sites include those described in Table 6 (Tables 6-1 to 6-3).
[01474] [01474] Table 6-2 is a continuation of Table 6-1.
[01475] [01475] Table 6-3 is a continuation of Table 6-2.
[01476] [01476] For pH conditions to measure the binding activity of the Fc region and the Fcγ receptor contained in the antigen binding molecule of the present invention, conditions in an acidic pH range or a pH range neutral can be used appropriately. The neutral pH range, as a condition for measuring the binding activity of the Fc region and the Fcγ receptor contained in the antigen binding molecule of the present invention, generally indicates pH 6.7 at pH 10.0. Preferably, it is a range indicated with pH values arbitrated between pH 7.0 and 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 particularly preferably, it is pH 7.4, which is close to the plasma (blood) pH in vivo. In this order, the acidic pH range, as a condition to have a binding activity of the Fc region and the Fcγ receptor contained in the antigen binding molecule of the present invention, generally indicates pH 4.0 at pH 6, 5. Preferably, it indicates pH 5.5 to pH 6.5, and particularly preferably, it indicates pH 5.8 to pH 6.0, which is close to the pH in the first endosome in vivo. As for the temperature used as a measurement condition, the binding affinity between an Fc region and an Fcγ receptor can be evaluated at any temperature between 10 ° C and 50 ° C. Preferably, a temperature between 15 ° C and 40 ° C is used to determine the binding affinity between an Fc region and an Fcγ receptor. More preferably, any temperature between 20 ° C and 35 ° C, like any single temperature of 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C , 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, and 35 ° C, can be similarly used to determine the binding affinity between an Fc region and an Fcγ receptor. A temperature of 25 ° C is a non-limiting example in a mode of the present invention.
[01477] [01477] In this application, "the Fcγ region with modified FcγR binding has a higher Fcγ receptor binding activity than the native Fc region" means that the human Fcγ receptor binding activity of the Fc region with modified FcγR that binds towards any of the human Fcγ receptors of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb is higher than the binding activity of the native Fc region towards these receive-
[01478] [01478] In the present invention, a native human IgG Fc region in which the sugar chain linked to the amino acid at position 297 (EU numbering) is a sugar chain containing fucose, is properly used as a native Fc region of human IgG to be used as a control. If the sugar chain linked to the amino acid at position 297 (EU numbering) is a sugar chain containing fucose, it can be determined using the technique described in Non-Patent Document 6. For example, it is possible to determine whether the sugar chain linked with native human IgG Fc region is a sugar chain containing fucose by a method like the one below. The sugar chain is dissociated from native human IgG to be tested
[01479] [01479] As an antigen-binding molecule containing an Fc region of a native antibody of the same subclass, which should be used as a control, an antigen-binding molecule having an Fc region of a monoclonal IgG antibody may be the - properly used. The structures of the Fc regions are described in SEQ ID NO: 13 (A is added to the N terminal of the Access No. of RefSeq AAC82527.1), SEQ ID NO: 14 (A is added to the N terminal of the Access No. RefSeq AAB59393.1), SEQ ID NO: 15 (RefSeq Access No. CAA27268.1) and SEQ ID NO: 16 (A is added to the N terminal of RefSeq Access No. AAB59394.1). In addition, when an antigen-binding molecule containing an Fc region of a particular antibody isotype is used as the test substance, the effect of the antigen-binding molecule containing the Fc region tests on the receptor activity of Fcγ is tested using as an control an antigen binding molecule having an Fc region of a monoclonal IgG antibody of that particular isotype. Thus, the antigen-binding molecules containing a region of
[01480] [01480] Examples of Fc regions suitable for use in the present invention include regions of Fc having a higher binding activity to a particular Fcγ receptor than to other Fcγ receptors (regions of Fc having a binding activity selective to an Fcγ receptor). When an antibody is used as the antigen-binding molecule, a single antibody molecule can bind only to a single Fcγ receptor molecule. Therefore, a single antigen-binding molecule cannot bind to other activating FcγRs in a state bound to the Fcγ receptor, and cannot bind to other Fcγ activation receptors or inhibitory Fcγ receptors. rivers in a state linked to the Fcγ activation receptor.
[01481] [01481] As described above, suitable examples of activating Fcγ receptors include FcγRI (CD64) which includes FcγRIa, FcγRIb and FcγRIc; FcγRIII (CD16) which includes the FcγRIIIa (including allotypes V158 or F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and FcγRIIa (including allotypes H131 or R131). However, suitable examples of inhibitory Fcγ receptors include FcγRIIb (including FcγRIIb-1 or FcγRIIb-2).
[01482] [01482] The Fc region comprised of an antigen-binding molecule of the present invention that contains a selective FcγR binding domain, and an antigen-binding molecule comprising this Fc region may have maintained binding activity or reduced to activation FcγR (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb-NA1, FcγRIIIb allotype including the FYRIII allotype, FcγRIII FcγRIIa including allotype
[01483] [01483] In comparison to a wild Fc region and an antigen binding molecule comprising a wild Fc region, an Fc region comprised in an antigen binding molecule of the present invention that contains a binding domain selective FcγR, and an antigen-binding molecule comprising this Fc region is reduced in its above-mentioned activation FcγR-binding activity to a level of, 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, 88% or less, 86% or less , 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 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, 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, 5% or less, 4% or less, 3% or less, 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.
[01484] [01484] The Fc region comprised in an antigen binding molecule of the present invention containing a selective FcγR binding domain, and an antigen binding molecule comprising this Fc region may have an increased inhibitory FcγR binding activity ( FcγRIIb-1 and / or FcγRIIb-2) when compared to the Fc region of IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16) human (hereinafter called the wild Fc region), and an antigen-binding molecule comprising the wild Fc region.
[01485] [01485] In comparison to a wild Fc region and an antigen binding molecule comprising a wild Fc region, the Fc region comprised in an antigen binding molecule of the present invention containing a selective FcγR binding domain, and an antigen binding molecule comprising this Fc region, is increased in its above-mentioned inhibitory FcγR binding activity to a level of, for example, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 112% or greater, 114% or greater, 116% or greater, 118 % or greater, 120% or greater, 122% or greater, 124% or greater, 126% or greater, 128% or greater, 130% or greater, 132% or greater, 134% or greater, 136% or greater , 138% or greater, 140% or greater, 142% or greater, 144% or greater, 146% or greater, 148% or greater, 150% or greater, 155% or greater, 160% or greater, 165% or greater , 170% or greater, 175% or greater, 180% or greater, 185% or greater , 190% or greater, 195% or greater, 2 times or greater, 3 times or greater, 4 times or greater, times or greater, 6 times or greater, 7 times or greater, 8 times or greater, 9 times or greater, 10 times or greater, 20 times or greater, 30 times or greater, 40 times or greater, 50 times or greater, 60 times or greater, 70 times or greater, 80 times or greater, 90 times or greater, 100 times or greater, 200 times or greater, 300 times or greater, 400 times or greater, 500 times or greater, 600 times or greater, 700 times or greater, 800 times or greater, 900 times or greater, 1000 times or greater, 10,000 times or greater, or 100000 times or greater.
[01486] [01486] The Fc region comprised of an antigen binding molecule of the present invention containing a selective FcγR binding domain, and an antigen binding molecule comprising this Fc region may have
[01487] [01487] a maintained or reduced binding activity for activating FcγR (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the V158 allotype, FcγRIIIa including the F158 allotype, FcγRIIIb including the FcγRIIIb- NA1, FcγRIIIb allotype including the FγRI including the H131 allotype, FcγRIIa including the R131 allotype, and / or FcγRIIc), when compared to the IgG1 Fc region (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15 ), or human IgG4 (SEQ ID NO: 16) (hereinafter referred to as the wild Fc region), and an antigen binding molecule comprising the wild Fc region; and
[01488] [01488] an increased binding activity for inhibitory FcγR (FcγRIIb-1 and / or FcγRIIb-2) when compared to the Fc region of IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 ( SEQ ID NO: 15), or human IgG4 (SEQ ID NO: 16) (hereinafter referred to as the wild Fc region), and an antigen binding molecule comprising the wild Fc region.
[01489] [01489] Compared to the human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or human IgG4 (SEQ ID NO: 16) Fc region hereinafter referred to as the wild Fc region) and an antigen binding molecule comprising the wild Fc region, the Fc region comprised of an antigen binding molecule of the present invention containing a selective FcγR binding domain, and a molecule binding antigen comprising this region of Fc, has a higher level of increase in its binding activity to inhibitory FcγR (FcγRIIb-1 and / or FcγRIIb-2) than the level of increase in its binding activity to activation Fcγ receptor (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including the allotype
[01490] [01490] In the present invention, it is possible to add at least one other modification to the Fc region whose amino acid at position 238 (EU numbering) is Asp or to the Fc region whose amino acid at position 328 (EU numbering) is Glu by modalities and as described in the section above on amino acid modifications. In addition to these modifications, additional modifications can also be included. Additional modifications can be selected, for example, from any of the substitutions, deletions, and modifications of amino acids and combinations thereof. For example, modifications that increase the binding activity of FcγRIIb by maintaining or reducing the binding activity to FcγRIIa (type H) and FcγRIIa (type R) can be added. The addition of such modifications improves the selectivity of binding to FcγRI-Ib over FcγRIIa.
[01491] [01491] Furthermore, if the binding activities of the polypeptides of the present invention towards various FcγRs are maintained, increased or decreased, they can be determined by increasing or decreasing the amount of binding of various FcγRs to the polypeptides of the present invention, which were determined according to the examples described above. In this order, the amount of binding of several FcγRs to the polypeptides refers to values obtained by determining the difference in the RU values of sensorgrams that were modified before and after interaction of several FcγRs such as the analyte with each polypeptide and division of them by differences in the RU values of sensorgrams that were modified before and after capturing polypeptides in the sensor chips.
[01492] [01492] It is possible to determine whether the FcγRIIa binding activity (type R and type H) of the polypeptides of the present invention is maintained or reduced, and whether the peptides are peptides with increased FcγRIIb binding activity can be determined from the amount of binding of polypeptides to FcγRIIa and the amount of binding of polypeptides to FcγRIIb following the above examples.
[01493] [01493] An exemplary case is when the amount of a polypeptide of the present invention bound to FcγRIIb is increased compared to the amount of FcγRIIb binding to the parent polypeptide, and the amount of the polypeptide of the present invention bound to FcγRIIa (ti - po R and type H) is equivalent to (maintained) or preferably reduced as to the amount of binding of the parental polypeptide to FcγRIIa (type R and type H). It is also possible to determine by appropriately combining the amount of FcγRIIa binding and the amount of FcγRIIa binding the determined polypeptide by following the above examples.
[01494] [01494] In a non-limiting embodiment of the present invention, an example of an Fc region having a higher inhibitory Fcγ receptor binding activity than an activating Fcγ receptor binding activity (having selective binding activity towards an inhibitory Fcγ receptor) is the Fc region presented in US2009 / 0136485 or WO 2012/115241; and another suitable example is an Fc region in which the amino acid at position 238 or 328 as indicated by the EU numbering in the Fc region mentioned above has been modified to an amino acid different from that of the native Fc region.
[01495] [01495] In a non-limiting embodiment of the present invention, a suitable example of an Fc region that has a higher binding activity towards an inhibitory Fcγ receptor than towards an Fcγ activation receptor (i.e. , having a selective binding activity towards an inhibitory Fcγ receptor) is an Fc region with one or more of the following
[01496] [01496] In a non-limiting embodiment of the present invention, a suitable example is an Fc region in which one or more of the amino acids indicated by the EU number at positions 238 and 328 according to the EU number are respectively modified to Asp or Glu at mentioned Fc region.
[01497] [01497] Furthermore, in a non-limiting embodiment of the present invention, suitable examples of the Fc regions are those with the substitution of Asp for Pro at position 238 (EU numbering), and one or more modifications selected from Trp for the amino acid of position 237, Phe for the amino acid of position 237, Val for the amino acid of position 267, Gln for the amino acid of position 267, Asn for the amino acid of position 268, Gly for the amino acid of position 271, Leu for amino acid of position 326, Gln for amino acid of position 326, Glu for amino acid of position 326, Met for amino acid of position 326, Asp for amino acid of position 239, Ala for amino acid of position 267, Trp for the amino acid at position 234, Tyr for the amino acid at position 234, Ala for the amino acid at position 237, Asp for the amino acid at position 237, Glu for the amino acid at position 237, Leu for the amino acid at position 237 , Met for the amino acid at position 237, Tyr for the amino acid at position 237, Lys for the amino acid at position 330, Arg for the amino acid at position 330, Asp for the amino acid at position 233, Asp for the amino acid at position 268, Glu for the amino acid at position 268, Asp for the amino acid of position 326, Ser for the amino acid of position 326,
[01498] [01498] In the present invention, an "antigen-binding molecule" is used in the broadest sense to refer to a molecule that contains an antigen-binding domain and an Fc region. Specifically, antigen-binding molecules include several types of molecules while exhibiting antigen-binding activity. Molecules in which an antigen binding domain is linked to an Fc region include, for example, antibodies. Antibodies can include single monoclonal antibodies (including agonistic antibodies and antagonistic antibodies), human antibodies, humanized antibodies, chimeric antibodies and such. Alternatively, when used as antibody fragments, they preferably include antigen binding domains and antigen binding fragments (for example, Fab, F (ab ') 2, scFv and Fv). Framework molecules where three-dimensional structures, such as the already known stable α / β barrel protein structure, are used as a framework (base) and only some portions of the structures are made in libraries to build binding domains to the antigen are also included in antigen-binding molecules of the present invention.
[01499] [01499] An antigen binding molecule of the present invention can contain at least some portions of an Fc region that mediates binding to 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 that comprises a polypeptide that has a first amino acid sequence that is linked to a polypeptide that has a second amino acid sequence that would not naturally bind in nature. For example, a fusion protein may comprise the amino acid sequence of at least a portion of an Fc region (for example, a portion of an Fc region responsible for binding to FcRn or a portion of a region of Fc Fc responsible for binding to the Fcγ receptor) and a non-immunoglobulin polypeptide that contains, for example, the amino acid sequence of the ligand-binding domain of a receptor or a receptor-binding domain of a ligand. Amino acid sequences can be present in separate proteins that are transported together with a fusion protein, or they can generally be present in a single protein; however, they are included in a new rearrangement in a fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by genetic recombination techniques to express a polynucleotide that encodes peptide regions in a desired arrangement.
[01500] [01500] Each of the domains of the antigen binding domain, Rc region, and the like of the present invention can be linked together via linkers or directly via the polypeptide link. The ligands comprise arbitrated peptide ligands that can be introduced by genetic engineering, synthetic ligands and ligands described in, for example, Protein Engineering (1996) 9 (3), 299-305. However, peptide ligands are preferred in the present invention. The length of the peptide ligands is not particularly limited and can be appropriately selected by those skilled in the art according to the objective. The length is preferably five amino acids or more (without particular limitation, the upper limit is usually 30 amino acids or less, preferably
[01501] [01501] For example, such peptide linkers preferably include:
[01502] [01502] Be
[01503] [01503] Gly · Be
[01504] [01504] Gly · Gly · Be
[01505] [01505] Ser · Gly · Gly
[01506] [01506] Gly · Gly · Gly · Ser (SEQ ID NO: 29)
[01507] [01507] Ser · Gly · Gly · Gly (SEQ ID NO: 30)
[01508] [01508] Gly · Gly · Gly · Gly · Ser (SEQ ID NO: 31)
[01509] [01509] Ser · Gly · Gly · Gly · Gly (SEQ ID NO: 32)
[01510] [01510] Gly · Gly · Gly · Gly · Gly · Being (SEQ ID NO: 33)
[01511] [01511] Being · Gly · Gly · Gly · Gly · Gly (SEQ ID NO: 34)
[01512] [01512] Gly · Gly · Gly · Gly · Gly · Gly · Being (SEQ ID NO: 35)
[01513] [01513] Being · Gly · Gly · Gly · Gly · Gly · Gly (SEQ ID NO: 36)
[01514] [01514] (Gly · Gly · Gly · Gly · Ser (SEQ ID NO: 31)) n
[01515] [01515] (Ser · Gly · Gly · Gly · Gly (SEQ ID NO: 32)) n
[01516] [01516] where n is an integer of 1 or greater. The length or sequences of peptide ligands can be selected accordingly by those skilled in the art depending on the objective.
[01517] [01517] Synthetic ligands (chemical interconnecting agents) are routinely used to interlink peptides, and for example:
[01518] [01518] N-hydroxy succinimide (NHS),
[01519] [01519] dissuccinimidyl suberate (DSS),
[01520] [01520] bis (sulfosuccinimidyl) suberate (BS3),
[01521] [01521] dithiobis (succinimidyl propionate) (DSP),
[01522] [01522] dithiobis (sulfosuccinimidyl propionate) (DTSSP),
[01523] [01523] ethylene glycol bis (succinimidyl succinate) (EGS),
[01524] [01524] ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS),
[01525] [01525] disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
[01526] [01526] bis [2- (succinimidoxicarbonyloxy) ethyl] sulfone (BSOCOES),
[01527] [01527] and bis [2- (sulfosuccinimidoxicarbonyloxy) ethyl] sulfone (sulfo-BSOCOES). These interconnecting agents are commercially available.
[01528] [01528] When multiple ligands to link the respective domains are used, they can all be of the same type, or they can be of different types.
[01529] [01529] In addition to the linkers exemplified above, linkers with peptide markers such as His marker, HA marker, myc marker and FLAG marker can also be used appropriately. In addition, hydrogen bonding, disulfide bonding, covalent bonding, ionic interaction and bonding properties as a result of the combination of these can be used appropriately. For example, the affinity between CH1 and CL of the antibody can be used, and the Fc regions that originate from the bispecific antibodies described above can also be used for the association of hetero Fc region. In addition, disulfide bonds formed between domains can also be used appropriately.
[01530] [01530] In order to link the respective domains via peptide bond, polynucleotides that encode the domains are linked in structure. Known methods for ligating polynucleotides in structure include techniques such as restriction fragment ligation, fusion PCR, and overlap PCR. Such methods can be appropriately used alone or in combination to produce the antigen binding molecules of the present invention. In the present invention, the terms "bonded" and "fused", or "bonded" and "fused" are used interchangeably. These terms mean that two or more elements or components such as polypeptides are linked together to form a single structure by any means including the chemical bonding and recombination techniques described above. When two or more domains, elements or components are polypeptides, linkage in structure means linking two or more units of reading phases to form a longer continuous reading phase while maintaining the correct reading phases of the polypeptides. When two Fab molecules are used as the antigen binding domain, an antibody that is an antigen binding molecule of the present invention in which the antigen binding domain is linked in structure to an Fc region by a binding peptide and not via a linker, can be used as a suitable antigen-binding molecule in this patent application. Complex containing two or more antigen binding molecules and two or more antigen binding units
[01531] [01531] As described in Example 1 (as shown in WO 2011/122011), Fv4-IgG1, that the binding activity of sIL-6R changes depending on the pH condition much more than that of H54 / L28-IgG1, accelerates the elimination of sIL-6R but does not accelerate elimination any more than when sIL-6R is used alone. To accelerate elimination more than when sIL-6R is used alone, antigen binding molecules comprising an Fc region with increased FcRn binding in the neutral pH range (for example, Fv4-IgG1-v2 and the like in Example 1) should be used.
[01532] [01532] However, surprisingly, GA2-IgG1, of which the human IgA binding activity varies depending on the Ca ion concentration, has been revealed to accelerate the elimination of human IgA more than when human IgA is used only, although contains a native IgG1-derived Fc region, that FcRn binding in the neutral pH range is not increased. Similarly, clone 278 of which human IgE binding activity varies depending on the pH condition, has been shown to accelerate the elimination of human IgE more than when human IgE is used only, although it contains a native IgG1-derived Fc region, which has no increased FcRn binding in the neutral pH range. Without being restricted to a particular theory, it is thought that the following exemplary mechanism considers what happened to GA2-IgG1 and clone 278.
[01533] [01533] When the antigen binding unit is a unit (that is, a homomonomer), as in sIL-6R, two molecules (that is, two antigen binding units) of antigens bind to a molecule A single antibody containing a divalent antigen-binding domain, and a complex of an anti-sIL-6R antibody molecule and two antigen molecules that contain two antigenic binding units is formed. antibody-antigen has only one Fc region (native IgG1 Fc region) as shown in Fig. 1. Since this complex binds to two FcRn molecules or one FcγR molecule via a region of Fc only, the affinity towards these receptors is the same as that of a general IgG antibody, and absorption in cells is thought to occur mostly not specifically.
[01534] [01534] On the other hand, when the antigenic binding unit is two units as in human IgA, which is a dimer of a hetero complex of heavy chains and light chains, there are two units of epitopes to which the antigen binding domains will connect to the antigen binding unit. However, when a bivalent anti-IgA antibody (that is, antigen-binding domains contained in an anti-IgA antibody molecule bind to the same epitope) binds to its antigen, IgA, it is believed that the binding of each the divalent antigen-binding domains contained in the unique anti-IgA antibody molecule to each of the two epitope units present in a single IgA molecule is difficult in view of the position of the epitopes. As a result, it is believed that separate anti-IgA antibody molecules bind to two antigenic binding epitope units present on the two IgA molecules that bind to the bivalent antigen-binding domains present on a single anti-IgA antibody molecule , and consequently, anti-body-antigen complexes (immune complexes) containing at least four molecules (that is, two IgA molecules that are the antigen molecule and two anti-IgA antibody molecules that are molecules binding to antigen) are formed.
[01535] [01535] When an antigen-binding molecule, such as an antibody that binds to an antigen molecule that contains two or more antigenic binding units, forms a large immune complex that is at least one tetramer, the immune complex can bind strongly with avidity for at least two or more multivalent Fc regions to FcγR, FcRn, complement receptors and the like. Therefore, as shown in Fig. 7, the complex is absorbed into cells that express these receptors with a higher efficiency than native IgG1. On the other hand, since the affinity mediated by the Fc region towards these immune complex receptors formed from antigen molecules and antigen binding molecules that, for example, bind to antigen molecules (monomeric) ) containing an antigenic binding unit is insufficient as mentioned above, immune complexes are mostly not specifically absorbed (less efficiently compared to avidity-mediated binding absorption) in cells expressing these receptors, as shown in Fig. 1. In this way, absorption is more inefficient than absorption mediated by binding greed.
[01536] [01536] When the antigen-binding molecule such as an antibody that binds to an antigen molecule that contains two or more antigenic binding units is an antibody that contains
[01537] [01537] As described above, if an antibody containing a native IgG1 constant region against a multimeric antigen that contains two or more antigenic binding units and shows pH- or Ca-dependent binding can form a large immune complex and binding to FcγR, FcRn, complement receptors, and similarly avidly, it is believed that antigen elimination alone can be selectively and enormously accelerated. It is believed that when GA2-IgG1 that binds to human IgA is administered, such large immune complexes are formed. In fact, as shown in Example 3, GA2-IgG1- FcγR (-) formed by the introduction of GA2-IgG1 modifications that impair binding to mouse FcγR cannot substantially accelerate the elimination of human IgA as GA2-IgG1 when compared to human IgA only, and showed an equivalent level of elimination as human IgA only. Therefore, the reason that GA2-IgG1 can accelerate the elimination of human IgA is because the immune complex containing GA2-IgG1 and
[01538] [01538] In order to exhibit the effect of further accelerating the elimination of multimeric antigens comprising two or more plasma antigen binding units as described above, it is considered preferable that a large immune complex of molecules be formed binding to antigen and antigens, and the Fc regions comprised in the antigen binding molecules strongly bind to FcγR and / or FcRn avidly. When the antigen contains two or more antigen binding units, a large immune complex comprising two or more antigen binding molecules and two or more antigen binding units can be formed. For this reason, by tracing by antigen-binding molecules of which the activity of binding to multimeric antigens that contain two or more antigenic binding units is modified according to an ionic concentration and that avidly bind to the aforementioned receptors, antigen-binding molecules that further accelerate the elimination of multimeric antigens that contain two or more antigenic plasma binding units can be obtained. Method for assessing complex formation
[01539] [01539] Examples of methods for assessing the formation of immune complexes comprising antigen-binding molecules and antigens include techniques in analytical chemistry, including methods that use the property that immune complexes become larger molecules than a molecule of antigen binding only or an antigen molecule only as size exclusion chromatography (gel filtration), ultracentrifugation analysis method, light scattering method, electron microscopy and mass spectrometry (Molecular Immunology ( 2002), 39, 77-84; Molecular Immunology (2009), 47, 357-364). For example, when size exclusion chromatography (gel filtration) is used as shown in Fig. 9, whether an immune complex is formed is assessed by looking at whether there are molecular species that are larger than those in analyzes of the antigen only molecule or antigen binding molecule only.
[01540] [01540] In addition, when the antigen-binding molecule or antigen has an immunoglobulin constant region, examples include immunochemical methods including methods that use the immune complex's ability to bind more strongly to a protein receptor. Fc or a complement component than the antigen-binding molecule alone or the antigen only, as ELI-
[01541] [01541] As described above, when the antigen is a multimeric antigen (a non-limiting example is an immunoglobulin like IgA or IgE or a member of the TNF superfamily like TNF or CD154), a large immune complex comprising two or more molecules of antigen binding and two or more antigen binding units can be formed. On the other hand, even when the antigen is monomeric, a mixture of two or more appropriate antigen-binding molecules in which each binds to a separate epitope present in the monomeric antigen, where the binding to the epitopes varies depending on an ionic concentration condition (such as pH or Ca concentration) it can also form a large immune complex that contains two or more antigen-binding molecules and two or more antigenic binding units (monomeric antigens). In this application, the mixture of two or more appropriate antigen-binding molecules in which each binds to a separate epitope present in a monomeric antigen, where binding to the epitopes varies depending on an ionic concentration condition (such as pH or concentration Ca) is called the antigen-binding molecule cocktail. Among these antigen-binding molecules, at least one (the antigen-binding domains comprised of
[01542] [01542] Even in the case of a monomeric antigen, an antigen binding molecule containing antigen binding domains in which each binds to a separate epitope present in the monomeric antigen, where the binding of the antigen binding domains to respective epitopes varies depending on an ionic concentration condition (such as pH or Ca concentration) can also form a large immune complex that contains two or more antigen binding molecules and two or more antigen binding units (monomeric antigens). Examples of a non-limiting modality of such an antigen-binding molecule are multiparatopic antibodies or multispecific antibodies that contain appropriate variable regions that bind to the epitopes present in the monomeric antigen that differ from one another. As a non-limiting modality of such multispecific antibodies or multiparatopic antibodies, antigen-binding molecules whose variable regions show pH- or Ca-dependent binding (bispecific antibodies or biparatopic antibodies that contain a variable region of the right arm that recognizes the epitope A and a variable left arm region that recognizes epitope B, as shown in Fig. 8) can also form a large immune complex that contains two or more antibodies and two or more antigenic binding units (antigens monomeric).
[01543] [01543] Antigen-binding molecules that still accelerate the elimination of monomeric antigens from plasma can be obtained by tracking combinations of antigen-binding domains that target different epitopes of a monomeric antigen, where binding activity to each one of the epitopes varies depending on a condition of ionic concentration, which can bind avidly to the aforementioned receptors.
[01544] [01544] An antigen binding molecule containing at least two antigen binding domains, where at least one of the antigen binding domains binds to a first epitope on an antigen molecule and at least the other of the binding domains the antigen binds to a second epitope on the antigen molecule, it is called a multispecific molecule that binds the antigen from the point of view of its specificity of reaction. When a single antigen-binding molecule binds to two different epitopes by two types of antigen-binding domains contained in the antigen-binding molecule, this antigen-binding molecule is called a bispecific antigen-binding molecule. When a single antigen-binding molecule binds to three different epitopes by three types of antigen-binding domains contained in the antigen-binding molecule, this antigen-binding molecule is called a specific antigen-binding molecule. antigen.
[01545] [01545] The paratope in an antigen-binding domain that binds to a first epitope on the antigen molecule has a different structure than that of the paratope in an antigen-binding domain that binds to a second epitope that is structurally different from the first epitope. Therefore, an antigen binding molecule containing at least two antigen binding domains, in which at least one of the antigen binding domains binds to a first epitope on an antigen molecule and at least the other of the - antigen-binding mines binds to a second epitope in the antigen molecule, it is called a multiparatopic molecule of antigen binding from the point of view of its structure and specificity. When a single antigen binding molecule binds to two different epitopes by two types of antigen binding domains contained in the antigen binding molecule, this antigen binding molecule is called a biparatopic antigen binding molecule. When a single antigen binding molecule binds to three different epitopes by three types of antigen binding domains contained in the antigen binding molecule, this antigen binding molecule is called a triparatopic molecule binding to the antigen. antigen.
[01546] [01546] Multivalent multispecific or multiparatopic antigen binding molecules comprising one or more antigen binding domains and methods for preparing them have been described in Non-Patent Documents such as Conrath et al. (J.Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302), and Kontermann RE (2011) Bispecific Antibodies (Springer-Verlag) and in Patent Documents such as WO 1996/034103 and WO 1999/023221. The antigen-binding molecules of the present invention can be produced using the multispecific or multiparatopic antigen-binding molecules and methods for preparing them described in these documents.
[01547] [01547] The bispecific antibodies and production methods of these are presented below as the examples of the modality of the aforementioned multispecific or multiparatopic antigen binding molecules and methods of producing them. Bispecific antibodies are antibodies that comprise two types of variable regions that specifically bind to different epitopes. Bispecific IgG-like antibodies can be secreted from a hybrid hybridoma (quadroma) produced by fusing two types of hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).
[01548] [01548] When a bispecific antibody is produced using recombination techniques such as those described in the aforementioned section on antibodies, a method can be adopted that introduces genes encoding heavy chains containing the two types of variable regions of interest in cells to coexpress them. However, even when only the combination of the heavy chain is considered, such a coexpression method will produce a mixture of (i) a combination of a pair of heavy chains in which the heavy chains contain a variable region that binds to a first epitope and another heavy chain contains a variable region that binds to a second epitope, (ii) a combination of a pair of heavy chains that includes only heavy chains that contain a variable region that binds to the first epitope, and (iii) a combination of a pair of heavy chains that include only heavy chains that contain a variable region that binds to the second epitope, which are present in a 2: 1: 1 molecular proportion. It is difficult to purify antigen-binding molecules that contain the desired combination of heavy chains from the mixture of three types of heavy chain combinations.
[01549] [01549] When producing bispecific antibodies using such recombination techniques, bispecific antibodies that contain a combination
[01550] [01550] In addition, there are also known techniques for producing a bispecific antibody by applying methods to control the association of polypeptides or the association of heteromeric multimers formed from the polypeptide to the association between heavy chains. Specifically, methods for controlling heavy chain formation can be employed to produce a bispecific antibody (International Publication WO No. 2006/106905), in which the amino acid residues that form the interface between heavy chains are high. - designed to inhibit the association between heavy chains that have the same sequence and allow the formation of heavy chains of different sequences. Such methods can be used to generate bispecific antibodies.
[01551] [01551] In a non-limiting embodiment of the present invention, two polypeptides that form an Fc region derived from a bispecific antibody described above can be appropriately used as the Fc region contained in an antigen binding molecule.
[01552] [01552] In another non-limiting embodiment of the present invention, two polypeptides that form an Fc region, in which, of the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to the EU number is Asp, and of the amino acid sequence of the other polypeptides, the amino acid at position 399 according to the EU numbering is Lys, can be appropriately used as the Fc region. In the above-mentioned embodiment, the amino acid at position 409 can be Glu instead of Asp, and the amino acid at position 399 can be Arg instead of Lys. In addition, in addition to the amino acid Lys at position 399, Asp can be appropriately added as an amino acid at position 360 or Asp can be appropriately added as an amino acid at position 392.
[01553] [01553] In yet another non-limiting embodiment of the present invention, two polypeptides that form an Fc region, in which, from the amino acid sequence of one of the polypeptides, the amino acid at position 370 according to the EU number is Glu, and from the amino acid sequence of the other polypeptides, the amino acid at position 357 according to EU numbering is Lys, can be appropriately used as the Fc region.
[01554] [01554] In yet another non-limiting embodiment of the present invention, two polypeptides that form an Fc region, in which, from the amino acid sequence of one of the polypeptides, the amino acid at position 439 according to the EU number is Glu, and from the amino acid sequence of the other polypeptides, the amino acid at position 356 according to the EU number is Lys, can be appropriately used as the Fc region.
[01555] [01555] In yet another non-limiting modality of the present invention, any of the modalities indicated below, in which those mentioned above have been combined, can be appropriately used as the Fc region:
[01556] [01556] two polypeptides that form an Fc region, in which, of the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to the EU number is Asp and the amino acid at position 370 is Glu, and the sequence of amino acids of the other polypeptides, the amino acid at position 399 according to EU numbering is Lys and the amino acid at position 357 is Lys (in this embodiment, the amino acid at position 370 according to EU numbering may be Asp instead of Glu, and the amino acid Asp at position 392 according to EU numbering can be used instead of the amino acid Glu at position 370 according to EU numbering);
[01557] [01557] two polypeptides that form an Fc region, in which, of the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to EU numbering is Asp and the amino acid at position 439 is Glu, and the sequence of amino acids of the other polypeptides, the amino acid at position 399 according to EU numbering is Lys and the amino acid at position 356 is Lys (in this modality, the amino acid Asp at position 360 according to EU numbering, the amino acid Asp at position 392 according to EU numbering, or the Asp amino acid at position 439 according to EU numbering can be used instead of the amino acid Glu at position 439 according to EU numbering);
[01558] [01558] two polypeptides that form an Fc region, in which,
[01559] [01559] two polypeptides that form an Fc region, in which, of the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to the EU number is Asp, the amino acid at position 370 is Glu, and the amino acid at position 439 is Glu, and of the amino acid sequence of the other polypeptides, the amino acid at position 399 according to EU numbering is Lys, the amino acid at position 357 is Lys, and the amino acid at position 356 is Lys (in this modality , the amino acid at position 370 according to EU numbering cannot be replaced by Glu and furthermore, when the amino acid at position 370 is not replaced by Glu, the amino acid at position 439 can be Asp instead of Glu , or the amino acid Asp in position 392 can be used instead of the amino acid Glu in position 439).
[01560] [01560] Still, in another non-limiting embodiment of the present invention, two polypeptides that form an Fc region, in which, from the amino acid sequence of one of the polypeptides, the amino acid at position 356 according to the EU number is Lys , and the amino acid sequence of the other polypeptides, the amino acid at position 435 according to EU numbering is Arg and the amino acid at position 439 is Glu, can also be used appropriately.
[01561] [01561] In yet another non-limiting embodiment of the present invention, two polypeptides that form an Fc region, in which, from the amino acid sequence of one of the polypeptides, the amino acid at position 356 according to the EU number is Lys and the amino acid at position 357 are Lys, and from the amino acid sequence of the other polypeptides, the amino acid at position 370 according to the
[01562] [01562] In addition to the aforementioned technique of associating heterologous heavy chains, the CrossMab technology which is known as a technology for associating heterologous light chains, in which a light chain that forms a variable region that binds to a first epitope and a light chain that forms a variable region that binds to a second epitope are associated respectively with a heavy chain that forms a variable region that binds to the first epitope and a heavy chain that forms a variable region that binds to the second epitope (Scaefer et al. (Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192)), can also be used to produce the multispecific or multiparatopic antigen binding molecules provided by this invention. The use of an antigen binding molecule to eliminate antigens that contain two or more antigen binding units from the plasma
[01563] [01563] The present invention provides the use of an antigen binding molecule comprising (i) an Fc region and (ii) an antigen binding domain on which antigen binding activity varies depending on of a condition of ionic concentration;
[01564] [01564] wherein the antigen binding molecule can form an immune complex comprising (a) two or more of the antigen binding molecules and (b) two or more antigen molecules as long as the antigens comprise two or more antigenic binding units to eliminate antigens from the plasma.
[01565] [01565] In the present invention, the modalities of using an antigen binding molecule are not particularly limited as long as the antigens can be eliminated from the plasma. Examples of a non-limiting modality of such use are pharmaceutical compositions
[01566] [01566] In the present invention, the "ability to eliminate antigens in plasma" refers to the ability to eliminate plasma antigens that are present in plasma when antigen-binding molecules are administered in vivo or when molecules of li - antigens are secreted in vivo. Thus, in the present invention, the phrase "the ability of an antigen-binding molecule to eliminate an antigen in plasma is increased" can be used when the rate of antigen elimination from plasma is accelerated when the antigen binding is administered, as compared to the administration of antigen binding molecules that cannot form the immune complexes described in the present invention, antigen binding molecules that contain antigen binding domains from which antigen binding activity is independent of ionic concentration or antigen-binding molecules that contain Fc regions with compromised binding activity towards FcγR or FcRn. If the ability of an antigen-binding molecule to eliminate an antigen in plasma is increased,
[01567] [01567] In this application, "increased pharmacokinetics", "improved pharmacokinetics", and "superior pharmacokinetics" can be declared again as "increased plasma (blood) retention", "improved plasma (blood) retention", "higher plasma retention (blood)", and "prolonged plasma retention (blood)". These terms are synonymous.
[01568] [01568] In this application, "improved pharmacokinetics" means not only extending the period until elimination from plasma (for example, until the antigen binding molecule is degraded intracellularly or similar and cannot return to plasma) after admittance - administration of the antigen-binding molecule to humans or non-human animals such as mice, rats, monkeys, rabbits, and dogs, but also prolonging the plasma retention of the antigen-binding molecule in a form that allows for binding of the antigen (for example, in an antigen-free form of the antigen-binding molecule) during the period of administration to elimination due to degradation. Human IgG that has a wild Fc region can bind to FcRn of non-human animals. For example, the mouse can preferably be used to be administered to confirm the property of the antigen-binding molecule of the invention since human IgG having the wild-type Fc region can bind to the stronger mouse FcRn than than to human FcRn (Int. Immunol (2001) 13 (12): 1551-1559). As another example, the mouse in which their native FcRn genes are disrupted and a human FcRn gene transgene is hidden to be expressed (Methods Mol Biol. 2010; 602: 93-104) can also be used preferentially to be administered in order to confirm the property of the antigen-binding molecule of the invention described below.
[01569] [01569] In this application, "improved pharmacokinetics" also includes extending the period that an antigen is bound to an antigen-binding molecule after administration of the antigen-binding molecule. If the period that an antigen is bound to the antigen-binding molecule after administration of the antigen-binding molecule is prolonged, it can be assessed by determining the plasma concentration of the free antigen. The prolongation can be judged based on the determined plasma concentration of the free antigen or the period of time required for an increase in the proportion of the concentration of free antigen to the concentration of total antigen.
[01570] [01570] The plasma concentration of the free antigen not bound to the antigen binding molecule or the ratio of the concentration of free antigen to the total antigen concentration can be determined by methods known to those skilled in the art, for example, by the method used in Pharm Res 2006 Jan; 23 (1): 95-103. Alternatively, when an antigen exhibits a particular function in vivo, whether the antigen is bound to an antigen binding molecule that neutralizes the antigen function (antagonistic molecule) can be assessed by testing whether the antigen function is neutralized. If the function of the antigen is neutralized it can be assessed by analyzing a marker in vivo that reflects the function of the antigen. If the antigen is linked to an antigen-binding molecule that activates the antigen function (agonistic molecule) can be assessed by analyzing a marker in vivo that reflects the function of the antigen.
[01571] [01571] The determination of the plasma concentration of the free antigen and the ratio of the amount of the free antigen in the plasma to the amount of the total antigen in the plasma, in vivo marker assay and such measures is not particularly limited; however, the assays are preferably carried out after a certain period of time has passed after administration of the antigen-binding molecule. In the present invention, the period after the administration of the antigen-binding molecule is not particularly limited; those skilled in the art can determine the appropriate period depending on the properties and the like of the 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 molecule of antigen binding, and 28 days after administration of the antigen binding molecule. In this application, the concept "plasma antigen concentration" comprises both "total antigen concentration in plasma" which is the sum of the antigen binding molecule bound to the antigen and concentration of unbound antigen or "concentration of free antigen in plasma "which is the concentration of unbound antigen from the antigen binding molecule.
[01572] [01572] The concentration of total antigen in plasma can be reduced by administering the antigen-binding molecule of the present invention as an antigen-binding molecule 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1,000 times, or even more compared to the administration of an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity is independent of the concentration of ions or a molecule antigen binding pathway containing an Fc region with impaired FcγR binding activity, or in comparison when the antigen binding domain molecule of the present invention is not administered.
[01573] [01573] The molar ratio of antigen / antigen binding molecule can be calculated as shown below:
[01574] [01574] A value: the molar concentration of antigen at each time point
[01575] [01575] B value: the molar concentration of the antigen binding molecule at each time point
[01576] [01576] C value: the 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.
[01577] [01577] The lowest C value indicates the highest efficiency of antigen elimination per antigen binding molecule while the highest C value indicates the lowest efficiency of antigen elimination per antigen binding molecule.
[01578] [01578] The molar ratio of antigen / antigen binding molecule can be calculated as described above.
[01579] [01579] Administer a molecule that binds to the antigen of the
[01580] [01580] In the present invention, as a reference for comparison with the antigen binding molecules of the present invention, antigen binding molecules that cannot form the immune complexes described in the present invention, antigen binding molecules which contain antigen-binding domains of which antigen-binding activity is independent of ionic concentrations or antigen-binding molecules that contain regions of Fc with binding activity towards FcγR or FcRn.
[01581] [01581] When an FcRn-mediated pathway is used in the incorporation of antigen-binding molecules of the present invention from plasma into cells, the reduction in plasma total antigen concentration or the antigen / antibody molar ratio can also be evaluated using strain 32 or strain 276 of human FcRn transgenic mice (Jackson Laboratories, Methods Mol Bi-2010. 602: 93-104) by the antibody-antigen co-injection model or model permanent antigen infusion model when the antigen-binding molecule does not cross-react with the antigen of the mouse counterpart. When the antigen-binding molecule cross-reacts with the mouse counterpart, it can also be assessed by simply injecting the antigen-binding molecule into strain 32 or strain 276 of human FcRn transgenic mice (Jackson Laboratories). In the co-injection model, the mixture of the antigen-binding molecule and antigen is administered to mice. In the permanent antigen infusion model, the infusion pump filled with an antigen solution is introduced into mice to achieve a constant plasma antigen concentration, and then the antigen-binding molecule is injected into the mice. The test antigen binding molecules are administered in the same dose. The concentration of total antigen in plasma, the concentration of free antigen in plasma and the concentration of antigen-binding molecule in plasma are measured in the method using appropriate time points known to those skilled in the art.
[01582] [01582] When an FcγR-mediated pathway is used in the incorporation of plasma-binding antigen molecules of the present invention into cells, the reduction in plasma total antigen concentration or antigen / antibody molar ratio can be assessed by the model of antibody-antigen co-injection or by the permanent antigen infusion model using conventionally used C57BL / 6J mice (Charles River Japan) when the antigen binding molecule does not cross-react with the antigen of the mouse counterpart . If the antigen-binding molecule cross-reacts with the mouse counterpart, the assessment can be performed simply by injecting the antigen-binding molecule into the conventionally used C57BL / 6J mice (Charles River Japan).
[01583] [01583] In the co-injection model, a mixture of the antigen-binding molecule and antigen is administered to mice. In the permanent antigen infusion model, an infusion pump filled with an antigen solution is introduced into mice.
[01584] [01584] The concentration of total or free antigen in plasma and molar proportion of antigen / antigen-binding molecule can be measured at 2, 4, 7, 14, 28, 56, or 84 days after administration to assess the effect terms of the present invention. In other words, a long-term plasma antigen concentration is determined by measuring the concentration of total or free antigen in the plasma and the molar ratio of antigen / antigen-binding molecule in 2, 4, 7, 14, 28, 56, or 84 days after administration of an antigen-binding molecule in order to assess the property of the antigen-binding molecule of the present invention. Whether the reduction in plasma antigen concentration or molar ratio of antigen / antigen binding molecule is achieved by the antigen binding molecule described in the present invention can be determined by assessing the reduction in either or more of the time points described above.
[01585] [01585] The concentration of total or free antigen in plasma and molar proportion of antigen / antigen binding molecule can be measured within 15 min, 1, 2, 4, 8, 12, or 24 hours after administration to assess the short-term effect of the present invention. In other words, a short-term plasma antigen concentration is determined by measuring the concentration of total or free antigen in the plasma and molar ratio of antigen / antigen-binding molecule in 15 min, 1, 2, 4 , 8, 12, or 24 hours after administration of an antigen-binding molecule in order to assess the property of the antigen-binding molecule of the present invention.
[01586] [01586] The route of administration of an antigen-binding molecule of the present invention can be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parental and intramuscular injection.
[01587] [01587] In the present invention, it is preferred that the pharmacokinetics of the antigen binding molecule in humans is improved. When plasma retention in humans is difficult to determine, it can be predicted based on plasma retention in mice (eg, normal mice, transgenic mice expressing human antigen, transgenic mice expressing human FcRn) or monkeys (eg, cinomolgus monkeys) ).
[01588] [01588] In this application, "improved pharmacokinetics and prolonged plasma retention of an antigen-binding molecule" means the improvement of any pharmacokinetic parameter (either prolonging plasma half-life, prolonging mean retention time in plasma, reduced plasma clearance and bioavailability) after in vivo administration of the antigen binding molecule or increase in the concentration of the antigen binding molecule in the plasma at an appropriate time after administration. It can be determined by measuring any parameter such as plasma half-life, mean plasma retention time, plasma clearance and bioavailability of the antigen binding molecule (Pharma-cokinetics: Enshu-niyoru Rikai (Understanding through practice) "Nanzando ), (Nanzando)) .For example, when an antigen-binding molecule is administered to mice (normal mice and transgenic human FcRn mice), rats, monkeys, rabbits, dogs, humans, and the like, and to concentration of the antigen binding molecule in plasma is determined and each parameter is calculated, it can be judged that the pharmacokinetics of the antigen binding molecule is improved when the plasma half-life or retention time plasma is prolonged. These parameters can be determined by methods known to those skilled in the art. For example, the parameters can be appropriately evaluated by non-compartmental analysis using the program. pharmacokinetic analysis system WinNonlin (Pharsight) according to the attached instruction manual.
[01589] [01589] Without being restricted to a particular theory, the following mechanism is an example of a mechanism that allows the elimination of antigenic binding units from the plasma by an antigen binding molecule of the present invention comprising (i) an Fc region and (ii) an antigen-binding domain whose antigen-binding activity varies depending on an ionic concentration condition, in which the antigen-binding molecule can form an immune complex that contains two or more of the antigen binding molecules and antigens that comprise two or more antigen binding units. When the antigen binding unit is a unit (i.e., a monomer), as in sIL-6R, two molecules ((i.e., two antigenic binding units) of antigens bind to a single antibody molecule that contains a divalent antigen-binding domain, and a complex of one anti-sIL-6R antibody molecule and two antigen molecules that contain two antigen binding units is formed, so this type of anti-body-antigen complex has only an Fc region (native IgG1 Fc region) as shown in Fig. 1. Since this complex binds to two FcRn molecules or one FcγR molecule via a single Fc region, the affinity towards these receptors is the same as that of a general IgG antibody, and it is thought that absorption in cells
[01590] [01590] On the other hand, when the antigen binding unit is two units as in human IgA, which is a dimer of a hetero complex of heavy chains and light chains, there are two units of epitopes to which the antigen binding domains will connect to the antigen binding unit. However, when a bivalent anti-IgA antibody (that is, the antigen-binding domains contained in an anti-IgA antibody molecule bind to the same epitope) binds to its antigen, IgA, it is believed that the binding of each one of the divalent antigen-binding domains contained in the unique anti-IgA antibody molecule to each of the two epitope units present in a single IgA molecule is difficult in view of the position of the epitopes. As a result, the separate anti-IgA antibody molecules are believed to bind to two antigenic binding units present in the two IgA molecules that bind to the divalent antigen-binding domains present in an anti-IgA molecule. unique anti-IgA body, and consequently, antibody-antigen complexes (immune complexes) containing at least four molecules (that is, two IgA molecules that are the antigen molecule and two anti-IgA antibody molecules that are binding molecules antigen) are formed.
[01591] [01591] When an antigen-binding molecule such as an antibody that binds to an antigen molecule that contains two or more antigenic binding units, forms a large immune complex that is at least one tetramer, the immune complex can bind - is strongly avid for at least two or more Fc regions multivalent to FcγR, FcRn, complement receptors and the like. Therefore, as shown in Fig. 7, the complex is efficiently absorbed into cells that express these receptors. On the other hand, once the affinity mediated by the Fc region towards these immune complex receptors formed from antigen molecules and antigen binding molecules that, for example, bind to antigen (monomeric) molecules that contain one antigen binding unit is insufficient as mentioned above, immune complexes are mostly not specifically absorbed (less efficiently when compared to binding-mediated absorption) in cells that express these receptors, as shown in Fig Thus, absorption is more inefficient than absorption mediated by binding greed.
[01592] [01592] When the antigen binding molecule as an antibody that binds an antigen molecule that contains two or more antigen binding units is an antibody that contains antigen binding domains from which the antigen binds varies depending on an ionic concentration condition such as pH- or Ca-dependent binding and which forms an antibody-antigen complex (immune complex) containing at least four molecules (two antigen molecules and two antibody molecules) in the plasma, once the immune complex is absorbed in the cells, the antigens dissociate from the antibodies in the endosomes where the ionic concentration conditions are different from those in the plasma. Therefore, the formation of the immune complex is dissolved in the endosomes of cells that have absorbed the immune complexes. Since the dissociated antigens cannot bind to FcRn in the endosomes, they are degraded after moving to the lysosomes. On the other hand, antibodies dissociated from the antigen are thought to be recycled to plasma after binding to FcRn in endosomes (Fig. 7).
[01593] [01593] As described above, if an antibody that contains a native IgG1 constant region against a multimeric antigen that contains two or more antigenic binding units and shows pH- or Ca-dependent binding can form a large immune complex and binding to FcγR, FcRn, complement receptors, and similarly avidly, it is believed that antigen elimination can only be selectively and enormously accelerated.
[01594] [01594] An example of a non-limiting modality of using an antigen binding molecule in the method provided by the present invention to eliminate antigens from plasma includes the use of the antigen binding molecule in a so-called ex vivo method for eliminate antigens from plasma, which includes the formation of an immune complex that contains two or more of the antigen binding molecules and two or more antigen molecules (as long as the antigens contain two or more antigen binding units) allowing the binding molecules to the antigen of the present invention to come into contact with the plasma isolated from an individual and allowing the immune complex to come into contact with cells that express FcRn and / or Fcγ receptors. The speed of elimination of antigen from plasma can also be promoted by replacing or combining a method for administering antigen-binding molecules in vivo with a so-called ex vivo method, in which plasma containing antigen-binding molecules and antigens that bind to antigen-binding molecules are temporarily absorbed outside the body and then placed in contact with cells that express FcRn and / or Fcγ receptors for a certain period of time and the plasma containing extracellularly recycled antigen-binding molecules (or re-secreted or recirculated) that are not linked to the antigen is returned to the body.
[01595] [01595] In addition, an example of a non-limiting embodiment of the use of an antigen binding molecule in the method provided by the present invention to eliminate antigens from plasma includes the use of the antigen binding molecule in a so-called ex vivo method to eliminate plasma antigens, which includes putting in contact with an immune complex that contains two or more of the antigen binding molecules and two or more antigen molecules (as long as the antigens comprise two or more antigen binding units) present in plasma isolated from an individual to whom the antigen-binding molecules of the present invention are administered with cells that express FcRn and / or Fcγ receptors.
[01596] [01596] If an antigen is eliminated from the plasma it can be confirmed, for example, by assessing whether the speed of elimination of the antigen from the plasma mentioned above is promoted using as a control, antigen-binding molecules that cannot form the complexes. immune compounds described in the present invention, antigen-binding molecules that contain antigen-binding domains with antigen-binding activity regardless of ionic concentration or antigen-binding molecules that contain an Fc region with impaired binding activity towards FcγR or FcRn, instead of the antigen-binding molecules of the present invention. Method of tracing antigen-binding molecules that contain an Fc region and an antigen-binding domain whose antigen-binding activity is dependent on ionic concentration
[01597] [01597] The present invention provides a method of tracking an antigen-binding molecule and has a function of removing antigens from plasma, the method comprising:
[01598] [01598] obtaining an antigen-binding domain whose antigen-binding activity varies depending on an ionic concentration condition;
[01599] [01599] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[01600] [01600] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[01601] [01601] culture of a host cell comprising the genes operably linked in (c) above;
[01602] [01602] isolation of an antigen-binding molecule from a culture solution obtained in (d) above;
[01603] [01603] put in contact the antigen-binding molecule obtained in (e) above with an antigen; and
[01604] [01604] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen.
[01605] [01605] In a non-limiting embodiment of the present invention, after isolating a polynucleotide that encodes an antigen-binding domain whose modifications of binding activity depending on the condition selected as described above, the polynucleotide is inserted into an appropriate expression vector . For example, when the antigen-binding domain is an antibody variable region, through a cDNA that encodes the variable region, the cDNA is digested with restriction enzymes that recognize the restriction sites inserted in the two ends of the cDNA. Preferably, restriction enzymes recognize and digest a nucleotide sequence that appears at a low frequency in the nucleotide sequence that makes up the gene for the antigen-binding molecule. In addition, restriction enzymes that provide cohesive ends are preferably inserted to insert a single copy of a fragment digested in the vector in the correct orientation. The cDNA encoding a variable region of a digested antigen binding molecule as described above is inserted into an appropriate expression vector to obtain an expression vector of the antigen binding molecule of the present invention. At this time, a gene encoding an antibody constant region (region C) can be fused into the structure with the gene encoding the variable region.
[01606] [01606] To produce an antigen-binding molecule of interest, a polynucleotide that encodes the antigen-binding molecule is inserted in a manner operationally linked to a regulatory sequence in an expression vector. The sequences regulated
[01607] [01607] For a nucleic acid, "operably linked" means that the nucleic acid has a functional relationship with another nucleic acid sequence. For example, DNA encoding a pre-sequence or secretory leader is operationally linked to DNA encoding a certain polypeptide if it is to be expressed as a precursor protein involved in secreting the polypeptide. A promoter or enabler is operationally linked to a coding sequence if it affects the transcription of the coding sequence. A ribosome binding site is operationally linked to a coding sequence if it is in a position that facilitates translation. Generally, "operationally linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader, it means that the linked DNA sequences are contiguous and in a reading phase. However, enhancers do not have to be contiguous. The league-
[01608] [01608] In a non-limiting embodiment of the present invention, after isolating a polynucleotide encoding the antigen-binding molecule described above whose binding activity varies depending on a selected condition, a variant of the polynucleotide is inserted into an appropriate expression vector. Such variants preferably include those prepared via humanization based on the polynucleotide sequence that encodes an antigen-binding molecule of the present invention obtained by screening as a library of random variable region, a synthetic library or an immune library constructed originating from non-human animals. The same methods as described above for producing the humanized antigen binding molecules described above can be used as a method for producing humanized antigen binding molecule variants.
[01609] [01609] In another embodiment, such variants preferably include those obtained by introducing a change that increases the antigen affinity (affinity maturation) of an antigen-binding molecule of the present invention in a polynucleotide sequence isolated from the molecule obtained by screening using a synthetic library or a naive library as a library of random variable region. Such variants can be obtained by several known procedures for affinity maturation, including CDR mutagenesis (Yang et al. (J. Mol. Biol. (1995) 254, 392-403)), chain mixture (Marks et al. (Bio / Technology (1992) 10, 779-783)), use of E. coli mutant strains (Low et al. (J. Mol. Biol. (1996) 250, 359-
[01610] [01610] As described above, antigen binding molecules that are produced by the production methods of the present invention include antigen binding molecules that have an Fc region. Several variants can be used as Fc regions. In one embodiment, the variants of the present invention preferably include polynucleotides that encode antigen-binding molecules that have a heavy chain in which a polynucleotide that encodes an Fc region variant as described above is linked in structure to a polynucleotide that encodes the antigen-binding domain described above whose binding activity varies depending on a selected condition.
[01611] [01611] In a non-limiting embodiment of the present invention, the Fc regions preferably include, for example, Fc constant regions of antibodies such as IgG1 of SEQ ID NO: 13 (Ala is added to the N-terminus of AAC82527.1 ), IgG2 of SEQ ID NO: 14 (Ala is added to the N-terminus of AAB59393.1), IgG3 of SEQ ID NO: (CAA27268.1) and IgG4 of SEQ ID NO: 16 (Ala is added to the terminal N of AAB59394.1). Plasma retention of IgG molecules is relatively long (plasma clearance is slow) since F-cRn, particularly human FcRn, functions as a rescue receptor for IgG molecules. The IgG molecules incorporated in endosomes by PEGcitosis bind under endosomal acidic condition to FcRn, particularly human FcRn, expressed in endosomes. IgG molecules that cannot bind to FcRn, particularly human FcRn, are transferred to lysosomes and degraded there. However, IgG molecules bound to FcRn, particularly human FcRn, are transferred to the cell surface, and then return to the plasma as a result of the dissociation of FcRn, particularly human FcRn, under neutral condition in the plasma.
[01612] [01612] Since antibodies comprising a typical Fc region do not have binding activity to FcRn, particularly to human FcRn, under condition of plasma neutral pH range, typical antibodies and antibody-antigen complexes are incorporated into cells by nonspecific endocytosis and transferred to the cell surface by binding to FcRn, particularly human FcRn, under the condition of an endosomal acidic pH range. FcRn, particularly human FcRn, carries antibodies from the endosome to the cell surface. In this way, it is thought that any FcRn, particularly human FcRn, are also present on the cell surface. However, antibodies are recycled to plasma, since they dissociate from FcRn, particularly human FcRn, in the condition of a neutral pH range on the cell surface.
[01613] [01613] Fc regions that have human FcRn binding activity in the neutral pH range, which may be included in the antigen binding molecules of the present invention, can be obtained by any method. Specifically, Fc regions that have human FcRn binding activity in the neutral pH range can be obtained by altering human IgG-like immunoglobulin amino acids as an initial Fc region. Preferred Fc regions of the human IgG-like immunoglobulin for alteration include, for example, those of human IgGs (IgG1, IgG2, IgG3, and IgG4 and variants thereof). Amino acids in any position can be changed to other amino acids while the resulting regions have human FcRn binding activity in the neutral pH range or increased human FcRn binding activity in the neutral range. When an antigen-binding molecule comprises the human IgG1 Fc region as the human Fc region, it is preferable that the resulting region comprises
[01614] [01614] Among those described above, appropriate changes that increase human FcRn that binds in the neutral pH range are selected for use in the present invention. Particularly preferred amino acids of such Fc region variants include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (indicated EU numbering). The human FcRn binding activity of the Fc region included in an antigen binding molecule can be increased in the neutral pH range by replacing at least one amino acid with a different amino acid.
[01615] [01615] Particularly preferred changes in the Fc region include, for example, at least one or more amino acid changes selected from the group of:
[01616] [01616] Met for the amino acid at position 237;
[01617] [01617] Ile for the amino acid at position 248;
[01618] [01618] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid
[01619] [01619] Phe, Trp or Tyr for the amino acid at position 252;
[01620] [01620] Thr for the amino acid at position 254;
[01621] [01621] Glu for the amino acid at position 255;
[01622] [01622] Asp, Asn, Glu or Gln for the amino acid at position 256;
[01623] [01623] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid at position 257;
[01624] [01624] His for the amino acid at position 258;
[01625] [01625] Wing for the amino acid at position 265;
[01626] [01626] Ala or Glu for the amino acid at position 286;
[01627] [01627] His for the amino acid at position 289;
[01628] [01628] Wing for the amino acid at position 297;
[01629] [01629] Wing for the amino acid at position 303;
[01630] [01630] Wing for the amino acid at position 305;
[01631] [01631] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid at position 307;
[01632] [01632] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid at position 308;
[01633] [01633] Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309;
[01634] [01634] Ala, His or Ile for the amino acid at position 311;
[01635] [01635] Ala or His for the amino acid at position 312;
[01636] [01636] Lys or Arg for the amino acid at position 314;
[01637] [01637] Ala, Asp or His for the amino acid at position 315;
[01638] [01638] Wing for the amino acid at position 317;
[01639] [01639] Val for the amino acid at position 332;
[01640] [01640] Read for the amino acid at position 334;
[01641] [01641] His for the amino acid at position 360;
[01642] [01642] Wing for the amino acid at position 376;
[01643] [01643] Wing for the amino acid at position 380;
[01644] [01644] Wing for the amino acid at position 382;
[01645] [01645] Ala for the amino acid at position 384;
[01646] [01646] Asp or His for the amino acid at position 385;
[01647] [01647] Pro for the amino acid at position 386;
[01648] [01648] Glu for the amino acid at position 387;
[01649] [01649] Ala or Ser for the amino acid at position 389;
[01650] [01650] Wing for the amino acid at position 424;
[01651] [01651] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid at position 428;
[01652] [01652] Lys for the amino acid at position 433;
[01653] [01653] Ala, Phe, His, Ser, Trp or Tyr for the amino acid at position 434; and
[01654] [01654] His, Ile, Leu, Phe, Thr or Val for the amino acid at position 436 in the EU numbering system. However, the number of altered amino acids is not particularly limited; such amino acid changes include a single amino acid change and a change in amino acids at two or more sites. Combinations of amino acid changes at two or more sites include, for example, those described in Tables 5-1 to 5-32.
[01655] [01655] In addition to the Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16), as the Fc regions included in the present invention, Fc regions with modified FcγR binding, which have a higher Fcγ receptor binding activity than the native human IgG Fc region in which the sugar linked in position 297 (EU numbering) is a sugar chain containing fucose, can be used appropriately. Such Fc regions with the modified FcγR binding can be produced by modifying amino acids in the Fc region of native human IgG. If the FcγR binding activity of an Fc region is higher than that of the native human IgG Fc region
[01656] [01656] In the present invention, "amino acid modification" or "amino acid modification" of an Fc region includes modification in an amino acid sequence that is different from that of the initial Fc region. The initial Fc region can be any Fc region, while a modified variant of the initial Fc region can bind to the human Fcγ receptor in a neutral pH range. In addition, a modified Fc region from an initial Fc region that had already been modified can also be used preferably as an Fc region of the present invention. The "initial Fc region" can refer to the polypeptide itself, a composition comprising the initial Fc region or amino acid sequence that encodes the initial Fc region. The initial Fc regions can comprise a known Fc region produced via recombination described briefly in the "Antibodies" section. The origin of early Fc regions is not limited, and can be obtained from humans or any non-human organism. Such organisms preferably include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels and organisms selected from non-human primates. In another embodiment, initial Fc regions can also be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The initial Fc regions can preferably be obtained 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 used appropriately as an initial Fc region, and in this application it also means that an Fc region of an arbitrated or subclassed IgG class of any organism described above can preferably be used as an initial Fc region. Examples of naturally occurring IgG variants or modified forms are described in published documents (Curr. Opin. Biotechnol. (2009) (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460- 470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202; International Publication WO No. 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338); however, they are not limited to examples.
[01657] [01657] Examples of changes include those with one or more mutations, for example, mutations by substituting different amino acid residues for amino acids from initial Fc regions, by inserting one or more amino acid residues in the initial Fc regions, or by deletion of one or more amino acids in the initial Fc region. Preferably, the amino acid sequences of altered Fc regions comprise at least a part of the amino acid sequence of a non-native Fc region. Such variants necessarily have less than 100% sequence identity or similarity to their initial Fc region. In a preferred embodiment, the variants have identity or similarity of amino acid sequence from approximately 75% to less than 100%, more preferably from approximately 80% to less than 100%, even more preferably from approximately 85% to less than 100% %, even more preferably from approximately 90% to less than 100%, and even more preferably from approximately 95% to less than 100% to the amino acid sequence of your initial Fc region. In a non-limiting fashion of the present invention, at least one amino acid is different between a modified FcγR-binding Fc region of the present invention and its initial Fc region. The amino acid differences between a modified FcγR binding Fc region of the present invention and its initial Fc region can also be appropriately specified based on the amino acid differences at the positions
[01658] [01658] The Fc region with the modified FcγR binding, which has a higher Fcγ receptor binding activity than that of the native human IgG Fc region in which the sugar chain linked at position 297 ( EU numbering) is a sugar chain containing fucose, contained in the antigen-binding molecules of this invention can be obtained by any method. Specifically, the Fc region with the modified FcγR binding can be obtained by modifying amino acids in a human IgG-like immunoglobulin and is used as the initial Fc region. The preferred Fc regions of IgG-like immunoglobulins for modification include, for example, the Fc regions of human IgGs (IgG1, IgG2, IgG3, IgG4 and variants thereof).
[01659] [01659] Amino acids of any position can be modified to other amino acids, while the binding activity towards the Fcγ receptor is higher than that of the native human IgG Fc region, in which the linked sugar at heading 297 (EU numbering) is a sugar chain containing fucose. When the antigen-binding molecule contains a human IgG1 Fc region such as the human Fc region, it preferably contains a modification that produces the effect of a higher Fcγ receptor binding activity than that of the Fc region of native human IgG, in which the sugar chain linked at position 297 (EU numbering) is a sugar chain containing fucose. Such amino acid modifications have been reported, for example, in international publications such as WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO2006 / 020114, WO2006 / 116260 and WO2006 / 023403.
[01660] [01660] Examples of such amino acids that can be modified include at least one or more amino acids selected from the group of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 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 ( EU numbering). The modification of these amino acids can produce Fc regions (Fc regions with modified FcγR binding) having a higher Fcγ receptor binding activity than the Fcγ receptor binding activity of an IgG Fc region. native human, in which the sugar chain linked at position 297 (EU numbering) is a sugar chain containing fucose.
[01661] [01661] Examples of particularly preferred modifications for use in the present invention include at least one or more amino acid changes in the Fc region selected from the group of:
[01662] [01662] Lys or Tyr for the amino acid of position 221;
[01663] [01663] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[01664] [01664] Phe, Trp, Glu or Lys for the amino acid of position 223;
[01665] [01665] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[01666] [01666] Glu, Lys or Trp for the amino acid of position 225;
[01667] [01667] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[01668] [01668] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[01669] [01669] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[01670] [01670] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[01671] [01671] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[01672] [01672] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[01673] [01673] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[01674] [01674] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235;
[01675] [01675] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[01676] [01676] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237;
[01677] [01677] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[01678] [01678] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239;
[01679] [01679] Ala, Ile, Met, or Thr for the amino acid at position 240;
[01680] [01680] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[01681] [01681] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[01682] [01682] His for the amino acid at position 244;
[01683] [01683] Ala for the amino acid of position 245;
[01684] [01684] Asp, Glu, His or Tyr for the amino acid of position 246;
[01685] [01685] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[01686] [01686] Glu, His, Gln or Tyr for the amino acid of position 249;
[01687] [01687] Glu or Gln for the amino acid of position 250;
[01688] [01688] Phe for the amino acid of position 251;
[01689] [01689] Phe, Met, or Tyr for the amino acid of position 254;
[01690] [01690] Glu, Leu or Tyr for the amino acid of position 255;
[01691] [01691] Ala, Met, or Pro for the amino acid of position 256;
[01692] [01692] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[01693] [01693] Asp, Glu, His or Tyr for the amino acid of position 260;
[01694] [01694] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[01695] [01695] Ala, Ile, Met, or Thr for the amino acid of position 263;
[01696] [01696] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[01697] [01697] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[01698] [01698] Ala, Ile, Met, or Thr for the amino acid of position 266;
[01699] [01699] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[01700] [01700] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[01701] [01701] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[01702] [01702] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[01703] [01703] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[01704] [01704] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[01705] [01705] Phe or Ile for the amino acid of position 273;
[01706] [01706] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[01707] [01707] Leu or Trp for the amino acid of position 275;
[01708] [01708] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[01709] [01709] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
[01710] [01710] Wing for the amino acid of position 279;
[01711] [01711] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[01712] [01712] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[01713] [01713] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282;
[01714] [01714] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[01715] [01715] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284;
[01716] [01716] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[01717] [01717] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[01718] [01718] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[01719] [01719] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[01720] [01720] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[01721] [01721] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[01722] [01722] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[01723] [01723] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[01724] [01724] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[01725] [01725] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[01726] [01726] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
[01727] [01727] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[01728] [01728] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 299;
[01729] [01729] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 300;
[01730] [01730] Asp, Glu, His or Tyr for the amino acid of position 301;
[01731] [01731] Ile for the amino acid of position 302;
[01732] [01732] Asp, Gly or Tyr for the amino acid of position 303;
[01733] [01733] Asp, His, Leu, Asn or Thr for the amino acid of position 304;
[01734] [01734] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[01735] [01735] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311;
[01736] [01736] Phe for the amino acid of position 313;
[01737] [01737] Read for the amino acid of position 315;
[01738] [01738] Glu or Gln for the amino acid of position 317;
[01739] [01739] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[01740] [01740] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 320;
[01741] [01741] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[01742] [01742] Ile for the amino acid of position 323;
[01743] [01743] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[01744] [01744] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[01745] [01745] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val,
[01746] [01746] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[01747] [01747] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[01748] [01748] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329;
[01749] [01749] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[01750] [01750] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 331;
[01751] [01751] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[01752] [01752] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the 333 amino acid;
[01753] [01753] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[01754] [01754] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[01755] [01755] Glu, Lys or Tyr for the amino acid of position 336;
[01756] [01756] Glu, His or Asn for the amino acid of position 337;
[01757] [01757] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[01758] [01758] Ala or Val for the amino acid of position 376;
[01759] [01759] Gly or Lys for the amino acid of position 377;
[01760] [01760] Asp for the amino acid of position 378;
[01761] [01761] Asn for the amino acid of position 379;
[01762] [01762] Ala, Asn or Ser for the amino acid of position 380;
[01763] [01763] Ala or Ile for the amino acid of position 382;
[01764] [01764] Glu for the amino acid of position 385;
[01765] [01765] Thr for the amino acid of position 392;
[01766] [01766] Read for the amino acid of position 396;
[01767] [01767] Lys for the amino acid of position 421;
[01768] [01768] Asn for the amino acid of position 427;
[01769] [01769] Phe or Leu for the amino acid of position 428;
[01770] [01770] Met for the amino acid of position 429;
[01771] [01771] Trp for the amino acid of position 434;
[01772] [01772] Ile for the amino acid of position 436; and
[01773] [01773] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[01774] [01774] as indicated by the EU numbering. However, the number of amino acids to be modified is not particularly limited, and an amino acid in only one site can be modified or the amino acids in two or more sites can be modified. Examples of combinations of amino acid modifications at two or more sites include those described in Table 6 (Tables 6-1 to 6-3).
[01775] [01775] Among the Fc regions suitable for use in the present invention, a suitable example of an Fc region that has a higher binding activity towards an inhibitory Fcγ receptor than towards an activation activation receptor Fcγ (ie, having a selective binding activity towards an inhibitory Fcγ receptor), which is used as a non-limiting modality of an Fc region with the property of having a higher binding activity towards a specific Fcγ receptor than towards other Fcγ receptors (ie, an Fc region that has selective Fcγ receptor binding activity), is an Fc region with one or more of the following changes in amino acids (indicated by EU numbering) from the Fc region mentioned above: the amino acid at position 238 is changed to Asp and the amino acid at position 328 is changed to Glu. The Fc regions and the modifications described in US2009 / 0136485 can be selected appropriately as the Fc region having selective binding activity to an inhibitory Fcγ receptor.
[01776] [01776] In a non-limiting embodiment of the present invention, a suitable example is an Fc region in which one or more of the amino acids indicated by the EU number at positions 238 and 328 according to the EU number are respectively modified to Asp or Glu at Fc region mentioned above.
[01777] [01777] In addition, in a non-limiting embodiment of the present invention, suitable examples of the Fc regions are those with the replacement of Asp by Pro at position 238 (EU numbering), and one or more modifications selected from Trp for the amino acid of position 237, Phe for the amino acid of position 237, Val for the amino acid of position 267, Gln for the amino acid of position 267, Asn for the amino acid of position 268, Gly for the amino acid of position 271, Leu for amino acid of position 326, Gln for amino acid of position 326, Glu for amino acid of position 326, Met for amino acid of position 326, Asp for amino acid of position 239, Ala for amino acid of position 267, Trp for the amino acid at position 234, Tyr for the amino acid at position 234, Ala for the amino acid at position 237, Asp for the amino acid at position 237, Glu for the amino acid at position 237, Leu for the amino acid at position 237 , Met for the amino acid at position 237, Tyr for the amino acid at position 237, Lys for the amino acid at position 330, Arg for the amino acid at position 330, Asp for the amino acid at position 233, Asp for the amino acid at position 268, Glu for the amino acid at position 268, Asp for the amino acid of position 326, Ser for the amino acid of position 326, Thr for the amino acid of position 326, Ile for the amino acid of position 323, Leu for the amino acid of position 323, Met for the amino acid of position 323, Asp for the amino acid at position 296, Ala for the amino acid at position 326, Asn for the amino acid at position 326, and
[01778] [01778] Methods known as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed to modify the amino acids from Fc regions. In addition, several known methods can also be used as an amino acid modification method to replace amino acids with those in addition to natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cellless translation system (Clover Direct (Protein Express)) containing tRNAs in which the amber suppressor tRNA, which is complementary to the UAG codon (amber codon) which is a stop codon, is linked with an unnatural amino acid , can be used appropriately.
[01779] [01779] As described above, antigen binding molecules isolated from the host cell culture solution containing operably linked genes that encode antigen binding molecules are brought into contact with antigens in the screening method of the present invention. In a non-limiting modality of the contact step, the conditions described, for example, in the section mentioned above in the antigen, binding antigens can be appropriately used.
[01780] [01780] Next, if the immune complexes containing the test antigen-binding molecule and the test antigen are formed and evaluated. Examples of methods for assessing the formation of immune complexes that contain antigen-binding molecules and antigens include methods that use the property that immune complexes become larger molecules than the antigen-binding molecule alone or the antigen molecule. only, such as size exclusion chromatography (gel filtration), ultracentrifuge analysis method, light scattering method, electron microscopy and mass spectrometry (Molecular Immunology (2002), 39, 77-84; Molecular Immunology ( 2009), 47, 357-364). For example, when size exclusion chromatography (gel filtration) is used as shown in Fig. 9, whether immune complexes are formed are evaluated based on whether the largest molecular species are observed when compared to when the antigen only molecule or antigen binding molecule are only analyzed.
[01781] [01781] In addition, when the antigen-binding molecule or antigen has an immunoglobulin constant region, examples also include ELISA and FACS that use the property of the immune complex to bind more strongly to a Fc or a complement component than the antigen binding molecule alone or antigen alone (The Journal of Biological Chemistry (2001) 276 (9), 6591-6604; Journal of Immunological Methods (1982) 50, 109 - 114). For example, when ELISA is performed by immobilizing an Fc receptor, the formation of an immune complex is evaluated by looking at whether the detected signal is increased compared to when an antigen alone or an antigen-binding molecule is only evaluated. Methods to produce an antigen-binding molecule containing an Fc region and an antigen-binding domain showing ionic concentration-dependent antigen-binding activity
[01782] [01782] The present invention provides a method for producing antigen binding molecules having a function of eliminating antigens from plasma, the method comprising:
[01783] [01783] bringing an antigen into contact with an antigen-binding molecule comprising an Fc region and two or more antigen-binding domains, in which at least one of the antigen-binding domains has a binding activity to the antigen that varies depending on a condition of ionic concentration;
[01784] [01784] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen;
[01785] [01785] culture of a host cell comprising a vector that carries a gene that encodes an antigen binding domain that is confirmed to form an immune complex in (b) above; and
[01786] [01786] isolation of the antigen-binding molecule from a culture solution obtained in (c) above.
[01787] [01787] The present invention provides a method for producing an antigen-binding molecule having a function of eliminating antigens from plasma, the method comprising:
[01788] [01788] obtaining an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition;
[01789] [01789] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[01790] [01790] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[01791] [01791] culture of a host cell comprising the genes operably linked in (c) above;
[01792] [01792] isolation of an antigen-binding molecule from a culture solution obtained in (d) above;
[01793] [01793] put in contact with the antigen binding molecule obtained in (e) above with an antigen;
[01794] [01794] evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen;
[01795] [01795] culture of a host cell comprising a vector that carries a gene that encodes an antigen binding domain that is confirmed to form an immune complex in (g) above; and
[01796] [01796] isolation of the antigen-binding molecule from a culture solution obtained in (h) above.
[01797] [01797] The present invention further provides a method for producing an antigen binding molecule having a function of eliminating antigens from plasma, the method comprising:
[01798] [01798] obtaining an antigen-binding domain from which the antigen-binding activity varies depending on an ionic concentration condition;
[01799] [01799] obtaining a gene that encodes the antigen-binding domain selected in (a) above;
[01800] [01800] operationally linking the gene obtained in (b) above with a gene encoding an Fc region;
[01801] [01801] culture of a host cell comprising the genes operably linked in (c) above; and
[01802] [01802] isolation of an antigen-binding molecule from a culture solution obtained in (d) above; and
[01803] [01803] in which the method also comprises contacting the antigen-binding molecule obtained by the production method with an antigen and assessing the formation of an immune complex comprising the antigen-binding molecule and the antigen.
[01804] [01804] In a non-limiting embodiment of the present invention, after isolating a polynucleotide that encodes an antigen-binding domain whose modifications of binding activity depending on the condition selected as described above, the polynucleotide is inserted into an appropriate expression vector . For example, when the antigen binding domain is an antibody variable region, once a cDNA encoding the variable region is obtained, the cDNA is digested with restriction enzymes that recognize the restriction sites inserted in the two ends of the cDNA. Preferably, restriction enzymes recognize and digest a nucleotide sequence that appears at a low frequency in the nucleotide sequence that makes up the gene for the antigen-binding molecule. In addition, restriction enzymes that provide cohesive ends are preferably inserted to insert a single copy of a fragment digested in the vector in the correct orientation. The cDNA encoding a variable region of a digested antigen binding molecule as described above is inserted into an appropriate expression vector to obtain an expression vector of the antigen binding molecule of the present invention. At this time, a gene encoding an antibody constant region (region C) can be fused in structure with the gene encoding the variable region.
[01805] [01805] To produce an antigen-binding molecule of interest, a polynucleotide that encodes the antigen-binding molecule is inserted in a manner operationally linked to a regulatory sequence in an expression vector. Regulatory sequences include, for example, enhancers and promoters. In addition, an appropriate signal sequence can be linked to the N-terminus so that the expressed antigen-binding molecule is secreted outside the cells. As a signal sequence, for example, a peptide that has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 5) is used; however, it is also possible to link other appropriate signal sequences. The expressed polypeptide is cleaved at the carboxyl terminus of the sequence described above, and the cleaved polypeptide is secreted as a mature polypeptide outside the cells. Then, appropriate host cells are transformed with this expression vector so that recombinant cells that express the polynucleotide that encodes the antigen-binding molecule of interest can be obtained. The antigen-binding molecules of the present invention can be produced from cells
[01806] [01806] For a nucleic acid, "operably linked" means that the nucleic acid has a functional relationship with another nucleic acid sequence. For example, a DNA that encodes a pre-sequence or a secretory leader is operationally linked to DNA that encodes a certain polypeptide if it is to be expressed as a precursor protein involved in the secretion of the polypeptide. A promoter or enhancer is operationally linked to a coding sequence if it affects the transcription of the coding sequence. A ribosome binding site is operationally linked to a coding sequence if it is in a position that facilitates translation. Generally, "operationally linked" means that the linked DNA sequences are contiguous, and in the case of a secretarial leader, it means that the linked DNA sequences are contiguous and in a reading phase. However, enhancers do not have to be contiguous. The connection is carried out by connecting to suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or ligands are used in accordance with conventional practice. In addition, linked nucleic acids can be produced by the aforementioned overlap extension PCR technique.
[01807] [01807] In a non-limiting embodiment of the present invention, after isolating a polynucleotide encoding the antigen binding molecule described above whose binding activity varies depending on a selected condition, a variant of the polynucleotide is inserted into an appropriate expression vector. Such variants preferably include those prepared via humanization based on the polynucleotide sequence that encodes an antigen-binding molecule of the present invention obtained by screening as a library of the random variable region, a synthetic library or an i-
[01808] [01808] In another embodiment, such variants preferably include those obtained by introducing a change that increases the antigen affinity (affinity maturation) of an antigen-binding molecule of the present invention in a polynucleotide sequence isolated from the molecule obtained by tracking using a synthetic library or a naive library as a random variable region library. Such variants can be obtained by several procedures known for affinity maturation, including CDR mutagenesis (Yang et al. (J. Mol. Biol. (1995) 254, 392-403)), chain mixing (Marks et al. (Bio / Technology (1992) 10, 779-783)), use of mutant strains of E. coli (Low et al. (J. Mol. Biol. (1996) 250, 359-368)) , DNA mixing (Patten et al. (Curr. Opin. Biotechnol. (1997) 8, 724-733)), phage display (Thompson et al. (J. Mol. Biol. (1996) 256, 77 -88)), and sexual PCR (Clameri et al. (Nature (1998) 391, 288-291)).
[01809] [01809] As described above, antigen binding molecules that are produced by the production methods of the present invention include antigen binding molecules that have an Fc region. Several variants can be used as Fc regions. In one embodiment, variants of the present invention preferably include polynucleotides that encode antigen-binding molecules that have a heavy chain in which a polynucleotide that encodes an Fc region variant as described above is linked in structure to a polynucleotide that encodes the antigen-binding molecule described above whose binding activity varies depending on a selected condition.
[01810] [01810] In a non-limiting embodiment of the present invention, the Fc regions preferably include, for example, Fc-containing regions of antibodies such as IgG1 of SEQ ID NO: 13 (Ala is added to the N-terminus of AAC82527. 1), IgG2 of SEQ ID NO: 14 (Ala is added to the N terminal of AAB59393.1), IgG3 of SEQ ID NO: 15 (CAA27268.1) and IgG4 of SEQ ID NO: 16 (Ala is added to the N terminal of AAB59394.1). Plasma retention of IgG molecules is relatively long (plasma clearance is slow) since FcRn, particularly human FcRn, functions as a rescue receptor for IgG molecules. IgG molecules incorporated in endosomes by PEGcitosis bind under endosomal acidic conditions to FcRn, particularly human FcRn, expressed in endosomes. IgG molecules that cannot bind to FcRn, particularly human FcRn, are transferred to lysosomes and degraded there. However, IgG molecules bound to FcRn, particularly human FcRn, are transferred to the cell surface, and then return to the plasma as a result of the dissociation of FcRn, particularly human FcRn, under neutral condition in the plasma.
[01811] [01811] Since antibodies comprising a typical Fc region do not have binding activity to FcRn, particularly to human FcRn, under the condition of plasma neutral pH range, typical antibodies and antibody-antigen complexes are incorporated in cells by nonspecific endocytosis and transferred to the cell surface by binding to FcRn, particularly human FcRn, under the condition of the endosomal acidic pH range. FcRn, particularly human FcRn, carries binding molecules to the endosome antigen to the cell surface. Thus, it is thought that some FcRn, particularly human FcRn, are also present on the cell surface. However, the antibodies are recycled to plasma, since they dissociate from FcRn, particularly human FcRn, in the condition of a neutral pH range on the cell surface.
[01812] [01812] Fc regions having human FcRn binding activity in the neutral pH range, which can be included in the antigen binding molecules of the present invention, can be obtained by any method. Specifically, Fc regions having human FcRn binding activity in the neutral pH range can be obtained by altering human IgG-like immunoglobulin amino acids as an initial Fc region. The preferred Fc regions of the human IgG-like immunoglobulin for alteration include, for example, those of human IgGs (IgG1, IgG2, IgG3, and IgG4 and variants thereof). Amino acids in any position can be altered by other amino acids although the resulting regions have human FcRn binding activity in the neutral pH range or increased human FcRn binding activity in the neutral range. When an antigen-binding molecule comprises the human IgG1 Fc region as a human Fc region, it is preferred that the resulting region comprises a change that results in the effect of increasing the binding of human FcRn in the neutral pH range when compared to binding activity of the initial Fc region of human IgG1. The amino acids that allow such changes include, for example, at least one amino acid selected from the group of positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442 (indicated by the EU numbering). More specifically, such amino acid changes include those listed in Table 5. Changing these amino acids increases the binding of human FcRn from the IgG-like immunoglobulin Fc region to the neutral pH range.
[01813] [01813] Among those described above, appropriate changes that increase the binding of human FcRn in the neutral pH range are selected for use in the present invention. Particularly preferred amino acids of such Fc region variants include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (indicated EU numbering). The human FcRn binding activity of the Fc region included in an antigen binding molecule can be increased in the neutral pH range by replacing at least one amino acid with a different amino acid.
[01814] [01814] Particularly preferred changes in the Fc region include, for example, at least one or more amino acid changes selected from the group of:
[01815] [01815] Met for the amino acid at position 237;
[01816] [01816] Ile for the amino acid at position 248;
[01817] [01817] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid at position 250;
[01818] [01818] Phe, Trp or Tyr for the amino acid at position 252;
[01819] [01819] Thr for the amino acid at position 254;
[01820] [01820] Glu for the amino acid at position 255;
[01821] [01821] Asp, Asn, Glu or Gln for the amino acid at position 256;
[01822] [01822] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid at position 257;
[01823] [01823] His for the amino acid at position 258;
[01824] [01824] Wing for the amino acid at position 265;
[01825] [01825] Ala or Glu for the amino acid at position 286;
[01826] [01826] His for the amino acid at position 289;
[01827] [01827] Wing for the amino acid at position 297;
[01828] [01828] Wing for the amino acid at position 303;
[01829] [01829] Wing for the amino acid at position 305;
[01830] [01830] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid at position 307;
[01831] [01831] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid at position 308;
[01832] [01832] Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309;
[01833] [01833] Ala, His or Ile for the amino acid at position 311;
[01834] [01834] Ala or His for the amino acid at position 312;
[01835] [01835] Lys or Arg for the amino acid at position 314;
[01836] [01836] Ala, Asp or His for the amino acid at position 315;
[01837] [01837] Wing for the amino acid at position 317;
[01838] [01838] Val for the amino acid at position 332;
[01839] [01839] Read for the amino acid at position 334;
[01840] [01840] His for the amino acid at position 360;
[01841] [01841] Wing for the amino acid at position 376;
[01842] [01842] Wing for the amino acid at position 380;
[01843] [01843] Ala for the amino acid at position 382;
[01844] [01844] Wing for the amino acid at position 384;
[01845] [01845] Asp or His for the amino acid at position 385;
[01846] [01846] Pro for the amino acid at position 386;
[01847] [01847] Glu for the amino acid at position 387;
[01848] [01848] Ala or Ser for the amino acid at position 389;
[01849] [01849] Wing for the amino acid at position 424;
[01850] [01850] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid at position 428;
[01851] [01851] Lys for the amino acid at position 433;
[01852] [01852] Ala, Phe, His, Ser, Trp or Tyr for the amino acid at position 434; and
[01853] [01853] His, Ile, Leu, Phe, Thr or Val for the amino acid at position 436 in the EU numbering system. However, the number of altered amino acids is not particularly limited; such amino acid changes include the single amino acid change and the amino acid change.
[01854] [01854] In addition to the Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16), as the Fc regions included in the present invention, Fc regions with modified FcγR binding, which have a higher Fcγ receptor binding activity than the native human IgG Fc region in which the sugar linked in position 297 (EU numbering) is a sugar chain containing fucose, can be used appropriately. Such Fc regions with the modified FcγR binding can be produced by modifying amino acids in the Fc region of native human IgG. If the FcγR binding activity of an Fc region is higher than that of the native human IgG Fc region, in which the linked sugar chain at position 297 (EU numbering) is a sugar chain containing fucose , can be appropriately determined using methods such as those described above.
[01855] [01855] In the present invention, "amino acid modification" or "amino acid modification" of an Fc region includes modification in an amino acid sequence that is different from that of the initial Fc region. The initial Fc region can be any Fc region, while a modified variant of the initial Fc region can bind to the human Fcγ receptor in a neutral pH range. In addition, a modified Fc region from an initial Fc region that had already been modified can also be used preferably as an Fc region of the present invention. The "initial Fc region" can refer to the polypeptide itself, a composition comprising the initial Fc region or amino acid sequence that encodes the initial Fc region. The initial Fc regions may comprise known Fc regions produced via recombination described briefly in the "Antibodies" section. The origin of early Fc regions is not limited, and can be obtained from humans or any non-human organism. Such organisms preferably include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels and organisms selected from non-human primates. In another modality, initial Fc regions can also be obtained from cynomolgus monkeys, saquers, rhesus monkeys, chimpanzees or humans. The initial Fc regions can be obtained preferably 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 used appropriately as an initial Fc region, and in this application it also means that an Fc region of an arbitrated IgG class or subclass derived from any organism described - above can preferably be used as an initial Fc region. Examples of naturally occurring IgG variants or modified forms are described in published documents (Curr. Opin. Bio-technol. (2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4) , 460-470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202; International Publication WO No. 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338); however, they are not limited to examples.
[01856] [01856] Examples of changes include those with one or more mutations, for example, mutations by substituting amino acid residues other than amino acids from initial Fc regions, by inserting one or more amino acid residues in the initial Fc regions, or by deletion of one or more amino acids in the initial Fc region. Preferably, the amino acid sequences of altered Fc regions comprise at least a part of the amino acid sequence of a non-native Fc region. Such variants
[01857] [01857] The Fc region with the modified FcγR binding, which has a higher Fcγ receptor binding activity than that of the native human IgG Fc region in which the sugar chain linked at position 297 ( EU numbering) is a sugar chain containing fucose, contained in the antigen-binding molecules of this invention can be obtained by any method. Specifically, the Fc region with the modified FcγR binding can be obtained by modifying amino acids in a human IgG-like immunoglobulin and is used as the initial Fc region. The preferred IgG-like immunoglobulin Fc regions of the modification include, for example, human IgG Fc regions (IgG1, IgG2, IgG3, IgG4 and variants thereof).
[01858] [01858] Amino acids of any position can be modified
[01859] [01859] Examples of such amino acids that can be modified include at least one or more amino acids selected from the group of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 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 ( EU numbering). By modifying these amino acids, Fc regions (Fc regions with modified FcγR binding) can be obtained with a higher binding activity towards the Fcγ receptor than the binding activity towards the receptor of Fcγ from the Fc region of native human IgG, in which the
[01860] [01860] Examples of particularly preferred modifications for use in the present invention include at least one or more amino acid changes in the Fc region selected from the group of:
[01861] [01861] Lys or Tyr for the amino acid of position 221;
[01862] [01862] Phe, Trp, Glu or Tyr for the amino acid of position 222;
[01863] [01863] Phe, Trp, Glu or Lys for the amino acid of position 223;
[01864] [01864] Phe, Trp, Glu or Tyr for the amino acid of position 224;
[01865] [01865] Glu, Lys or Trp for the amino acid of position 225;
[01866] [01866] Glu, Gly, Lys or Tyr for the amino acid of position 227;
[01867] [01867] Glu, Gly, Lys or Tyr for the amino acid of position 228;
[01868] [01868] Ala, Glu, Gly or Tyr for the amino acid of position 230;
[01869] [01869] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;
[01870] [01870] Glu, Gly, Lys or Tyr for the amino acid of position 232;
[01871] [01871] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233;
[01872] [01872] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234;
[01873] [01873] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235;
[01874] [01874] Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236;
[01875] [01875] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237;
[01876] [01876] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238;
[01877] [01877] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,
[01878] [01878] Ala, Ile, Met, or Thr for the amino acid at position 240;
[01879] [01879] Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of position 241;
[01880] [01880] Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243;
[01881] [01881] His for the amino acid at position 244;
[01882] [01882] Ala for the amino acid of position 245;
[01883] [01883] Asp, Glu, His or Tyr for the amino acid of position 246;
[01884] [01884] Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247;
[01885] [01885] Glu, His, Gln or Tyr for the amino acid of position 249;
[01886] [01886] Glu or Gln for the amino acid of position 250;
[01887] [01887] Phe for the amino acid of position 251;
[01888] [01888] Phe, Met, or Tyr for the amino acid of position 254;
[01889] [01889] Glu, Leu or Tyr for the amino acid of position 255;
[01890] [01890] Ala, Met, or Pro for the amino acid of position 256;
[01891] [01891] Asp, Glu, His, Ser or Tyr for the amino acid of position 258;
[01892] [01892] Asp, Glu, His or Tyr for the amino acid of position 260;
[01893] [01893] Ala, Glu, Phe, Ile or Thr for the amino acid of position 262;
[01894] [01894] Ala, Ile, Met, or Thr for the amino acid of position 263;
[01895] [01895] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264;
[01896] [01896] Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265;
[01897] [01897] Ala, Ile, Met, or Thr for the amino acid of position 266;
[01898] [01898] Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267;
[01899] [01899] Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268;
[01900] [01900] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
[01901] [01901] Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270;
[01902] [01902] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271;
[01903] [01903] Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272;
[01904] [01904] Phe or Ile for the amino acid of position 273;
[01905] [01905] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274;
[01906] [01906] Leu or Trp for the amino acid of position 275;
[01907] [01907] Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276;
[01908] [01908] Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 278;
[01909] [01909] Wing for the amino acid of position 279;
[01910] [01910] Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280;
[01911] [01911] Asp, Lys, Pro, or Tyr for the amino acid of position 281;
[01912] [01912] Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282;
[01913] [01913] Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283;
[01914] [01914] Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284;
[01915] [01915] Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285;
[01916] [01916] Glu, Gly, Pro, or Tyr for the amino acid of position 286;
[01917] [01917] Asn, Asp, Glu or Tyr for the amino acid of position 288;
[01918] [01918] Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid of position 290;
[01919] [01919] Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid of position 291;
[01920] [01920] Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292;
[01921] [01921] Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293;
[01922] [01922] Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294;
[01923] [01923] Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, T-hr, Val, Trp or Tyr for the amino acid of position 295;
[01924] [01924] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296;
[01925] [01925] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297;
[01926] [01926] Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298;
[01927] [01927] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 299;
[01928] [01928] Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid of position 300;
[01929] [01929] Asp, Glu, His or Tyr for the amino acid of position 301;
[01930] [01930] Ile for the amino acid of position 302;
[01931] [01931] Asp, Gly or Tyr for the amino acid of position 303;
[01932] [01932] Asp, His, Leu, Asn or Thr for the amino acid of position 304;
[01933] [01933] Glu, Ile, Thr or Tyr for the amino acid of position 305;
[01934] [01934] Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of the position
[01935] [01935] Phe for the amino acid of position 313;
[01936] [01936] Read for the amino acid of position 315;
[01937] [01937] Glu or Gln for the amino acid of position 317;
[01938] [01938] His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318;
[01939] [01939] Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 320;
[01940] [01940] Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322;
[01941] [01941] Ile for the amino acid of position 323;
[01942] [01942] Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324;
[01943] [01943] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325;
[01944] [01944] Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326;
[01945] [01945] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327;
[01946] [01946] Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328;
[01947] [01947] Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329;
[01948] [01948] Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 330;
[01949] [01949] Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 331;
[01950] [01950] Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332;
[01951] [01951] Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the amino acid of position 333;
[01952] [01952] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
[01953] [01953] Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335;
[01954] [01954] Glu, Lys or Tyr for the amino acid of position 336;
[01955] [01955] Glu, His or Asn for the amino acid of position 337;
[01956] [01956] Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339;
[01957] [01957] Ala or Val for the amino acid of position 376;
[01958] [01958] Gly or Lys for the amino acid of position 377;
[01959] [01959] Asp for the amino acid of position 378;
[01960] [01960] Asn for the amino acid of position 379;
[01961] [01961] Ala, Asn or Ser for the amino acid of position 380;
[01962] [01962] Ala or Ile for the amino acid of position 382;
[01963] [01963] Glu for the amino acid of position 385;
[01964] [01964] Thr for the amino acid of position 392;
[01965] [01965] Read for the amino acid of position 396;
[01966] [01966] Lys for the amino acid of position 421;
[01967] [01967] Asn for the amino acid of position 427;
[01968] [01968] Phe or Leu for the amino acid of position 428;
[01969] [01969] Met for the amino acid of position 429;
[01970] [01970] Trp for the amino acid of position 434;
[01971] [01971] Ile for the amino acid of position 436; and
[01972] [01972] Gly, His, Ile, Leu or Tyr for the amino acid of position 440;
[01973] [01973] as indicated by the EU numbering. In addition, the number of amino acids to be modified is not particularly limited, and the amino acid in only one site can be modified or the amino acids in two or more sites can be modified. Examples of combinations of amino acid modifications at two or more sites include those described in Table 6 (Tables 6-1 to 6-3).
[01974] [01974] Among the Fc regions suitable for use in the present invention, a suitable example of an Fc region that has a higher binding activity towards an inhibitory Fcγ receptor than towards an activation activation receptor Fcγ (ie, having a selective binding activity towards an inhibitory Fcγ receptor), which is used as a non-limiting modality of an Fc region with the property of having a higher binding activity towards a specific Fcγ receptor than towards other Fcγ receptors (ie, an Fc region that has selective Fcγ receptor binding activity), is an Fc region with one or more of the following changes in amino acids (indicated by EU numbering) from the Fc region mentioned above: the amino acid at position 238 is changed to Asp and the amino acid at position 328 is changed to Glu. The Fc regions and the modifications described in US2009 / 0136485 or WO 2012/115241 can be appropriately selected as the Fc region having selective binding activity for an inhibitory Fcγ receptor.
[01975] [01975] In a non-limiting embodiment of the present invention, a suitable example is an Fc region in which one or more of the amino acids indicated by the EU number at positions 238 and 328 according to the EU number are respectively modified to Asp or Glu at mentioned Fc region.
[01976] [01976] Furthermore, in a non-limiting embodiment of the present invention, suitable examples of the Fc regions are those with the substitution of Asp for Pro at position 238 (EU numbering), and one or more modifications selected from Trp for the amino acid of position 237, Phe for the amino acid of position 237, Val for the amino acid of position 267, Gln for the amino acid of position 267, Asn for the amino acid of position 268, Gly for the amino acid of position 271,
[01977] [01977] Appropriate methods known as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be applied to alter the amino acids in Fc regions. In addition, several known methods can also be used as an amino acid alteration method to replace amino acids with those in addition to natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is also preferable to use a cell-free translation system (Clover Direct (Protein Express)) comprising tRNAs in which an unnatural amino acid is linked to an amber suppressor tRNA, which is complementary to the UAG stop codon (amber codon) ).
[01978] [01978] In a variant embodiment of the present invention, polynucleotides that encode antigen-binding molecules that have a heavy chain where a polynucleotide that encodes a modified Fc region by having an amino acid mutation as described above is linked in structure to a polynucleotide that encodes the antigen-binding domain described above whose binding activity varies depending on a selected condition.
[01979] [01979] The present invention provides methods for producing antigen-binding molecules, comprising collecting antigen-binding molecules from cell culture media introduced with vectors in which a polynucleotide encoding an Fc region is operably linked in structure to a polynucleotide that encodes an antigen-binding domain whose binding activity varies depending on the condition of ionic concentration. In addition, the present invention also provides methods for producing antigen-binding molecules, comprising collecting the antigen-binding molecules from cell culture media introduced with constructed vectors operably binding to a polynucleotide that encodes an antigen-binding domain. whose binding activity varies depending on the condition of ionic concentration to a polynucleotide that encodes an Fc region that is operationally linked in advance to a vector.
[01980] [01980] In the production methods of the present invention, appropriate modifications to the domain can be added after obtaining a "confirmed antigen binding domain to form an immune complex", while the formation of the immune complex is allowed. In addition, a method for producing an antigen-binding molecule is also provided, which includes collecting the antigen-binding molecule from a cell culture solution that contains a vector that carries a polynucleotide that encodes an antigen-binding molecule which has a heavy chain in which a polynucleotide encoding an antigen-binding domain capable of forming an immune complex and modified in this way is linked in structure to a polynucleotide encoding an Fc region variant in which mutations of the aforementioned amino acids were added. Pharmaceutical compositions
[01981] [01981] When a conventional neutralizing antibody against a soluble antigen is administered, the plasma retention of the antigen is expected to be prolonged by binding to the antibody. In general, antibodies have a long half-life (one week to three weeks) while the antigen's half-life is usually short (one day or less). However, antibody-bound antigens have a significantly longer half-life in plasma compared to when antigens are present alone. For this reason, administration of the existing neutralizing antibody results in an increased plasma antigen concentration. Such cases have been reported with several neutralizing antibodies that target soluble antigens including, for example, IL-6 (J. Immunotoxicol. (2005) 3, 131-139), amyloid (mAbs (2010) 2 (5 ), 1-13), MCP-1 (ARTHRITIS & RHEU-MATISM (2006) 54, 2387-2392), hepcidin (AAPS, J. (2010) 4, 646-657) and sIL-6 receptor (Blood (2008 ) 112 (10), 3959-64). Administration of existing neutralizing antibodies has been reported to increase the total antigen concentration in plasma to about 10 to 1,000 times (the level of increase varies depending on the antigen) to baseline. Here, the concentration of total antigen in plasma refers to a concentration as a total amount of antigen in plasma, that is, the sum of concentrations of antigens bound to antibody bound and unbound antibody. An increase in the concentration of antigen in the total plasma is undesirable for such pharmaceutical antibody agents that target a soluble antigen. The reason is that the concentration
[01982] [01982] Without being restricted to a particular theory, the following mechanism is an example of a mechanism that can cause the elimination of antigenic binding units from plasma by an antigen-binding molecule of the present invention, which contains (i) an Fc region and (ii) an antigen-binding domain from which antigen-binding activity varies depending on an ionic concentration condition, and where the antigen-binding molecule can form an immune complex that contains two or more of the antigen binding molecules and antigens that have two or more antigen binding units. When the antigenic binding unit is a unit (ie, a homomonomer), as in sIL-6R, two molecules (ie, two antigenic binding units) of antigens bind to a single antibody molecule that contains a divalent domain that binds to the antibody, and a complex of an anti-sIL-6R antibody molecule and two antigen molecules that contain two antigenic binding units is formed. Therefore, this type of antibody complex
[01983] [01983] On the other hand, when the antigen binding unit is two units as in human IgA, which is a dimer of a hetero complex of heavy chains and light chains, there are two units of epitopes to which the antigen binding domains will connect to the antigen binding unit. However, when a bivalent anti-IgA antibody (that is, the antigen-binding domains contained in an anti-IgA antibody molecule bind to the same epitope) binds to its antigen, IgA, it is believed that the binding of each one of the divalent antigen-binding domains contained in the unique anti-IgA antibody molecule to each of the two epitope units present in a single IgA molecule is difficult in view of the position of the epitopes. As a result, the separate anti-IgA antibody molecules are believed to bind to two antigenic binding units present in the two IgA molecules that bind to the divalent antigen-binding domains present in an anti-IgA molecule. unique anti-IgA body, and consequently, antibody-antigen complexes (immune complexes) containing at least four molecules (that is, two IgA molecules that are the antigen molecule and two anti-IgA antibody molecules that are binding molecules antigen) are formed.
[01984] [01984] When an antigen-binding molecule, such as an antibody that binds to an antigen molecule that contains two or more antigenic binding units, forms a large immune complex that is at least one tetramer, the immune complex can bind -
[01985] [01985] When the antigen binding molecule as an antibody that binds to an antigen molecule that contains two or more antigen binding units is an antibody that contains antigen binding domains from which the antigen binds varies depending on an ionic concentration condition such as pH- or Ca-dependent binding and which forms an antibody-antigen complex (immune complex) containing at least four molecules (two antigen molecules and two antibody molecules) in the plasma, once the immune complex is absorbed in the cells, the antigens dissociate from the antibodies in the endosomes where the ionic concentration conditions are different from those in the plasma. Therefore, the formation of an immune complex is dissolved in the endosomes of cells that have absorbed the immune complexes. Since the dissociated antigens cannot bind to FcRn in the endosomes, they are degraded after moving to the lysosomes. On the other hand, antibodies dissociated from the antigen are thought to be recycled to plasma after binding to FcRn in endosomes (Fig. 7).
[01986] [01986] As described above, if an antibody that contains a native IgG1 constant region against a multimeric antigen that contains two or more antigenic binding units and shows pH- or Ca-dependent binding can form a large immune complex and binding to FcγR, FcRn, complement receptors, and similarly avidly, it is believed that antigen elimination can only be selectively and enormously accelerated. It is believed that when GA2-IgG1 that binds to human IgA is administered, such large immune complexes are formed. In fact, as shown in Example 3, GA2-IgG1-FcγR (-) formed by introducing modifications of GA2-IgG1 that impair binding to mouse FcγR cannot substantially accelerate the elimination of human IgA as GA2-IgG1 when compared to human IgA only, and showed an equivalent level of elimination as human IgA only. Therefore, the reason that GA2-IgG1 could accelerate the elimination of human IgA is because the immune complex containing GA2-IgG1 and human IgA, which is a multimeric antigen that contains two or more antigenic binding units, avidly binds to FcγR and is rapidly absorbed in cells that express FcγR. IgA that dissociates from the immune complex in the endosomes of cells that absorbed the immune complex is degraded in lysosomes. At the same time, the IgA-dissociated antibody, which was bound to FcRn in endodesomes, is later recycled to plasma and can bind to IgA in plasma again. The elimination of human IgA in plasma is thought to be greatly accelerated in this way. A method using an amino acid variant of the Fc region that binds to FcRn in the neutral pH range is described in WO2011 / 122011 as a method to accelerate the elimination of antigens from plasma. The present invention is useful as a method to accelerate the plasma elimination of multimeric antigens that contain two or more antigenic binding units without using the aforementioned variants, and as shown in GA2-N434W, can further accelerate the elimination of multimé antigens - rich containing two or more antigenic plasma binding units in combination with the aforementioned variants. In addition, multimeric antigens that contain two or more antigenic binding units can be eliminated, in addition to plasma, interstitial fluid, synovial fluid, peritoneal fluid, pleural fluid, and pericardial fluid, while cells that come into contact with interstitial fluid, synovial fluid, peritoneal fluid, pleural fluid or pericardial fluid express FcγR or FcRn. A non-limiting modality of such cells includes immune cells and the like present in interstitial fluid, synovial fluid, peritoneal fluid, pleural fluid and pericardial fluid.
[01987] [01987] Specifically, the present invention also relates to pharmaceutical compositions comprising antigen binding molecules of the present invention, antigen binding molecules produced by altering the methods of the present invention or antigen binding molecules produced using the production methods of the present invention. The antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention are useful as pharmaceutical compositions since, when administered, they have the strong effect of reducing the concentration of antigen in the compared plasma with typical antigen-binding molecules, and exhibit the immune response in vivo, pharmacokinetics and others enhanced in animals administered with the molecules. The pharmaceutical compositions of the present invention can comprise pharmaceutically acceptable carriers.
[01988] [01988] In the present invention, pharmaceutical compositions generally refer to agents for the treatment or for the prevention or for the testing and diagnosis of diseases.
[01989] [01989] The pharmaceutical compositions of the present invention can be formulated using 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 unit dose required in generally approved drug manufacturing practice by combining, appropriately, with pharmacologically acceptable vehicles or means, specifically with sterile water, physiological saline. , vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder or similar. In such formulations, the amount of active ingredient is adjusted to obtain an appropriate amount in a predetermined range.
[01990] [01990] Sterile compositions for injection can be formulated using vehicles such as distilled water for injection, according to standard formulation practice.
[01991] [01991] Aqueous solutions for injection include, for example, physiological saline and isotonic solutions containing dextrose or other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride). It is also possible to use in combination suitable solubilizers, for example, alcohols (ethanol and the like), polyalcohols (propylene glycol, polyethylene glycol and the like), non-ionic surfactants (polysorbate 80 (TM), HCO-50 and the like).
[01992] [01992] Oils include sesame oil and soy oils. Benzyl benzoate and / or benzyl alcohol can be used in combination as solubilizers. It is also possible to combine buffers (for example, phosphate buffer and sodium acetate buffer), softening agents (for example, procaine hydrochloride), stabilizers (for example,
[01993] [01993] The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, compositions in dosage form for injections, transnasal administration, transpulmonary administration or transdermal administration are administered. For example, they can be administered systemically or locally through intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or similar.
[01994] [01994] The methods of administration can be appropriately selected considering the age and symptoms of the patient. A dose of a pharmaceutical composition containing an antigen binding molecule can be, for example, from 0.0001 to 1,000 mg / kg for each administration. Alternatively, the dose can be, for example, from 0.001 to 100,000 mg per patient. However, the present invention is not limited by the numerical values described above. Doses and methods of administration vary depending on weight, age, patient's symptoms and the like. Those skilled in the art can determine appropriate doses and methods of administration considering the factors described above.
[01995] [01995] Furthermore, the present invention provides kits for use in the methods of the present invention that comprise at least one antigen binding molecule of the present invention. In addition to the above, pharmaceutically acceptable carriers, means, instruction manuals describing the use of the method and the like can be packaged in the kits.
[01996] [01996] In addition, the present invention relates to pharmaceutical products for the elimination, of plasma, of complexes that contain two or more antigenic binding units and two or more antigen-binding molecules present in the plasma, which contain the antigen-binding molecules of the present invention or the antigen-binding molecules produced by the production methods of the present invention as an active ingredient.
[01997] [01997] The present invention relates to methods for treating a disease, which includes the administration in individuals (patients, humans, etc.) of the antigen binding molecules of the present invention or of the antigen binding molecules produced by the production methods of the present invention. A non-limiting example of the disease includes cancer and inflammatory diseases.
[01998] [01998] The present invention also relates to the use of the antigen binding molecules of the present invention or the antigen binding molecules produced by the production methods of the present invention in the production of a pharmaceutical agent to eliminate plasma complexes which contain two or more antigenic binding units and two or more antigen-binding molecules present in the plasma.
[01999] [01999] The present invention also relates to the use of the antigen-binding molecules of the present invention or the antigen-binding molecules produced by the production methods of the present invention for eliminating, from plasma, complexes containing two or more antigen binding units and two or more antigen binding molecules present in the plasma.
[02000] [02000] Furthermore, the present invention relates to antigen binding molecules of the present invention and antigen binding molecules produced by the production methods of the present invention for use in the methods of the present invention.
[02001] [02001] The amino acids contained in the amino acid sequences of the present invention can be modified after translation (for example, the modification of an N-terminal glutamine into a pyro-glutamic acid via pyroglutamylation is well known to experts.
[02002] [02002] All prior art documents cited in this specification are incorporated herein by reference. EXAMPLES
[02003] [02003] Below, the present invention will be specifically described with reference to the examples, however, it should not be interpreted as being limited thereto. Example 1 Preparation of antigen-binding molecules that bind to human IgA in a calcium-dependent manner Preparation of human IgA (hIgA)
[02004] [02004] Human IgA (hereinafter also abbreviated as "hIgA") was prepared as an antigen using the following recombinant techniques. HIgA was expressed by culturing host cells that carry recombinant vectors inserted with H (WT) -IgA1 (SEQ ID NO: 49) and L (WT) (SEQ ID NO: 50) and purified by a method known to those skilled in the art. technique using ion exchange chromatography and gel filtration chromatography. Antigen-binding molecules with calcium-dependent binding
[02005] [02005] H54 / L28-IgG1 described in International Publication WO No. 2009/125825 is a humanized IL-6 anti-receptor antibody. Fv4-IgG1 is a humanized IL-6 anti-receptor antibody that results in H54 / L28-IgG1 conferring with the property of binding to the soluble human IL-6 receptor in a pH dependent manner (i.e., binding under condition neutral and dissociation under acidic condition). The in vivo test described in International Publication WO No. 2009/125825 using mice demonstrated that elimination of the soluble human IL-6 receptor is greatly accelerated in a group administered with a mixture of Fv4-IgG1 and soluble human IL-6 receptor as the antigen when compared to a group administered a mixture of H54 / L28-IgG1 and human IL-6 receptor soluble as antigen.
[02006] [02006] The soluble human IL-6 receptor bound to a general antibody that binds to the soluble human IL-6 receptor is recycled to plasma along with the antibody via FcRn. However, an antibody that binds to the soluble human IL-6 receptor in a pH-dependent manner dissociates under acidic conditions in the endosome of the soluble human IL-6 receptor that was bound to the antibody. The dissociated soluble human IL-6 receptor is degraded in the lysosome. Thus, this can greatly accelerate the elimination of soluble human IL-6 receptor from the plasma. In addition, the antibody that binds to the soluble human IL-6 receptor in a pH-dependent manner is recycled to plasma via FcRn after it has dissociated itself from the soluble human IL-6 receptor, so that the recycled antibody can bind to a soluble human IL-6 receptor again. By repeating this cycle (the antibody that has bound to the antigens is absorbed into the cells> the antigens are dissociated from the antibody> the antigens are degraded and the antibody is recycled back to the plasma), a single antibody molecule can bind repeatedly to the soluble human IL-6 receptors multiple times (Fig. 1).
[02007] [02007] However, as described in International Publication WO No. 2009/125825, H54 / L28-IgG1 is a humanized IL-6 anti-receptor antibody and Fv4-IgG1 is a humanized IL-6 anti-receptor antibody that results from H54 / L28-IgG1 with the property of binding to the soluble human IL-6 receptor in a pH-dependent manner (ie binding under neutral condition and dissociation under acidic condition). Fv4-IgG1-v2 is a humanized IL-6 anti-receptor antibody in which FcRn binding is increased over Fv4-IgG1 under neutral conditions. The in vivo test described in the International Publication
[02008] [02008] In the actions of antibodies that bind to antigens in a pH dependent manner shown in Figs. 1 and 2, the property of antibodies to bind strongly to antigens in plasma and to detach from antigens on endosomes based on the environmental difference between plasma and endosomes, that is, pH difference (pH 7.4 in plasma; pH 6.0 in endosomes), is used. The properties of environmental factors in plasma and endosomes as well as the degree of their difference are important for using such differences in the ability to bind to the antigen of the antibody that binds in a pH-dependent manner in plasma and endosomes. A difference in pH corresponds to a difference in the concentration of protons. Specifically, the concentration of protons in the plasma (pH 7.4) is approximately 40 nM, while the concentration of protons in the endosome (pH 6.0) is approximately 1,000 nM; thus, the concentration of protons that is considered an environmental factor in the plasma and endosome differs up to 25 times.
[02009] [02009] In addition, the present inventors understood that, in order to achieve the actions illustrated in Figures 1 and 2 by different modalities or to achieve these modalities in combination, it would be beneficial to use an antibody that binds antigens depending on a fa - environmental tor with a large difference in plasma and endosome, in addition to the difference in the concentration of protons. In this way, the inventors sought an environmental factor whose concentration is considerably different between the plasma and the endosome and as a result, they found calcium. The ionized calcium concentration is approximately 1.1 mM to 1.3 mM in the plasma and approximately 3 M in the endosome; thus, the difference in calcium ion concentration, which is considered to be an environmental factor in plasma and endosome, is approximately 400 times and was found to be greater than the difference in proton concentration (25 times). Thus, it was considered that, using an antibody that binds to an antigen under a condition of high calcium concentration (1.1 mM to 1.3 mM) and dissociates from the antigen under a condition of low concentration of calcium (3 M), the antibody can dissociate from the antigen on the endosome at an equivalent or higher level compared to an antibody that binds to the antigen in a pH-dependent manner. Expression and purification of antibodies that bind to hIgA
[02010] [02010] GA1-IgG1 (heavy chain SEQ ID NO: 37; light chain SEQ ID NO: 38) and GA2-IgG1 (heavy chain SEQ ID NO: 39; SEQ ID NO: light chain 40) are antibodies that bind the hIga. The DNA sequences encoding GA1-IgG1 (heavy chain SEQ ID NO: 37; light chain SEQ ID NO: 38) and GA2-IgG1 (heavy chain SEQ ID NO: 39; SEQ ID NO: light chain 40) they were inserted into animal cell expression plasmids by a method known to those skilled in the art. The antibodies were expressed by the method described below. Cells from the lineage derived from the human fetal renal cell FreeStyle 293-F (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen). The cell suspension was seeded
[02011] [02011] Isolated antibodies as described in 1-3 were evaluated for their hIgA binding activity (KD dissociation constant (M)) using Biacore T200 (GE Healthcare). Running pads used in the measure were:
[02012] [02012] 0.05% tween20 / 20 mmol / L ACES / 150 mmol / L NaCl (pH 7.4 or 5.8) containing 3 M or 1.2 mM CaCl2; and
[02013] [02013] 0.05% tween20 / 20 mmol / L ACES / 150 mmol / L NaCl (pH 8.0) containing 0.1 M or 10 mM CaCl2.
[02014] [02014] The antibody was allowed to bind to the Sensor CM5 chip (GE Healthcare) immobilized with an adequate amount of recombinant Protein A / G (Thermo Scientific) by the method of amino coupling. Then, an appropriate concentration of hIgA (described in 1-1) was injected as an analyte to allow interaction with the antibody on the sensor chip. The measurement was performed at 37 ° C. After measurement, glycine-HCl 10 mmol / L (pH 1.5) was injected to regenerate the sensor chip. The dissociation constant KD (M) was calculated from the measurement result by analyzing the curve fit and analyzing the equilibrium parameter using the Biacore T200 Evaluation Program (GE Healthcare). The result and the obtained sensorgrams are shown in Table 7 and Fig. 3, respectively. It was revealed that GA2-IgG1 bound strongly to hIgA at a concentration of Ca2 + of 1.2 mM whereas the antibody weakly bound to hIgA at a concentration of Ca2 + of 3 M. In addition, at a concentration of Ca2 + of 1.2 mM, GA2-IgG1 was shown to bind to human IgA strongly at pH 7.4 but weakly at pH 5.8. More specifically, GA2-IgG1 has been shown to bind to human IgA in a pH and calcium dependent manner. Table 7 Name of Conditions Adjustment ka kd KD [M] antibody GA1-IgG1 pH 8.0 Ca 10 mM Binding model 1: 1 1.2E + 06 1.2E-01 1.0E-07 pH 8.0 Ca 0 , 1 µM Connection model 1: 1 1.1E + 06 2.4E-01 2.2E-07 pH 7.4 Ca 1.2 mM Connection model 1: 1 5.7E + 05 8.4E-01 1 , 5E-07 pH 7.4 Ca 3 µM Connection model 1: 1 6.4E + 05 1.2E-01 1.9E-07 pH 5.8 Ca 1.2 mM Connection model 1: 1 6.8E +05 9.9E-02 1.4E-07 pH 5.8 Ca 3 µM Connection model 1: 1 7.1E + 05 1.1E-01 1.5E-07 GA2-IgG1 pH 7.4 Ca 1, 2 mM Connection model 1: 1 4.0E + 05 1.6E-02 3.9E-08 pH 7.4 Ca 3 µM Permanent affinity - 6.7E-06 pH 5.8 Ca 1.2 mM Affinity in permanent state - 4.0E-06 pH 5.8 Ca 13 µM Affinity in permanent state - 5.0E-06 [Example 2] Preparation of modified antibodies that bind to HIGA in a calcium-dependent manner
[02015] [02015] Next, to further increase antigen (hI-gA) elimination from plasma, GA2-N434W (heavy chain SEQ ID NO: 41; SEQ ID NO: light chain 40) was built introducing the amino acid substitution N434W in GA2-IgG1, which binds hIgA in a calcium-dependent manner, to potentiate binding to mouse FcRn at pH 7.4. In addition, GA2-FcγR (-) (heavy chain SEQ ID NO: 42; light chain SEQ ID NO: 40) was constructed by introducing amino acid substitutions L235R and S239K in GA2-IgG1 to eliminate
[02016] [02016] In vivo kinetics of hIgA and anti-hIgA antibody was evaluated after administration of hIgA (human IgA; prepared as described in Example 1) alone or in combination with an anti-hIgA antibody to normal mice (C57BL / 6J mice; Charles River Japan). A hIgA solution (80 g / ml) or a mixture of hIgA and anti-hIgA antibody was administered once in a dose of 10 ml / kg via the caudal vein. The anti-hIgA antibodies used were GA1- IgG1, GA2-IgG1, GA2-N434W and GA2-FcγR (-) described above.
[02017] [02017] In each mixture, the hIgA concentration was 80 g / mL. However, the concentration of anti-hIgA antibody varied depending on the antibody affinity for hIgA. GA1-IgG1 was prepared at 10 mg / ml; GA2-IgG1 at 2.69 mg / ml; GA2-N434W in 1 mg / ml; and GA2- FcγR (-) at 2.69 mg / ml. Under the conditions described above, most hIgA is predicted to bind to the antibody since the anti-hIgA antibody is present sufficiently in excess over hIgA. Blood was collected from the mice five minutes, seven hours, one day, two days, three days, and seven days after administration. The collected blood was immediately centrifuged at 12,000 rpm and 4 ° C for 15 minutes to obtain the plasma. The separated plasma was stored in a refrigerator at -20 ° C or below until measurement. 3-2. Determination of plasma anti-hIgA antibody concentration in normal mice by ELISA
[02018] [02018] Anti-hIgA antibody concentrations in mouse plasma were determined by ELISA. First, immobilized human Anti-IgG plates were prepared by aliquoting Fragment F (ab ') 2 of the anti-human IgG antibody (specific γ chain) (SIGMA) to each of the wells of the Nunc-Immuno plate, MaxiSorp (Nalge nunc International) and allowing the plates to rest at 4 ° C overnight. The standard curve samples of anti-hIgA antibody prepared as standard solutions in plasma concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0.07813 g / mL and Test samples prepared by diluting mouse plasma samples 100 times or more were aliquoted on immobilized anti-human IgG plates, and then the plates were incubated at 25 ° C for one hour. Next, human anti-Ig G conjugate (specific γ chain) Biotin (BIOT) (Southern Biotechnology Associations Inc.) was aliquoted in each of the wells of the plates, and then the plates were incubated at 25 ° C for an hour. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added to each well of the plates, after which the plates were incubated at 25 ° C for one hour. The chromium gene reaction using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate was quenched with 1N sulfuric acid (Showa Chemistry), and then the reaction mixture in each well was measured using a reader microplate to measure absorbance at 450 nm. The concentration of anti-hIgA antibody in the mouse plasma was calculated from the absorbance of the standard curve using the SOFTmax PRO analysis program (Molecular Devices). The time course of plasma antibody concentrations of GA1-IgG1, GA2-IgG1, GA2-N434W, and GA2-FcγR (-) in normal mice, which were determined by the method described above, is shown in Fig. 4. 3- 3. Determination of hIgA concentration in plasma by ELISA
[02019] [02019] Mouse plasma hIgA concentrations were measured by ELISA. Firstly, immobilized anti-human IgA plates were prepared by aliquoting Goat anti-human IgA antibody (BETHYL) in each of the Nunc-Immuno, MaxiSorp (Nalge nunc International) wells and leaving the plates stand at 4 ° C overnight. The standard curve samples of hIgA prepared as standard solutions in plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 g / mL and test samples prepared by diluting mouse plasma samples 100 times or more, were aliquoted at 100 L / well on the anti-human IgA-mobilized plates, after which hsIL-6R 500 ng / mL was added at 200 L / well. The resulting plates were allowed to rest at room temperature for one hour. Then, after adding Biotinylated Human Anti-IL-6R (R&D) Antibody to each well of the plates, the plates were incubated at room temperature for one hour. Then, after aliquoting Streptavidin-PolyHRP80 (Steerospecific Detection Technologies) in each of the wells of the plates, the plates were incubated at room temperature for one hour. The chromogenic reaction using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate was quenched with 1N sulfuric acid (Showa Chemical), and then the reaction mixture in each well was measured using a microplate to measure absorbance at 450 nm. The concentration in mouse plasma was calculated from the absorbance of the standard curve using the SOFTmax PRO analysis program (Molecular Devices). The time course of plasma hIgA concentrations in normal mice after intravenous administration, as determined by the method described above, is shown in Fig. 5.
[02020] [02020] The result showed that the elimination of hIgA was delayed when hIgA was administered in combination with GA1-IgG1, an antibody that exhibits poor Ca-dependent binding (the degree of dependence is low) when compared to when hIGA was administered alone. In contrast, when hIgA was administered in combination with GA2-IgG1 whose Ca-dependent binding activity is 100 or more times greater, the elimination of hIgA was considerably accelerated when compared to when hIgA was administered alone. The concentration of free hIgA in the plasma was determined from the plasma antibody concentration shown in Fig. 4, the plasma hIgA concentration shown in Fig. 5, and the KD value of each antibody shown in Table 7. The result is shown in Fig. 6. As shown in Fig. 6, the concentration of free antigen (hIgA) in the group administered with GA2-IgG1 that binds to hIgA in a calcium-dependent manner was lower than that in the group administered with GA1-IgG1. This demonstrates that the antigen without antigen-binding molecules (hIgA) (unbound form of the antibody) can be reduced by accelerating antigen elimination using antibodies that bind in a calcium-dependent manner. In addition, GA2-N434W with pH 7.4-enhanced FcRn binding accelerated antigen elimination more than GA2-IgG1 did, and seven hours after administration, the plasma hIgA concentration was below the detection limit. Example 4 Preparation of pH 4-1 dependent anti-IgE antibody. Preparation of anti-human IgE antibody
[02021] [02021] Prepare pH-dependent anti-human IgE antibodies, human IgE (heavy chain SEQ ID NO: 43; SEQ ID NO: light chain 44) (the variable region is derived from a human anti-glypican3 antibody) as an antigen was expressed using FreeStyle293 (Life Technologies). Human IgE was prepared by purifying human IgE expressed using a conventional chromatographic method known to those skilled in the art.
[02022] [02022] An antibody that binds to human IgE in a pH-dependent manner and forms a large immune complex consisting of two or more anti-IgE antibody molecules and two or more IgE molecules has been selected from several antibodies obtained. The selected anti-human IgE antibody was expressed using the human IgG1 heavy chain constant region and the human light chain constant region, and then purified. The antibody produced was called clone 278 (heavy chain SEQ ID NO: 45; light chain SEQ ID NO: 46). 4-2. Evaluation of anti-human IgE antibodies for their binding activity and pH-dependent binding activity
[02023] [02023] Antibodies capable of dissociating antigens within the endosome can be created not only by designing them to bind to antigens in a pH-dependent manner, but also by designing them to bind to antigens in a manner dependent on Thus, clone 278 and the Xolair control (omalizumab; Novartis) whose IgE binding activity does not depend on pH / Ca were evaluated for their pH dependence and the pH / Ca dependence on human IgE binding activity (hIgE).
[02024] [02024] More specifically, the hIgE binding activities (KD dissociation constant (M)) of clone 278 and Xolair were evaluated using Biacore T200 (GE Healthcare). The running buffers used in the trial were:
[02025] [02025] CaCl2 1.2 mmol / l / 0.05% tween20, ACES 20 mmol / l, NaCl 150 mmol / l, pH 7.4;
[02026] [02026] CaCl2 1.2 mmol / l / 0.05% tween20, ACES 20 mmol / l, NaCl 150 mmol / l, pH 5.8; and
[02027] [02027] CaCl2 3 mol / l / 0.05% tween20, ACES 20 mmol / l, NaCl 150 mmol / l, pH 5.8.
[02028] [02028] The chemically synthesized peptide having a sequence derived from human glypican 3 protein (SEQ ID NO: 47) whose C-terminal Lys is biotinylated (hereinafter abbreviated "biotinylated GPC3 peptide") was added in an appropriate amount and immobilized to the Sensor SA chip (GE Healthcare) based on the affinity between biotin and streptavidin. Human IgE was immobilized on the chip by injecting it at an appropriate concentration to be captured by the biotinylated GPC3 peptide. As an analyte, clone 278 was injected at an appropriate concentration and allowed to interact with human IgE on the sensor chip. Then, glycine-HCl 10 mmol / L (pH 1.5) was injected to regenerate the sensor chip. The interaction was always measured at 37 ° C. The measurement result was analyzed by adjusting the curve using the Biacore T200 Evaluation Program (GE Healthcare) to calculate the association rate constant ka (1 / Ms) and dissociation rate constant kd (1 / s). The dissociation constant KD (M) was calculated from the constants described above. In addition, the proportions of KD in each antibody under the conditions of [H 5.8, Ca 1.2 mM] to [H 7.4, Ca 1.2 mM] was calculated to assess pH dependent binding, while the KD proportions in each antibody under the conditions of [H 5.8, Ca 3 M] to [H 7.4, Ca 1.2 mM] were calculated to assess the pH / Ca dependent binding. The result is shown in Table 8.
[02029] [02029] If the clone 278 forms large immune complexes of 2: 2 or more with human IgE under neutral condition (pH 7.4) and if the immune complexes dissociate under acidic condition (pH 5.8) were evaluated using gel filtration chromatography. Clone 278 that was dialyzed against 100 mM NaCl was diluted using a Tris-HCl mM, 150 mM NaCl, 1.2 mM CaCl2, pH 7.4 buffer for samples under neutral condition and using a Bis-tris-HCl 20 buffer mM, 150 mM NaCl, 3 M CaCl2, pH 5.8 for samples under acidic condition. The mixtures in which hIgE (Asp6) 100 g / mL (0.60 M) which is human IgE (prepared in Example 5) and clone 278 were mixed in a 1: 1 or 1: 6 molar ratio were left for two hours or more at room temperature or in a self-sampler at 25 ° C and then analyzed by gel filtration chromatography. A mobile phase of 20 mM Tris-HCl, 300 mM NaCl, 1.2 mM CaCl2, pH 7.4 was used under neutral condition and a mobile phase of 20 mM Bis-tris-HCl, 300 mM NaCl, 3 M CaCl2, pH 5.8 was used under acidic conditions. The analyzes were performed using a G4000SWxl (TOSOH) column and under conditions of a flow rate of 0.5 mL / min and 25 ° C. The results are shown in Fig. 9. As shown in Fig. 9, it was confirmed that clone 278 and human IgE formed large immune complexes con-
[02030] [02030] From these results, clone 278 was considered to be able to accelerate the elimination of human IgE, similarly to the anti-IgA antibody GA2-IgG1 mentioned above. Example 5 In vivo evaluation of clone 278 and Xolair 5-1. Preparation of human IgE (hIgE (Asp6)) for in vivo evaluation
[02031] [02031] hIgE (Asp6) (the variable region is derived from a human antiglipican3 antibody), which is human IgE for in vivo evaluation consisting of a heavy chain (SEQ ID NO: 48) and a light chain (SEQ ID NO : 44), was produced by the same method as that described in Example 1. hIgE (Asp6) is a modified molecule that results from asparagine alteration by aspartic acid in six N-linked glycosylation sites in human IgE so that heterogeneity the human IgE N-linked sugar chain is not affected by time-dependent changes in the plasma concentration of human IgE as an antigen. 5-2. Evaluation of clone 278 and Xolair for the effect of accelerating the elimination of human IgE using normal mice
[02032] [02032] In vivo kinetics of hIgE (Asp6) and anti-human IgE antibody was evaluated after administration of hIgE (Asp6) alone or in combination with an anti-hIgE antibody (clone 278 and Xolair) to C57BL / 6J mice ( Charles River Japan). A solution of hIgE (Asp6) (20 - g / mL) or a mixture of hIgE (Asp6) and anti-human IgE antibody (concentrations are shown in Table 9) was administered once in a dose of 10 mL / kg via caudal vein. Under the conditions described above, hIgE (Asp6) is predicted to bind almost completely to the antibody since each antibody is sufficiently present in excess on hIgE (Asp6). Blood was collected from the mice five minutes, two hours, seven hours, one day, two days, four or five days, seven days, 14 days, 21 days, and 28 days after administration. The blood collected was immediately centrifuged at 15,000 rpm and 4 ° C for 5 minutes to obtain the plasma. The separated plasma was stored in a refrigerator at -20 ° C or below until measurement. Table 9 Antibody hIgE concentration (Asp6) Concentration of anti-hIgE anti-hIgE antibody in the administered solution (g / mL) in the administered solution (g / mL) Clone 278 20 100 Xolair 20 308 5-3. Determination of plasma hIgE concentration (Asp6) in normal mice
[02033] [02033] Concentrations of hIgE (Asp6) in mouse plasma were determined by ELISA. Standard curve samples were prepared at plasma concentrations of 192, 96, 48, 24, 12, 6, and 3 ng / mL. Xolair (Novartis) was added at 10 g / mL to standard curve samples and mouse plasma assay samples to match the hIgE immune complex (Asp6) and anti-hIgE antibody. After minutes of incubation at room temperature, standard curve samples and mouse plasma assay samples were aliquoted on immunoplates (MABTECH) immobilized with anti-human IgE antibody or immunoplates (Nunc F96 MicroWell Plate (Nalge nunc International)) immobilized with the anti-human IgE antibody (clone 107; MABTECH). The plates were allowed to stand at room temperature for two hours or at 4 ° C during the night. So, human GPC3 main protein (SEQ ID NO: 51), anti-
[02034] [02034] Anti-hIgE antibody concentrations in mouse plasma were determined by ELISA. Standard curve samples were prepared in plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 g / mL. hIgE (Asp6) was added at 1 g / mL to standard curve samples and mouse plasma assay samples to match the hIgE (Asp6) immune complex and anti-hIgE antibody. After 30 minutes of incubation at room temperature, standard curve samples and mouse plasma assay samples were aliquoted on immunoplates (Nunc-Immuno Plate, MaxiSorp (Nalge nunc International)) immobilized with Anti-
[02035] [02035] The result showed that the elimination of human IgE was delayed when human IgE was administered in combination with Xolair, an anti-IgE control antibody, when compared to when human IgE was administered alone. However, the elimination of human IgE was markedly accelerated when administered in combination with clone 278, which has a strong pH-dependent human IgE binding activity when compared to when human IgE was administered alone. Specifically, it has been shown that not only in the case of IgA, but also in the case of IgE, antigen ejaculation has been accelerated by administering an antibody that forms a large immune complex when compared to when the antigen is administered alone. Example 6 Preparation of antibody variants showing calcium-dependent hIgA binding
[02036] [02036] Next, for the purpose of further increasing the elimination of antigen (hIgA) from plasma, Leu at position 328 (EU numbering) in GA2-IgG1, which shows calcium-dependent hIgA binding, was replaced by Tyr for increase its binding to mouse FcγR, producing GA2-F1087 (heavy chain SEQ ID NO: 52). A DNA sequence encoding GA2-F1087 (heavy chain SEQ ID NO: 52, and light chain SEQ ID NO: 40) was inserted into an animal expression plasmid by a method known to those skilled in the art. These antibody variants were expressed using the plasmid according to the aforementioned method, and their concentrations were determined after purification. Antibodies containing this modification showed very increased binding to mouse FcγR as shown in Reference Example 5. Example 7 Evaluation of the effect on plasma antigen retention in normal mice administered with hIgA-dependent antibodies of Ca 7-1. in vivo testing using normal mice
[02037] [02037] Normal mice (mouse C57BL / 6J; Charles River Japan) were administered with hIgA (human IgA: produced in Example (1-1)) alone or co-administered with hIgA and an anti-hIgA antibody, and then evaluated for in vivo dynamics of hIgA and the anti-hIgA antibody. A solution of hIgA (80 g / ml) or mixed solution of hIgA and an anti-hIgA antibody was administered once in a dose of 10 ml / kg in the tail vein. The anti-hIgA antibodies used were GA2-IgG1 and GA2-F1087 described above.
[02038] [02038] The hIgA concentration was 80 g / ml in all cases, and the anti-hIgA antibody concentration was 2.69 mg / ml in the mixed solutions. The anti-hIgA antibody was sufficiently present in excess of hIgA, and therefore it was considered that almost all hIgA was bound by the antibody. From the group administered with GA-
[02039] [02039] The concentration of anti-hIgA antibody in mouse plasma was determined by ELISA. First, Fragment of the anti-human IgG Antibody (specific γ chain) F (ab ') 2 (SIGMA) was dispensed into each of the wells of Nunc-Immuno Plates, MaxiSorp (Nalge nunc International), and left to stand overnight at 4 ° C to prepare immobilized anti-human IgG plates. Standard samples of anti-hIgA antibodies to be used as calibration curve samples were prepared in plasma concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0.007813 g / ml; and mouse plasma assay samples diluted 100 times or more were prepared and aliquoted on the above immobilized Human Anti-IgG plates, and then the plates were incubated at 25 ° C for one hour. Then, Goat anti-human IgG Conjugate (specific γ-chain) Biotin (BIOT) (Southern Biotechnology Associats Inc.) was dispensed in each of the wells of the plates mentioned above, and then the plates were incubated to leave the reaction occurs at 25 ° C for one hour. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was dispensed into each of the wells of the aforementioned plates, and then the plates were incubated to allow the reaction to occur at 25 ° C for one hour. A re-
[02040] [02040] The concentration of hIgA in mouse plasma was determined by ELISA. First, Goat's anti-human IgA antibody (BETHYL) was aliquoted in each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International). The plate was left to stand at 4 ° C overnight to prepare an immobilized anti-human IgA plate. The standard hIgA samples to be used as calibration curve samples were prepared in plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 g / mL. The mouse plasma test samples were prepared at 100-fold dilution or greater. 200 l of hsIL-6R 500 ng / ml were added to 100 L of each of the calibration curve samples and plasma samples. The resulting mixtures were allowed to rest at room temperature for one hour, and then the mixed solutions were aliquoted in 100 l on the above immobilized human anti-IgA plate; and this was left to stand at room temperature for an hour. Next, a bi-tinylated human Anti-IL-6R (R&D) antibody was aliquoted in each of the wells of the aforementioned plate, and then the plate was incubated at room temperature for one hour to allow the reaction to occur. In addition, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was aliquoted in each well of the aforementioned plate, and the plate was incubated at room temperature for one hour to allow the reaction to occur. A color development reaction was performed using the TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After the reaction was quenched with 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction solutions in each well at 450 nm was measured using a microplate reader. Mouse plasma concentrations were calculated based on the absorbance of the calibration curve using the SOFTmax PRO analytical program (Molecular Devices). The time course changes in plasma hIgA concentration determined by this method in normal mice submitted to intravenous administration are shown in Fig. 13.
[02041] [02041] The results showed that when hIgA was co-administered to mice with GA2-IgG1, which exhibits Ca-dependent binding activity of 100 times or more, the elimination of hIgA was accelerated compared to when hIgA was administered alone . In addition, when hIgA and GA2-F1087, which increased FcγR binding, were administered to mice, the plasma hIgA concentration was reduced below the measurement range (0.006 g / mL or more) one day after administration, and the elimination of plasma hIgA was significantly accelerated compared to when GA-IgG1 was administered to mice. The aforementioned indicates that in mice administered an anti-hIgA and hIgA antibody forming an immune complex, the effect of an antibody with
[02042] [02042] Next, in order to further increase the elimination of antigen (human IgE) from plasma, Leu at position 328 (EU numbering) on 278-IgG1, which shows pH-dependent human IgE binding, was replaced by Tyr to increase its binding to mouse FcγR, producing 278-F1087 (heavy chain SEQ ID NO: 53 and light chain SEQ ID NO: 46). A DNA sequence encoding 278-F1087 was inserted into an animal expression plasmid by a method known to those skilled in the art. Antibody variants were expressed according to the aforementioned method using animal cells introduced with the plasmid, and their concentrations were determined after purification. Example 9 In vivo evaluation of 278-IgG1 9-1. Preparation of human IgE (hIgE (Asp6)) for in vivo evaluation
[02043] [02043] A method similar to the method described in Example 5-1 was used to prepare hIgE (Asp6) (in which the variable region is that of human anti-Glypican antibody 3), which is human IgE for use in in vivo evaluation. Human IgE (Asp6) is a molecule in which asparagine has been modified to aspartic acid at six human IgE N-glycosylation sites, so that the heterogeneity of human IgE N-glycosides is not affected by the change in concentration Plasma of the human IgE antigen. 9-2. Verification of the effect of accelerated removal of human hIgE from normal mouse plasma administered with clone 278
[02044] [02044] It was demonstrated in Example 7 that the plasma concentration
[02045] [02045] C57BL / 6J mice (Charles River Japan) were administered with hIgE (Asp6) alone or co-administered with hIgE (Asp6) and an anti-hIgE antibody (278-IgG1 or 278-F1087), and then evaluated for in vivo dynamics of hIgE (Asp6) and the anti-human IgE antibody. A solution of hIgE (Asp6) (20 g / ml) or a mixed solution of hIgE (Asp6) and an anti-human IgE antibody (all antibody concentrations were adjusted to be the same concentrations as shown in the Table 10) was administered once in 10 mL / kg in the tail vein. In this case, since each antibody was sufficiently present in excess in relation to hIgE (Asp6), it was considered that almost all hIgE (Asp6) was bound by the antibody. Blood was collected from the mice 5 minutes, 2 hours, 7 hours, 1 day, 2 days, 4 days, 5 days, 7 days, 14 days, and 21 days after administration in the group administered with clone 278 (278- IgG1) . Blood was collected from the mice 5 minutes, 30 minutes, 1 hour, 2 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after administration in the group administered with 278-F1087. The blood samples collected were immediately centrifuged at 4 ° C and 15,000 rpm for minutes to obtain plasma samples. The separated plasma samples were stored in a refrigerator set at -20 ° C or below until the test was performed. Table 10 Antibody concentration of hIgE (Asp6) in the concentration of anti-hIgE antibody nti-hIgE solution administered (g / mL) in the solution administered (g / mL) 278-IgG1 20 100 278-F1087 20 100 9-3. Determination of the concentration of anti-human IgE antibody in normal mouse plasma
[02046] [02046] The concentration of anti-hIgE antibody in mouse plasma was determined by ELISA. The calibration curve samples were prepared in plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 g / mL. To make the immune complex formed between hIgE (Asp6) and the homogeneous anti-hIgE antibody, hIgE (Asp6) was added at 1 g / mL to the calibration curve samples and mouse plasma test samples. ; and the group administered with 278-hIgG1 and the corresponding calibration curve samples were left to stand at room temperature for 30 minutes. In addition, the group administered with 278-F1087 and the corresponding calibration curve samples were stirred overnight at 37 ° C. Calibration curve samples and mouse plasma analysis samples that were allowed to stand or shake were aliquoted on the immobilized human Kappa Anti-Light Chain Antibody immunoplate (Bethyl Laboratories) (Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) ), and were left to stand / stir at room temperature for two hours (samples from the group administered with 278-F1087 and samples from the 278-F1087 calibration curve) or left overnight at 4 ° C (samples from the group administered with 278-hIgG1 and samples of the 278-hIgG1 calibration curve). Then, the Secondary anti-human rabbit IgG (Fc) antibody, Biotin conjugate (Pierce Biotechnology) and Streptavidin-
[02047] [02047] The concentration of hIgE (Asp6) in mouse plasma was determined by ELISA. The calibration curve samples were prepared at plasma concentrations of 192, 96, 48, 24, 12, 6, and 3 ng / mL. To make the immune complex formed between hIgE (Asp6) and the homogeneous anti-hIgE antibody, Xolair (Novartis) was added at 10 g / mL to the calibration curve samples and the mouse plasma test samples from the group administered with 278 -hIgG1, and the samples were left to stand at room temperature for 30 minutes. In the group administered with 278-F1087, 278-F1022 (heavy chain SEQ ID NO: 54 and light chain SEQ ID NO: 46, prepared in a similar manner to Example or 278-F760 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 46, prepared in a similar manner to Example 8) was added at 20 g / ml, and then mixed at 37 ° C for 60 hours. The mouse plasma assay samples were aliquoted on the immobilized immunoplate of human anti-IgE (MABTECH) or immobilized human anti-IgE immunoplate (clone 107, MABTECH) (Nunc F96 MicroWell Plate (Nalge nunc International)), and the samples were allowed to stand or stir at room temperature for two hours, or were left to stand overnight at 4 ° C. Then, the human GPC3 main protein a (SEQ ID NO: 51), an anti-GPC3 antibody (internally prepared) biotinylated with NHS-PEG4-Biotin (Thermo Fisher Scientific), and Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was each reacted sequentially by one hour. The concentration in the mouse plasma was determined by the method of carrying out a color development reaction using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate, stopping the reaction with 1 N sulfuric acid (Showa Chemical), and then measuring the color development by the absorbance at 450 nm measured in a microplate reader; or the method of performing a color development reaction using SuperSignal (r) ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) as a substrate, and then measuring luminescence intensity in a microplate reader. The concentrations in the mouse plasma were calculated from the luminescence intensity or based on the absorbance of the calibration curve using the analytical program SOFT-max PRO (Molecular Devices). The time course changes in plasma hIgE concentration (Asp6) after intravenous administration determined by this method are shown in Fig. 15.
[02048] [02048] As a result, unlike elimination in the case of human IgE alone, the elimination of human IgE was accelerated in mice co-administered with human IgE and 278-IgG1, which has a strong pH-dependent binding activity, compared with Human IgE only. In addition, the elimination of human IgE was significantly accelerated in mice administered with human IgE and 278-F1087, which is produced by increasing the binding of 278-IgG1 FcγR, compared to mice administered with human IgE alone and coadministered mice. with human IgE and 278-IgG1. That is, antigen elimination was shown to be accelerated not only in mice administered with anti-IgA antibodies with increased FcγR binding discussed so far, but also in mice administered with anti-IgE antibodies with increased FcγR binding . The aforementioned results showed that antigen elimination can be accelerated further by increasing the FcγR binding in each pair of the formafor antibody of the hIgA-anti-hIgA immune complex and pair of the hIgE-anti-hIgE antibody. Example 10 Preparation of antibody variants showing calcium-dependent hIgA binding
[02049] [02049] Next, in order to increase the elimination of antigen (hIgA) from plasma, variants with increased binding to mouse FcRn were produced from GA2-IgG1, which shows calcium-dependent hIgA binding. First, in order to reduce the binding of FcγR by the Fc region, GA2-F760 (heavy chain SEQ ID NO: 57) was produced by replacing Arg with Leu at position 235 and Lys with Ser at position 239 as indicated by the EU numbering in GA2-IgG1. In addition, GA2-F1331, a variant showing stronger FcRn binding at pH 7.4 than GA2-F760, was produced by introducing the following substitutions in GA2-F760: Arg by Gly at position 236, Tyr by Met in position 252, Thr for Ser in position 254, Glu for Thr in position 256, Tyr for Asn in position 434, Val for Tyr in position
[02050] [02050] Transgenic human FcRn mice (mouse line B6.mFcRn - / -. HFcRn Tg 32 + / +, Jackson Laboratories; Methods Mol Biol. (2010) 602, 93-104) were administered with hIgA (human IgA : produced in Example (1-1)) alone or co-administered with hIgA and an anti-hIgA antibody; and then in vivo dynamics of hIgA and anti-hIgA antibody was evaluated. A solution of hIgA (80 g / ml) or a mixed solution of hIgA and anti-hIgA antibody was administered once in a dose of 10 ml / kg into the tail vein. Any of the aforementioned GA2-IgG1, GA2-F760 and GA2-F1331 was used as an anti-hIgA antibody for administration.
[02051] [02051] The concentration of hIgA in the mixed solution was 80 g / ml in all cases, and the concentration of anti-hIgA antibody was 2.69 mg / ml. Since the anti-hIgA antibody was sufficiently present in excess of hIgA, most hIgA was considered to be bound by the antibody. Blood was collected from the mice 15 minutes, 1 hour, 2 hours, 7 hours, 1 day, 3 days, 7 days, and 14 days after administration of the antibody. The blood samples collected were immediately centrifuged at 4 ° C and 12,000 rpm for minutes to obtain the plasma samples. The separated plasma samples were stored in a refrigerator at -20 ° C or below until measurement. 11-2. ELISA determination of anti-hIgA antibody concentration in human FcRn transgenic mouse plasma
[02052] [02052] The concentration of anti-hIgA antibody in mouse plasma was determined by ELISA. Firstly, the F (ab ') 2 anti-human IgG Antibody Fragment (specific γ chain) (SIGMA) was dispensed into each of the Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) wells, and that it was left to stand overnight at 4 ° C to prepare immobilized human Anti-IgG plates. Standard samples of anti-hIgA antibodies to be used as calibration curve samples were prepared at plasma concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0, 007813 g / ml; and mouse plasma assay samples diluted 100 times or more were prepared and aliquoted on immobilized human Anti-IgG plates, and then the plates were incubated at 25 ° C for one hour. Then, Goat anti-human IgG Conjugate (specific γ-chain) Biotin (BIOT) (Southern Biotechnology Associates Inc.) was dispensed in each of the wells of the plates mentioned above, and then the plates were incubated at 25 ° C for one hour to let the reaction take place. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was dispensed into each of the wells of the aforementioned plates, and then the plates were incubated at 25 ° C for one hour to allow the reaction to occur. A re-
[02053] [02053] The concentration of hIgA in mouse plasma was determined by ELISA. First, the Goat anti-human IgA antibody (BETHYL) was aliquoted in each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International). The plate was left to stand overnight at 4 ° C to prepare an immobilized anti-human IgA plate. The standard hIgA samples to be used as calibration curve samples were prepared in plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 - g / ml. The mouse plasma test samples were prepared at a dilution of 100 times or greater. 200 l of hsIL6R 500 ng / mL were added to 100 L of each of the calibration curve samples and plasma samples. The resulting mixtures were allowed to stand at room temperature for one hour, and then the mixed solutions were aliquoted in 100 l on the aforementioned immobilized anti-human IgA plate; and the plate was left to stand at room temperature for one hour. Next, the Biotinylated Human Anti-IL-6R (R&D) Antibody was aliquoted in each of the wells of the aforementioned plate, and then the plate was incubated in RT for one hour to let the reaction take place. In addition, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was aliquoted in each well of the aforementioned plate, and then the plate was incubated at room temperature for one hour to allow the reaction to occur. A color development reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After the reaction was quenched 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 calculated based on the absorbance of the calibration curve using the SOFTmax PRO analytical program (Molecular Devices). The time course changes in plasma hIgA concentration in transgenic human FcRn mice determined by this method after intravenous administration are shown in Fig. 17.
[02054] [02054] The results showed that the elimination of hIgA from plasma was markedly accelerated in mice co-administered with hIgA and GA2-F1331, which increased the binding of human FcRn, compared with plasma elimination of hIgA in ca - mice co-administered with hIgA and GA2-F760, which have a low human FcRn binding activity. Example 12 Preparation of antibody variants showing pH-dependent human IgE binding
[02055] [02055] Next, in order to increase the elimination of antigen (human IgE) from plasma, variants with increased binding to mouse FcRn were produced from 278-IgG1, which shows the pH-dependent human IgE binding. First, in order to decrease the binding of mouse FcγR, 278-F760 (SEQ ID NO: 55) was produced by replacing Arg with Leu at position 235 and Lys with Ser at position 239 as indicated by the EU number 278- IgG1. In addition, 278-F1331, a variant showing stronger FcRn binding at pH 7.4 than 278-F760, was produced by introducing the following substitutions in the 278-F760: Arg by Gly at position 236, Tyr by Met at position 252, Thr for Ser in position 254, Glu for Thr in position 256, Tyr for Asn in position 434, Val for Tyr in position 436, Arg for Gln in position 438, and Glu for Ser in position 440 as indicated by EU numbering. A DNA sequence encoding 278-F1331 (heavy chain SEQ ID NO: 58, and light chain SEQ ID NO: 46) or 278-F760 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 46) it was inserted into an animal expression plasmid by a method known to those skilled in the art. These antibody variants were expressed by the aforementioned method using animal cells introduced with the plasmid, and their concentrations were determined after purification. Example 13 Evaluation of the effect on plasma antigen retention in transgenic human FcRn mice administered with a pH 13-1 dependent hIgE binding antibody. In vivo testing using human FcRn transgenic mice
[02056] [02056] Transgenic human FcRn mice (mouse B6.mFcRn - / -. HFcRn Tg lineage 32 + / +, Jackson Laboratories; Methods Mol Biol. (2010) 602, 93-104) were co-administered with hI- gE (Asp6) (human IgE (Asp6): produced in Example (5-1)), an anti-hIgE antibody (278-F760 or 278-F1331), and Sanglopor (human normal immunoglobulin, CSL Behring) ; and then in vivo dynamics of hIgE (the Asp6) and anti-hIgE antibody was evaluated. A mixed solution of hIgE (Asp6), an anti-hIgE antibody and Sanglopor (the concentrations are shown in Table 11) was administered once at a dose of 10 mL / kg into the tail vein. The aforementioned 278-F760 or 278-F1331 were used as the anti-hIgE antibody for administration
[02057] [02057] Since the anti-hIgE antibody was sufficiently present in excess of hIgE (Asp6), most hIgE (Asp6) was considered to be bound by the antibody. Blood was collected from the mice 5 minutes, 2 hours, 7 hours, 1 day, 2 days, 4 days, 5 days, 7 days, 14 days, 21 days, and 28 days after administration of the antibody. The blood samples collected were immediately centrifuged at 4 ° C and 12,000 rpm for 15 minutes to obtain plasma samples. The separated plasma samples were stored in a refrigerator at -20 ° C or below until measurement. Table 11 Antibody hIgE concentration Antibody concentration- San- anti-hIgE concentration (Asp6) in the admixed anti-hIgE solution in the glopor solution in the administered solution (g / mL) administered (g / mL) administered (g / mL) / mL) 278-F760 20 100 100 278-F1331 20 100 100 13-2. Determination of anti-hIgE antibody concentration in human FcRn transgenic mouse plasma by ELISA
[02058] [02058] The concentration of anti-hIgE antibody in mouse plasma was determined by electrochemiluminescence assay (ECL). Standard samples of anti-hIgE antibodies to be used as calibration curve samples were prepared in plasma concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 g / mL. Each of the calibration curve samples and mouse plasma samples was aliquoted in each of the wells of the ECL plate with immobilized hIgE (Asp6), and then the plate was incubated at 4 ° C for one hour / during night to let the reaction take place. Then, Goat anti-human IgG Conjugate (specific γ chain) Biotin (BI-OT) (Southern Biotechnology Associates Inc.) was dispensed into each of the wells of the above mentioned plate, and then the plate was incubated at 25 ° C for one hour to let the reaction run. In addition, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was dispensed in each of the wells of the aforementioned plate, and then the plate was incubated at 25 ° C for one hour to allow the reaction to occur. Then, each reaction solution on the plate was allowed to react with a goat anti-rabbit antibody labeled with SULFO (Meso Scale Discovery) at room temperature for one hour. Finally, Read T buffer (x4) (Meso Scale Discovery) was dispensed in each reaction solution, and this was immediately followed by the luminescence measurement of the reaction solution using the Sector Iger 2400 Reader (Meso Scale Discovery ). The concentration of anti-hIgE antibody in the mouse plasma was calculated based on the response of the calibration curve using the analytical program SOFTmax PRO (Molecular Devices). Time course changes in plasma concentrations of antibodies 278-F1331 and 278-F760 in transgenic human FcRn mice determined by this method after intravenous administration are shown in Fig. 18. 13-3. Determination of plasma hIgE (Asp6) concentration in transgenic human FcRn mice
[02059] [02059] The concentration of hIgE (Asp6) in mouse plasma was determined by ELISA. The calibration curve samples were prepared at plasma concentrations of 192, 96, 48, 24, 12, 6, and 3 ng / mL. To produce the immune complex formed between hIgE (Asp6) and a homogeneous anti-hIgE antibody, calibration curve samples and mouse plasma assay samples were prepared by adding Xolair (Novartis) at 10 g / mL to the group administered with 278-hIgG1, and the samples were left to stand at room temperature for 30 minutes. Mouse plasma assay samples were aliquoted on immobilized human anti-IgE immuno plate (MABTECH) or immobilized human anti-IgE immuno plate (clone
[02060] [02060] The results showed that plasma elimination of mouse hIgE was markedly accelerated in mice co-administered with hIgE and 278-F1331, which increased the binding of human FcRn, compared with plasma elimination of hIgE in mice co-administered with hIgE and 278-F760, which has a low FcγR binding activity in mice. That is, it was shown that antigen elimination was accelerated not only in mice
[02061] [02061] Fv4-IgG1 (heavy chain SEQ ID NO: 59, and light chain SEQ ID NO: 60) is an anti-human IL6 receptor antibody that has the property of binding to human IL6R in a pH-dependent manner (binds under a neutral condition and dissociates under an acidic condition) as described in WO 2011/122011. PHX-IgG1 (heavy chain SEQ ID NO: 61, and light chain SEQ ID NO: 62) is a human IL6R binding antibody. A DNA sequence encoding Fv4-IgG1 (heavy chain SEQ ID NO: 59, and light chain SEQ ID NO: 60), PHX-IgG1 (heavy chain SEQ ID NO: 61, and light chain SEQ ID NO: 62), or PHX-F29 (heavy chain SEQ ID NO: 63, and light chain SEQ ID NO: 62) produced by the preparation of amino acid modifications to the constant region of the PHX-IgG1 heavy chain was inserted into an animal expression plasmid by a method known to those skilled in the art. These antibody variants were expressed by the method mentioned above (described in Example 1) using plasmids, and their concentrations were determined after purification. 14-2. Evaluation of human PH6-IgG1 IL6 receptor binding
[02062] [02062] The interaction between IL-6R and PHX-IgG1 prepared in 14-1 was analyzed using Biacore T200 (GE Healthcare) to calculate the
[02063] [02063] The KD dissociation constant (mol / L) of PHX-IgG1 from IL-6R was calculated by performing a permanent affinity analysis in the sensorgrams obtained as results of Biaker measurement using the Biacore Evaluation Program. The dissociation constant (KD) of PHX-IgG1 from IL-6R at pH 7.4 calculated by this method was 1.4 E-7 (M).
[02064] [02064] Next, the pH dependence of PHX-IgG1 on hIL-6R binding was assessed using Biacore T100. A 10 mM ACES buffer, 150 mM NaCl, 0.05% Tween20, pH 7.4 and a 10 mM ACES buffer, 150 mM NaCl, 0.05% Tween20, pH 6.0 were used as running buffers to determine the binding of PHX-IgG1 to hIL-6R at 37 ° C under conditions of pH 7.4 and pH 6.0, respectively. Protein A / G (Thermo Scientific) was immobilized on the Sensor CM4 Series S chip (GE Healthcare) by an amine coupling method, and the chip was allowed to capture an antibody of interest. Then, the running buffer and IL-6R, which was diluted using the running buffer at 1000, 250, and 62.5 nM, were allowed to interact with the captured antibody chip.
[02065] [02065] The sensorgrams obtained through measurements made by this method at pH 7.4 and pH 6.0 are shown in Fig. 20. Fig. 20 shows the hIL-6R phase connected and the hIL-6R phase dissociated from PHX-IgG1 when the amount of antibodies captured is normalized to 100 RU. The comparison of the results shown in Fig. 20 indicates that the binding of PHX-IgG1 to hIL-6R is reduced at pH 6.0 when compared to the case at pH 7.4. 14-3. Evaluation of the property of two types of antibodies to simultaneously bind to the same antigen by an electro-chemiluminescence method
[02066] [02066] Whether two types of antigen-binding molecules can bind to a single antigen simultaneously has been evaluated by a method of electro-chemiluminescence. First, biotinylated Fv4-IgG1 (1 g / mL, 100 L) using EZ-Link Sulfo-NHS-Biotin (Thermo SCIENTIFIC) was added to the 96-well Streptavidin Gold MULTI-ARRAY (r) plate, which was incubated at room temperature for an hour to let the reaction take place. After washing, 100 L of the hsIL-6R solutions prepared in 0, 0.2, 1, 5, and 25 g / ml were added, and allowed to react at room temperature for one hour. After another wash, PHX-F29 subjected to ruthenium marking using SULFO-TAG NHS Ester (Meso Scale Discovery) (0, 0.2, 1, 5, 25, and 125 g / mL; 100 L) or Fv4- IgG1 subjected to ruthenium labeling by the same method (5 g / mL; 100 L) was added and allowed to react at room temperature for one hour. After washing, 150 L of Read T Buffer (x4) (Meso Scale Discovery) was dispensed in each of the wells, and this was immediately followed by the chemiluminescence measurement using the SECTOR IMAGER 2400 Reader (Meso Scale Discovery).
[02067] [02067] The results are shown in Fig. 21. When marked with ruthenium, Fv4-IgG1 was allowed to react, the reaction was not obtained
[02068] [02068] Normal mice (mouse C57BL / 6J; Charles River Japan) were administered with hsIL-6R (soluble human IL-6 receptor: prepared in Reference Example 1) alone or co-administered with hsIL-6R and antibodies anti-human IL-6 receptor, and then evaluated for in vivo dynamics of hsIL-6R and human IL-6 anti-receptor antibodies. A solution of hsIL-6R (5 g / ml) or a mixed solution of hsIL-6R and an anti-human IL-6 receptor antibody was administered once at 10 ml / kg into the tail vein. The anti-human IL-6 receptor antibodies used were Fv4-IgG1 and PHX-IgG1.
[02069] [02069] The concentration of hsIL-6R in the mixed solution was 5 - g / ml in each case and the concentrations of human IL-6 antireceptor antibody were different for each administration group as shown in Table 12. Since the antibody anti-human IL-6 receptor was sufficiently present in excess of hsIL-6R, most hsIL-6R was considered to be bound by the antibody. Blood was collected from the mice 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, and 21 days after administration. Blood was collected from the group administered (# 1) with Fv4-IgG1 (1 mg / kg) 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 15 days, and 22 days after administration of the antibody. The blood samples collected were immediately centrifuged at 4 ° C and 12.00 0 rpm for 15 minutes to obtain the plasma samples. The separated plasma samples were stored in a refrigerator at -20 ° C or below until measurement. Table 12 Administration group- Quantity of Fv4-IgG1 Quantity of PHX-IgG1 administered traction (mg / kg) administered (mg / kg) # 1 1 0 # 2 1 1 # 3 1 5 # 4 1 25 14-5. Determination of the concentration of human IL-6 anti-receptor antibody in normal mouse plasma by an electrochemiluminescence method
[02070] [02070] The concentration of human IL-6 antireceptor antibody in mouse plasma was determined by an electro-chemiluminescence method. First, a human anti-kappa capture antibody (Antibody Solutions) was dispensed onto 96-well MULTI-ARRAY plates (Meso Scale Discovery) and mixed at room temperature for one hour, and then a PBS- Tween containing 5% BSA (w / v) was used to block at room temperature for two hours to prepare plates with immobilized anti-human IgG. The calibration curve samples were prepared in plasma concentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549 g / mL; and the mouse plasma test samples prepared in dilution of 500 times or more were aliquoted in each well of the plates with immobilized anti-human IgG, and then the plates were shaken for one hour at room temperature. Then, a human anti-cap capture Biotin antibody conjugate (Antibody Solutions) was dispensed into each of the wells of the above-mentioned plates, and then the plates were stirred at room temperature for one hour to allow the reaction to occur. In addition, SULFO-labeled streptavidin (Meso Scale Dis- covery) was aliquoted in each of the wells of the plates, and then the plates were stirred at room temperature for one hour to allow the reaction to occur. After washing each well, Read T Buffer (x1) (Meso Scale Discovery) was dispensed into the wells, and the chemiluminescence of the reaction solutions was measured immediately using the Sector Imager 2400 Reader (Meso Scale Discovery). The mouse plasma concentrations were calculated based on the response of the calibration curve using the SOFTmax PRO analytical program (Molecular Devices). Time course changes in the concentration of human anti-IL-6R antibody are shown in Fig. 22. 14-6. Determination of the plasma concentration of hsIL-6R by an electrochemiluminescence method
[02071] [02071] The concentration of hsIL-6R in mouse plasma was determined by an electrochemiluminescence method. The hsIL-6R calibration curve samples were prepared in plasma concentrations of 12.5, 6.25, 3.13, 1.56, 0.791, 0.391, and 0.195 ng / mL. The mouse plasma test samples were prepared at a 50-fold or greater dilution. A mixed solution of the hsIL-6R sample or mouse plasma assay sample, a monoclonal human anti-IL-6R (R&D) antibody that has been labeled with ruthenium using the SULFO-NHS Ester marker (Meso Scale Disco- very), a biotinylated human anti-IL-6R antibody (R&D), and tocilizumab (heavy chain SEQ ID NO: 64, light chain SEQ ID NO: 65) was allowed to react overnight at 37 ° C. Then, a PBS-Tween solution containing 0.5% (w / v) BSA was used to block a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery) by incubating at 5 ° C overnight, and the mixed solution was al quoted in each of the wells on that plate. After another two-hour incubation of this plate at room temperature to allow the reaction to occur, each well of the plate was washed, and then Read T Buffer (x2) (Meso Scale Discovery) was dispensed in each well; and the chemiluminescence of the reaction solutions was measured immediately using the SECTOR Imager 2400 Reader (Meso Scale Discovery). The concentrations of hsIL-6R were calculated based on the response of the calibration curve using the SOFTmax PRO analytical program (Molecular Devices).
[02072] [02072] The calculated time course changes in the concentration of human IL6R (which is also described as hsIL6R or hsIL-6R and refers to the same protein) are shown in Fig. 23. Elimination of plasma IL6R was accelerated in mice administered with PHX-IgG1 in conjunction with Fv4-IgG1 compared to mice administered with Fv4-IgG1 alone.
[02073] [02073] Without being restricted to a particular theory, the following explanation can be given for the aforementioned acceleration of plasma antigen elimination. Even when administered alone, Fv4-IgG1 was present in sufficient quantity in relation to hsIL6R, and thus it was considered that the majority of hsIL6R were bound by Fv4-IgG1 in plasma. When PHX-IgG1, which has an epitope different from that of Fv4-IgG1 and can bind to hsIL6R with Fv4-IgG1, an immune complex is administered that comprises Fv4-IgG1 and PHX-IgG1 from a single hsIL6R molecule It is formed. As a result, absorption in cells was promoted by increased binding to an Fcγ and / or FcRn receptor, and this may have resulted in accelerated elimination of hsIL6R from plasma. Specifically, the formation of large immune complexes that comprise two or more antibodies and two or more antigenic binding units (monomeric antigens) by multispecific antibodies comprising appropriate variable regions that bind to epitopes that are different from one another and are present in a monomeric antigen or multiparatopic antibodies in a non-limiting modality, which are antibodies whose variable regions show pH- or Ca-dependent binding (bispecific antibodies or biparatopic antibodies that comprise a variable region of the right arm that recognizes the epitope A and a variable region of the left arm that recognizes epitope o B, as shown in Fig. 8) allows the acceleration of antigen elimination. Example 15 Evaluation of binding of an immune complex with FcγR using Biacore 15-1. Regarding the binding of an immune complex with FcγR
[02074] [02074] As methods to assess the formation of immune complexes comprising an antigen binding molecule and an antigen, gel filtration chromatography (size exclusion) was used in Example 4, and an electrochemiluminescence method (ECL) was used in Example 14. When the antigen or antigen binding molecule comprised in an immune complex contains an FcγR binding domain as an immunoglobulin constant region, the immune complex binds to FcγR avidly. Therefore, the formation of an immune complex comprising an FcγR binding domain can be confirmed by a method that uses the stronger FcγR binding property (especially, the slower dissociation property) than the antigen binding molecule. only or the antigen alone (The Journal of Biological Chemistry (2001) 276 (9), 6591-6604, mAbs (2009) 1 (5), 491-504). 15-2. Preparation of antibodies and antigens for evaluation and histidine-labeled human FcγR IIIaV
[02075] [02075] Xolair (Novartis), clone 278-IgG1 (prepared in Example
[02076] [02076] hIgE (hIgE (Asp6); prepared in Example 5) and IL6R (Reference Example 1) to be used for the evaluation were prepared by the methods mentioned above, respectively. A human IgA recombinant, hIgA-v2 (GC-hIgA), was prepared by the method below. A gene fragment encoding GC-hIgA-MYC (heavy chain SEQ ID NO: 66 and light chain SEQ ID NO: 67) was inserted into an animal cell expression vector. The constructed plasmid vector was introduced into FreeStyle 293 (Invitrogen) using 293Fectin (Invitrogen) together with an EBNA1 expression gene. Then, the transfected cells were cultured at 37 ° C under 8% CO 2 for six days, and the GC-hIgA protein was secreted in the culture supernatant. The cell culture containing GC-hIgA-MYC was filtered through a 0.22 m filter at the mouth of the bottle to obtain the culture supernatant. Purified GC-hIgA-MYC was obtained using ion exchange chromatography and gel filtration chromatography according to methods known to those skilled in the art.
[02077] [02077] Histidine-labeled human FcγR IIIaV was prepared by the method of Reference Example 2. 15-3. Assessment of binding of an immune complex with FcγR
[02078] [02078] The binding of an antibody-antigen immune complex with FcγR was assessed using Biacore T200 (GE Healthcare).
[02079] [02079] An adequate amount of Penta-His Antibody (QIA-GEN) was immobilized on the Sensor CM5 chip (GE Healthcare) by an amine coupling method; and an adequate concentration of His-labeled human FcγR IIIaV was injected. Human FcγR IIIaV was immobilized on the chip allowing the penta-His antibody immobilized on the chip to capture human FcγR IIIaV. A solution containing 200 nM antibody or a mixed solution containing 200 nM antibody and 200 nM antigen was injected as the analyte; and was allowed to interact with human FcγR IIIaV immobilized on the sensor chip. Subsequently, a 10 mmol / L, pH 2.5 Glycine-HCl solution was injected to regenerate the sensor chip. A 1.2 mmol / L / 0.05% tween20 CaCl2 buffer, 20 mmol / L ACES, 150 mmol / L NaCl, pH 7.4 was used as the racing buffer, and binding between the antibody-antigen immune complex and FcγR was measured at 25 ° C.
[02080] [02080] The results of reaction binding analyzes of a solution containing the antibody or an antibody-antigen solution mixed with human FcγR IIIaV are shown in Fig. 24. When focusing on dissociation, the Fig. 24 shows sensorgrams in which binding levels have been normalized to 100. Although Fv4-IgG1 binds to IL6R, it does not form an immune complex. Therefore, no difference was observed between dissociation in the reaction of a solution containing antibody and FcγR and dissociation when an antibody-antigen mixture was used. However, when a mixed solution of hIgE and Xolair (J. Pharmacol. Exp. Ther. (1996) 279 (2) 1000-1008), which were reported to form an immune complex, if bound to FcγR, was allowed observed that the dissociation of FcγR was slower than when Xolair alone was allowed to bind to FcγR. In addition, for clone 278-IgG1, which was confirmed to form an immune complex using size exclusion chromatography (gel filtration) in Example 4, when a mixed solution of clone 278-IgG1 and an antigen ( hIgE) if it bound to FcγR, the dissociation of FcγR was slower than when clone 278-IgG1 alone was allowed to bind to FcγR. In addition, when a mixed solution of GA2-IgG1 and hIgA was allowed to bind to FcγR, dissociation of FcγR was also slower than when GA2-
[02081] [02081] Consequently, when the antigen or antigen binding molecule comprised in an immune complex contains an FcγR binding domain as an immunoglobulin constant region, the immune complex binds to FcγR avidly. Therefore, it has been found that a method for assessing the delay in dissociation of FcγR when a mixed solution of an antigen-binding molecule is allowed containing an FcγR-like or similar domain and its antigen binds to FcγR, or when a mixed solution of an antigen containing an FcγR binding domain and an antigen binding molecule that binds to this antigen is allowed to bind to FcγR, when compared to when a molecule containing an FcγR binding domain as an immunoglobulin allowed the constant region to bind itself to FcγR, it was an effective method to evaluate the formation of immune complexes that comprise an antigen binding molecule and an antigen. The aforementioned assessment of immune complexes using FcγR dissociation as criteria was shown to be effective in the case of immune complexes formed by an antibody against an antigen bound by a plurality of monomers, and also in the case of immune complexes formed by a monomeric antigen and a plurality of antibodies that bind to different epitopes. Example 16 Evaluation of binding of an immune complex with FcRn using Biacore 16-1. Regarding the binding of an immune complex with FcRn
[02082] [02082] In a method to assess the formation of immune complexes
[02083] [02083] Fv4-F22 used in the evaluation is a variant of the constant region of the Fv4-IgG1 heavy chain (described in Example 14). Xolair- F22 is a variant of the Xolair (Novartis) heavy chain constant region. A DNA sequence encoding Fv4-F22 (heavy chain SEQ ID NO: 68, and light chain SEQ ID NO: 60) and a DNA sequence encoding Xolair-F22 (heavy chain SEQ ID NO: 69, and ca - light hay SEQ ID NO: 70) were inserted into an animal cell expression plasmid by a method known to those skilled in the art. The antibody variants were expressed according to the method described above (described in Example 1) using animal cells
[02084] [02084] The hIgE (hIgE (Asp6), prepared in Example 5) and IL6R (prepared in Reference Example 1) used for the evaluation were prepared by the respective methods mentioned above.
[02085] [02085] Human FcRn and mouse FcRn were prepared by the methods described in Reference Examples 3 and 4, respectively. 16-3. Assessment of binding of an immune complex to human FcRn
[02086] [02086] The binding of an antibody-antigen immune complex with human FcRn was assessed using Biacore T100 (GE Healthcare).
[02087] [02087] A solution containing an antibody alone or a mixed antibody-antigen solution shown in Table 13 was injected as an analyte on the Sensor CM4 chip (GE Healthcare) for which an adequate amount of human FcRn (hFcRn) had been immobilized by an amine coupling method, and the interaction was allowed to take place with hFcRn on the sensor chip. Subsequently, a solution of 20 mM Tris-HCl, 150 mM NaCl, pH 9.0 or pH 9.5 was injected to regenerate the sensor chip. A 50 mmol / L Na-phosphate buffer (phosphoric acid), 150 mmol / L NaCl, 0.05% Tween20, pH 7.4 was used as the running buffer, and binding between the antibody-antigen immune complex and FcRn was measured at 25 ° C. Table 13 Sample name Antibody Antigen Fv4-F22 Fv4-F22 (10 µg / mL) - Fv4-F22 + IL6R Fv4-F22 (10 µg / mL) hsIL6R (2.5 µg / mL) Xolair-F22 Xolair-F22 ( 10 µg / mL) - Xolair-F22 + IgE Xolair-F22 (10 µg / mL) hIgE (Asp6) (11.2 µg / mL)
[02088] [02088] The results of binding analyzes, when a solution containing the mixed antibody or antibody-antigen solution was allowed to react with hFcRn, are shown in Fig. 25. When focusing on dissociation, Fig. 25 shows sensorgrams in which binding levels were normalized to 100. Although Fv4-F22 binds with IL6R, it does not form an immune complex containing a plurality of molecules that carry constant regions of immunoglobulin; and thus no difference was observed between dissociation in the reaction of a solution containing antibody and FcRn and dissociation when a mixed antibody-antigen solution was used.
[02089] [02089] In 16-3, the FcRn binding of immune complexes containing variants of immunoglobulin constant regions with increased hFcRn binding was evaluated. Since native human IgG can bind more strongly to human FcRn than to mouse FcRn (Int. Immunol. (2001) 13 (12), 1551-1559), binding of an antibody containing constant region of human IgG native to mouse FcRn was evaluated to examine whether evaluation of an immune complex formation is possible without modifying the immunoglobulin constant region.
[02090] [02090] The binding of an immune antibody-antigen complex with FcRn was assessed using Biacore T100 (GE Healthcare). A solution containing only antibodies or a mixed antibody-antigen solution as shown in Table 14 was injected as an analyte for the Sensor CM4 chip (GE Healthcare) which was immobilized with an adequate amount of the mouse FcRn (mFcRn) for a amine coupling, and the interaction was allowed to take place with mFcRn on the sensor chip. Subsequently, a solution of 20 mM Tris-HCl, 150 mM NaCl, pH 9.0 or pH 9.5 was injected to regenerate the sensor chip. A 50 mmol / L Na-phosphate buffer (phosphoric acid), 150 mmol / L NaCl, 0.05% Tween20, pH 7.4 was used as the racing buffer, and binding between the antibody-antigen immune complex and FcRn was measured at 25 ° C.
[02091] [02091] The results of binding analyzes, when a solution containing only mixed antibodies or a mixed antibody-antigen solution was allowed to react with mFcRn, are shown in Figs. 26 and 27. By focusing on dissociation, Figures 26 and 27 show sensorgrams in which binding levels have been normalized to 100. When a mixed solution of an antigen (hIgE) and clone 278-IgG1 was allowed , which was confirmed to form an immune complex by size exclusion chromatography (gel filtration) in Example 4, if it bound with mFcRn, dissociation of mF-cRn was also slower than when clone 278-IgG1 was allowed alone if it binds to mFcRn. In addition, when a mixed solution of hIgE and Xolair, which has been reported to form an immune complex (J. Pharmacol. Exp. Ther. (1996) 279 (2) 1000-1008), was allowed to bind with mFcRn, it was observed that the dissociation of mFcRn was slower than when Xolair alone was allowed to bind to mFcRn (Fig. 26). On the other hand, no difference was observed between the dissociation of mFcRn when a solution containing Fv4-IgG1 was allowed to bind with mFcRn and dissociation of mF-cRn when a mixed solution of Fv4-IgG1 and
[02092] [02092] The aforementioned has shown that using mouse FcRn, even a native human IgG1 molecule without modifications that would affect the interaction with FcRn can be evaluated for binding FcRn under pH 7.4 condition which is the same as in in vivo condition. In addition, when an antigen-binding molecule or antigen contains an FcRn-binding domain as an immunoglobulin constant region, the immune complex binds strongly with FcRn; therefore, the formation of an immune complex can be confirmed by assessing the delay in the dissociation of FcRn from an immune complex comprising a plurality of molecules containing an FcRn binding domain as a constant region of immunoglobulin compared with the dissociation of FcRn of a molecule containing an FcRn binding domain of an immunoglobulin constant region. It has been confirmed that the aforementioned assessment of immune complex formation using FcRn can be used to verify the formation of both an immune complex formed from an antigen comprising a plurality of bound monomers and an antibody that binds to this antigen and an immune complex formed from a monomeric antigen and a plurality of antibodies having different binding epitopes that bind to this antigen. Reference Example 1 Preparation of soluble human IL-6 receptor (hsIL-6R)
[02093] [02093] A recombinant form of human IL-6 receptor, which is an antigen, was prepared as follows. A strain of CHO that constantly expresses a soluble human IL-6 receptor (hereinafter referred to as hsIL-6R) composed of the amino acid sequence from the first to the 357th N-terminal amino acid as reported in J. Immunol. (1994) 152, 4958-4968 was built using a method known to those skilled in the art. hsIL-6R was expressed by cultivating this CHO strain. hsIL-6R was purified from the resulting culture supernatant of the resulting CHO strain by a double step process involving Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction that eluted as the main peak in the final step was used as the final purified product. Reference Example 2 Preparation of histidine-labeled human FcγRIIIaV
[02094] [02094] Histidine-labeled human FcγRIIIaV was prepared by the following method. First, a gene from the FcγR extracellular domain was synthesized by a method well known to those skilled in the art. Then, the sequence of each FcγR was produced based on the information registered in the NCBI. Specifically, FcγRIIIa was produced based on the NCBI Accession No. NM_001127593.1 sequence, and a His marker was attached to the C-terminus. In addition, the polymorphism is known as FcγRIIIa; however, the production was carried out referring to J. Clin. Invest., 1997, 100 (5): 1059-1070 for FcγRIIIa.
[02095] [02095] The gene fragments obtained were inserted into an expression cell for animal cells, and expression vectors were produced. The expression vectors produced were introduced transiently into FreeStyle293 cells derived from the human embryonic kidney cancer cell line (Invitrogen) to express the proteins of interest. The cells were cultured, and after collecting the obtained culture supernatant, which was passed through a 0.22 m filter to obtain the culture supernatant. In principle, the culture supernatants obtained were purified in the following four steps. The steps performed were cation exchange column chromatography (SP Sepharose FF) in step 1, affinity column chromatography (HisTrap HP) for the His marker in step 2, gel filtration column chromatography (Superdex200) in step 3 and cro-
[02096] [02096] Human FcRn forms a complex with β2-microglobulin. Oligo-DNA primers were designed based on the reported human FcRn gene sequence (J Exp Med. 1994 Dec 1; 180 (6): 2377-81). A cDNA was amplified by PCR from a human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) using the designed primers. The amplified cDNA encoding the extracellular domain that contains the signal sequence (Met1-Leu290) was inserted into an animal cell expression vector. Similarly, primers were designed for β2-microglobulin based on the reported human β2-microglobulin sequence, and the cDNA was amplified by PCR. The Met1-Met119 cDNA fragment containing a signal sequence was amplified and inserted into an animal cell expression vector.
[02097] [02097] Soluble human FcRn (called hFcRn) was prepared according to the procedure below. The vector to express human FcRn (SEQ ID NO: 71) and the vector to express β2-microglobulin (SEQ ID NO: 72) were transduced into HEK293H cells (Invitrogen) by the lipofection method using PEI (Poliscience). The transduced cells were cultured, and the culture solution was collected. Human FcRn was purified from the culture collected by each of the chromatographic methods, IgG Sepharose 6 Fast Flow (Amersham Biosciences) and HiTrap Q HP (GE Healthcare) (J Immunol. 2002 Nov 1; 169 (9): 5171-80). Reference example 4 Preparation of mouse soluble FcRn (mFcRn)
[02098] [02098] Soluble mouse FcRn was prepared in a manner similar to human soluble FcRn. First, a gene that co-complicates the extracellular domain of mouse FcRn and a gene that encodes mouse β2-microglobulin has been synthesized by methods known to those skilled in the art. For this operation, mouse FcRn and mouse β2-microglobulin sequences were produced based on information registered with the NCBI. Specifically, for mouse FcRn, a gene fragment that encodes amino acids from positions 1 to 290 containing the signal sequence was produced as the extracellular domain based on the NCBI accession # NP_034319.2 (SEQ ID NO: 73). In addition, for β2-microglobulin, a gene fragment was produced based on the NCBI accession sequence # NP_033865 (SEQ ID NO: 74). An expression vector was produced by inserting the gene fragments obtained into an animal cell expression vector. The expression vector produced was introduced transiently into FreeStyle 293 cells derived from human embryonic kidney carcinoma cell (Invitrogen) to express the protein of interest. The transduced cells were cultured, and the culture solution was collected. The mouse FcRn was purified from the culture collected by each of the chromatographic methods, IgG Sepharose 6 Fast Flow (Amersham Biosciences) and Su- perdx 200 (GE Healthcare). Reference example 5 Production of an antigen binding molecule with higher FcγR binding activity than that of native human IgG Fc region and increased human FcRn binding activity under an acidic pH condition (5- 1) Production of a human IL-6 anti-receptor antibody with increased binding to mouse FcγR
[02099] [02099] VH3-IgG1-F1087 (SEQ ID NO: 75), which is formed by replacing Asp with Lys at position 326 (EU numbering) in VH3-IgG1 and
[02100] [02100] VH3 / L (WT) -IgG1-F1087 and VH3 / L (WT) - IgG1-F1182, which contain VH3-IgG1-F1087 and VH3-IgG1-F1182 respectively, were produced as the heavy chain and L ( WT) -CK (SEQ ID NO: 78) as the light chain. The mouse FcγR binding activities of these antibodies and VH3 / L (WT) -IgG1-F1022 were evaluated, and the results are shown in Table 15. The increased number of times in FcγR binding activity of mice from each of the variants compared to that of IgG1 before modification is shown in Table 16. Table 15 Variant name KD (M) mFc γ RI mFc γ RIIb mFc γ RIII mFc γ RIV IgG1 5.3E-08 9 , 8E-07 2,4E-06 8,6E-08 F1022 7,6E-09 1,0E-08 5,5E-09 1,4E-07 F1087 2,9E-08 5,6E-08 5,2E- 08 3,3E-07 F1182 2,4E-09 1,1E-07 4,8E-07 5,3E-10 Table 16 Name of variant Proportion of binding to IgG1 mFc γ RI mFc γ RIIb mFc γ RIII mFc γ RIV IgG1 1.0 1.0 1.0 1.0 F1022 7.0 93.6 440.5 0.6 F1087 1.8 17.5 46.2 0.3 F1182 22.1 9.1 5.0 162, 3 Industrial applicability
[02101] [02101] The antigen binding molecules of the present invention allow the acceleration of the elimination of antigens with two or more antigenic binding units from the plasma, being equipped with the attribute of forming large immune complexes that comprise antigens with two or more antigenic binding units (epitopes) and two or more antigen-binding molecules (eg, antibodies). The antigen-binding molecules of the present invention also allow for further acceleration of the elimination of antigens equipped with ion-dependent antigen-binding activity in addition to the aforementioned attributes.
Equipped with such technical attributes, the antigen-binding molecules of the present invention become very useful as pharmaceuticals to treat diseases or symptoms (such as cancer and inflammatory disease).

权利要求:
Claims (54)
[1]
1. Use of an antigen-binding molecule that comprises (i) an Fc region and (ii) two or more antigen-binding domains, where at least one of the domains is an antigen-binding domain of which antigen-binding activity varies depending on an ionic concentration condition, and in which the antigen-binding molecule can form an immune complex comprising (a) two or more of the antigen-binding molecules and (b) two or more antigens, in which the antigens comprise two or more units of antigenic binding, said use being characterized by the fact that it is in the preparation of a medication for the elimination of antigens from the plasma.
[2]
2. Use, according to claim 1, characterized by the fact that the ionic concentration condition is a calcium ion concentration condition.
[3]
3. Use according to claim 2, characterized by the fact that the antigen binding domain has an antigen binding activity under a condition of low calcium ion concentration which is lower than the antigen binding activity under a condition of high calcium ion concentration.
[4]
4. Use according to any one of claims 1 to 3, characterized by the fact that the condition of ionic concentration is a condition of pH.
[5]
5. Use according to claim 4, characterized by the fact that the antigen-binding domain has an antigen-binding activity in an acidic pH range that is lower than the antigen-binding activity in a condition neutral pH range.
[6]
Use according to any one of claims 1 to 5, characterized in that the antigens comprising two or more antigen binding units are multimers.
[7]
7. Use, according to claim 6, characterized by the fact that the antigen is any one of 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), TNF, TNF- alpha, TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL Apo-2 linker, TL2), TNFSF11 (TRANCE / RANK ODF linker, OPG linker), TNFSF12 (TWEAK Apo-3 linker, DR3 linker), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR, TL6), TNFSF1A (TNF-a Connect, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 linker gp34, TXGP1), TNFSF5 (CD40 link CD154, gp39, HIGM1, IMD3, TRAP), TNFS (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 CD70 ligand), TNFSF8 (CD30 CD153 ligand), TNFSF9 (4-1BB ligand CD137 ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta Pan Specific and IL-8.
[8]
Use according to any one of claims 1 to 5, characterized in that the antigens comprising two or more antigen binding units are monomers.
[9]
Use according to any one of claims 1 to 8, characterized by the fact that the antigen binding molecules are a multispecific or multiparatopic antigen binding molecule or cocktail of antigen binding molecules.
[10]
10. Use according to any one of claims 1 to 9, characterized by the fact that the Fc region is represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[11]
11. Use according to any one of claims 1 to 9, characterized by the fact that the Fc region is an Fc region with increased FcRn binding activity under an acidic pH range condition compared to that of Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[12]
12. Use according to claim 11, characterized by the fact that the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of the amino acids at positions 238, 244 , 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307 , 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387 , 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442 and 447 (EU numbering) in the amino acid sequence from the Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[13]
13. Use, according to claim 12, characterized by the fact that the Fc region comprises at least one or more amino acids selected from the group consisting of: Leu for the amino acid of position 238; Read for the amino acid at position 244; Arg for the amino acid at position 245; Pro for the amino acid of position 249; Gln or Glu for the amino acid of position 250; Arg, Asp, Glu or Leu for the amino acid of position 251; Phe, Ser, Thr or Tyr for the amino acid at position 252; Ser or Thr for the amino acid of position 254; Arg, Gly, Ile or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position 256;
Ala, Ile, Met, Asn, Ser or Val for the amino acid of position 257; Asp for the amino acid of position 258; Be for the amino acid of position 260; Read for the amino acid at position 262; Lys for the amino acid at position 270; Leu or Arg for the amino acid of position 272; Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 283; Asn for the amino acid of position 285; Phe for the amino acid of position 286; Asn or Pro for the amino acid of position 288; Val for the amino acid of position 293; Ala, Glu, Gln, or Met for the amino acid of position 307; Ala, Glu, Ile, Lys, Leu, Met, Ser, Val or Trp for the amino acid of position 311; Pro for the amino acid of position 309; Ala, Asp, or Pro for the amino acid of position 312; Ala or Leu for the amino acid at position 314; Lys for the amino acid of position 316; Pro for the amino acid of position 317; Asn or Thr for the amino acid at position 318; Phe, His, Lys, Leu, Met, Arg, Ser or Trp for the amino acid of position 332; Asn, Thr or Trp for the amino acid of position 339; Pro for the amino acid of position 341; Glu, His, Lys, Gln, Arg, Thr or Tyr for the amino acid of position 343;
Arg for the amino acid at position 375; Gly, Ile, Met, Pro, Thr or Val for the amino acid of position 376; Lys for the amino acid of position 377; Asp, Asn or Val for the amino acid of position 378; Ala, Asn, Ser or Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 382; Ala, Arg, Asp, Gly, His, Lys, Ser or Thr for the amino acid of position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr for the amino acid of position 386; Ala, Arg, His, Pro, Ser or Thr for the amino acid of position 387; Asn, Pro, or Ser for the amino acid of position 389; Asn for the amino acid of position 423; Asn for the amino acid of position 427; Leu, Met, Phe, Ser or Thr for the amino acid of position 428; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr for the amino acid of position 430; His or Asn for the amino acid of position 431; Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433; Ala, Gly, His, Phe, Ser, Trp or Tyr for the amino acid of position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid of position 436; Lys, Leu, Thr or Trp for the amino acid of position 438; Lys for the amino acid of position 440; and
Lys for the amino acid of position 442; Ile, Pro, or Thr for the amino acid of position 308; as indicated by the EU numbering following amino acids from the Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[14]
14. Use according to any one of claims 1 to 9, characterized by the fact that the Fc region is an Fc region with increased FcRn binding activity under a neutral pH range condition compared to that of Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[15]
15. Use, according to claim 14, characterized by the fact that the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of positions 237, 248, 250 , 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376 , 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino acid sequence of the Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[16]
16. Use according to claim 15, characterized by the fact that the Fc region comprises at least one or more amino acids selected from the group consisting of: Met for the amino acid at position 237; Ile for the amino acid of position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250; Phe, Trp or Tyr for the amino acid of position 252; Thr for the amino acid at position 254; Glu for the amino acid of position 255; Asp, Asn, Glu or Gln for the amino acid at position 256;
Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257; His for the amino acid of position 258; Wing for the amino acid of position 265; Ala or Glu for the amino acid of position 286; His for the amino acid of position 289; Wing for the amino acid of position 297; Wing for the amino acid of position 303; Wing for the amino acid of position 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308; Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309; Ala, His or Ile for the amino acid at position 311; Ala or His for the amino acid at position 312; Lys or Arg for the amino acid of position 314; Ala, Asp or His for the amino acid at position 315; Wing for the amino acid of position 317; Val for the amino acid of position 332; Read for the amino acid of position 334; His for the amino acid at position 360; Wing for the amino acid of position 376; Wing for amino acid at position 380; Wing for the amino acid of position 382; Wing for the amino acid of position 384; Asp or His for the amino acid of position 385; Pro for the amino acid of position 386; Glu for the amino acid of position 387;
Ala or Ser for the amino acid of position 389; Wing for the amino acid of position 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 428; Lys for the amino acid of position 433; Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and His, Ile, Leu, Phe, Thr or Val for the amino acid at position 436; as indicated by the EU numbering following amino acids from the Fc region represented by any of SEQ ID NOS: 13, 14, 15 and 16.
[17]
17. Use according to any of claims 1 to 13, characterized in that the Fc region includes an Fc region that has a higher Fcγ receptor binding activity than that of the IgG Fc region native human.
[18]
18. Use according to claim 17, characterized by the fact that the Fc region comprises in its amino acid sequence at least one or more amino acids that are different from the amino acids of the Fc region of native human IgG selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247 , 249, 250, 251, 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 (EU numbering).
[19]
19. Use according to claim 18, characterized by the fact that the Fc region comprises in its amino acid sequence at least one amino acid selected from the group consisting of: Lys or Tyr for the amino acid of position 221; Phe, Trp, Glu or Tyr for the amino acid of position 222; Phe, Trp, Glu or Lys for the amino acid of position 223; Phe, Trp, Glu or Tyr for the amino acid of position 224; Glu, Lys or Trp for the amino acid of position 225; Glu, Gly, Lys or Tyr for the amino acid of position 227; Glu, Gly, Lys or Tyr for the amino acid of position 228; Ala, Glu, Gly or Tyr for the amino acid of position 230; Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231; Glu, Gly, Lys or Tyr for the amino acid of position 232; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 238; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid at position 239; Ala, Ile, Met, or Thr for the amino acid at position 240; Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of the
tion 241; Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243; His for the amino acid at position 244; Wing for the amino acid of position 245; Asp, Glu, yours or Tyr for the amino acid of position 246; Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr for the amino acid of position 247; Glu, His, Gln or Tyr for the amino acid at position 249; Glu or Gln for the amino acid of position 250; Phe for the amino acid of position 251; Phe, Met, or Tyr for the amino acid at position 254; Glu, Leu or Tyr for the amino acid at position 255; Ala, Met, or Pro for the amino acid at position 256; Asp, Glu, His, Ser or Tyr for the amino acid of position 258; Asp, Glu, His or Tyr for the amino acid at position 260; Ala, Glu, Phe, Ile or Thr for the amino acid of position 262; Ala, Ile, Met, or Thr for the amino acid at position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid at position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265; Ala, Ile, Met, or Thr for the amino acid at position 266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267; Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid at position 270; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271; Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272; Phe or Ile for the amino acid of position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274; Leu or Trp for the amino acid at position 275; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid at position 278; Wing for the amino acid of position 279; Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283; Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284; Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285; Glu, Gly, Pro, or Tyr for the amino acid of position 286; Asn, Asp, Glu or Tyr for the amino acid of position 288; Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the amino acid at position 290;
Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid in position 291; Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293; Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297; Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the 299 amino acid; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid at position 300; Asp, Glu, His or Tyr for the amino acid of position 301; Ile for the amino acid of position 302; Asp, Gly or Tyr for the amino acid of position 303; Asp, His, Leu, Asn or Thr for the amino acid at position 304; Glu, Ile, Thr or Tyr for the amino acid of position 305; Ala, Asp, Asn, Thr, Val or Tyr for the amino acid of position 311; Phe for the amino acid at position 313; Read for the amino acid at position 315;
Glu or Gln for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr for the amino acid of position 318; Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid at position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322; Ile for the amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325; Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid at position 330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid at position 331; Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr for the amino acid of position 333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;
Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid at position 335; Glu, Lys or Tyr for the amino acid of position 336; Glu, His or Asn for the amino acid at position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339; Ala or Val for the amino acid of position 376; Gly or Lys for the amino acid of position 377; Asp for the amino acid of position 378; Asn for the amino acid of position 379; Ala, Asn or Ser for the amino acid of position 380; Ala or Ile for the amino acid of position 382; Glu for the amino acid of position 385; Thr for the amino acid at position 392; Read for the amino acid of position 396; Lys for the amino acid of position 421; Asn for the amino acid of position 427; Phe or Leu for the amino acid of position 428; Met for the amino acid of position 429; Trp for the amino acid of position 434; Ile for the amino acid of position 436; and Gly, His, Ile, Leu or Tyr for the amino acid at position 440; as indicated by the EU numbering.
[20]
20. Use according to any one of claims 1 to 16, characterized by the fact that the Fc region has a higher binding activity towards an inhibitory Fcγ receptor than towards an activating Fcγ receptor.
[21]
21. Use according to claim 20, characterized by the fact that the inhibitory Fcγ receptor is human FcγRIIb.
[22]
22. Use according to claim 20 or 21, characterized
by the fact that the activating Fcγ receptor is human FcγRII, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) or human FcγRIIIa (F).
[23]
23. Use according to any one of claims 22, characterized by the fact that the amino acid at position 238 or 328 (EU numbering) in the Fc region is different from the amino acid in the Fc region of native human IgG.
[24]
24. Use according to claim 23, characterized by the fact that the amino acid at position 238 of the Fc region is Asp or the amino acid at position 328 of the Fc region is Glu as indicated by the numbering EU.
[25]
25. Use according to claim 23 or 24, characterized by the fact that the amino acid sequence of the Fc region comprises at least one or more amino acids selected from the group consisting of: Asp for the amino acid of position 233; Trp or Tyr for the amino acid of position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp or Tyr for the amino acid of position 237; Asp for the amino acid at position 239; Ala, Gln or Val for the amino acid at position 267; Asn, Asp or Glu for the amino acid of position 268; Gly for the amino acid at position 271; Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser or Thr for the amino acid of position 326; Arg, Lys, or Met for the amino acid at position 330; Ile, Leu, or Met for the amino acid of position 323; and Asp for the amino acid at position 296; as indicated by the EU numbering.
[26]
26. Method of tracing an antigen-binding molecule with the function of eliminating an antigen from plasma, characterized by the fact that it comprises: (a) obtaining an antigen-binding domain from which antigen-binding activity varies depending on a condition of ionic concentration; (b) obtaining a gene that encodes the antigen-binding domain selected in (a) above; (c) operationally linking the gene obtained in (b) above with a gene encoding an Fc region; (d) culturing a host cell comprising the genes operably linked in (c) above; (e) isolating an antigen-binding molecule from a culture solution obtained in (d) above; (f) contact of the antigen-binding molecule obtained in (e) above with an antigen; and (g) assessing the formation of an immune complex comprising the antigen-binding molecule and the antigen.
[27]
27. Method for producing an antigen-binding molecule having the function of eliminating an antigen from plasma, characterized by the fact that it comprises: (a) contact of an antigen with an antigen-binding molecule comprising a Fc region and two or more antigen-binding domains, in which at least one of the antigen-binding domains has an antigen-binding activity that varies depending on an ionic concentration condition; (b) evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen; (c) culturing a host cell comprising a vector that carries a gene that encodes an antigen-binding molecule that is confirmed to form an immune complex in (b) above; and
(d) isolating the antigen-binding molecule from a culture solution obtained in (c) above.
[28]
28. Method for the production of an antigen-binding molecule having the function of eliminating an antigen from plasma, characterized by the fact that it comprises: (a) obtaining an antigen-binding domain from which the antigen-binding activity varies depending on a condition of ionic concentration; (b) obtaining a gene that encodes the antigen-binding domain selected in (a) above; (c) operationally linking the gene obtained in (b) above with a gene encoding an Fc region; (d) culturing a host cell comprising the genes operably linked in (c) above; (e) isolating an antigen-binding molecule from a culture solution obtained in (d) above; (f) contact of the antigen-binding molecule obtained in (e) above with an antigen; (g) evaluation of the formation of an immune complex comprising the antigen-binding molecule and the antigen; (h) culture of a host cell comprising a vector that carries a gene that encodes an antigen-binding molecule that is confirmed to form an immune complex in (g) above; and (i) isolating the antigen-binding molecule from a culture solution obtained in (h) above.
[29]
29. Method for producing an antigen-binding molecule having the function of eliminating an antigen from plasma, characterized by the fact that it comprises: (a) obtaining an antigen-binding domain from which the antigen-binding activity varies depending on a condition of ionic concentration; (b) obtaining a gene that encodes the antigen-binding domain selected in (a) above; (c) operationally linking the gene obtained in (b) above with a gene encoding an Fc region; (d) culturing a host cell comprising the genes operably linked in (c) above; and (e) isolating an antigen-binding molecule from a culture solution obtained in (d) above; and where the method also comprises contacting the antigen-binding molecule obtained by the production method with an antigen and assessing the formation of an immune complex comprising the antigen-binding molecule and the antigen.
[30]
30. Method according to any of claims 26 to 29, characterized by the fact that the ion concentration condition is a calcium ion concentration condition.
[31]
31. Method according to claim 30, characterized by the fact that the antigen-binding domain has an antigen-binding activity under a condition of low calcium ion concentration that is less than the activity of antigen binding under a high calcium ion concentration condition.
[32]
32. Method according to any one of claims 26 to 31, characterized by the fact that the ion concentration condition is a pH condition.
[33]
33. The method of claim 32, characterized by the fact that the antigen binding domain has an antigen binding activity in an acidic pH range that is lower than the antigen binding activity. under neutral pH range condition.
[34]
34. Method according to any of the claims
26 to 33, characterized by the fact that the antigens that comprise two or more antigenic binding units are multiples.
[35]
35. Method according to claim 34, characterized by the fact that the antigen is any one of 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 (), TNF, TNF-alpha MIC-1, TNF-alphabet, TNF-beta2, TNFSF10 (TRAIL ligand Apo-2, LT2), TNFSF11 (TRANCE / RANK ODF ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13b (BAFF BLyS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTG), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conec- tin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTB TNFC, p33), TNFSF4 (gp34 linker OX40, TXGP1), TNFSF5 (CD40 linker CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 Fas l Apo 1-l ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153 ligand), TNFSF9 (CD137 ligand 4-1BB), VEGF, IgE, IgA, IgG, IgM, RANKL , TGF-alpha, TGF-beta, specific TGF-beta Pan o and IL-8.
[36]
36. Method according to any one of claims 26 to 33, characterized by the fact that antigens comprising two or more antigenic binding units are monomers.
[37]
37. Method according to any one of claims 26 to 36, characterized in that the antigen-binding molecules are multispecific or multiparatopic antigen-binding molecules, or a mixture of antigen-binding molecules.
[38]
38. Method according to any of claims 26 to 37, characterized by the fact that the Fc region is represented
seated by any of SEQ ID NOs: 13, 14, 15, and 16.
[39]
39. Method according to any one of claims 26 to 37, characterized in that the Fc region is an Fc region with enhanced FcRn binding activity under an acidic pH range compared to that of the represented Fc region by any of SEQ ID NOs: 13, 14, 15, and 16.
[40]
40. Method according to claim 39, characterized by the fact that the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of amino acids at positions 238, 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) in the amino acid sequence of the Fc region represented by any of SEQ ID NOs: 13, 14, 15, and 16.
[41]
41. Method according to claim 40, characterized by the fact that the Fc region comprises at least one or more amino acids selected from the group consisting of: Leu for the amino acid of position 238; Read for the amino acid at position 244; Arg for the amino acid at position 245; Pro for the amino acid of position 249; Gln or Glu for the amino acid of position 250; Arg, Asp, Glu, Leu or for the amino acid of position 251; Phe, Ser, Thr, Tyr or for the amino acid of position 252; Ser or Thr for the amino acid of position 254; Arg, Gly, Ile, or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met, Asn, Ser, or Val for the amino acid of position 257; Asp for the amino acid of position 258; Be for the amino acid of position 260; Read for the amino acid at position 262; Lys for the amino acid at position 270; Leu or Arg for the amino acid of position 272; Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of position 279; Ala, Asp, Phe, Gly, His, lie, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of position 283; Asn for the amino acid of position 285; Phe for the amino acid of position 286; Asn or Pro for the amino acid of position 288; Val for the amino acid of position 293; Ala, Glu, Gln, or Met for the amino acid of position 307; Ala, Glu, Ile, Lys, Leu, Met, Ser, Vai, or Trp for the amino acid of position 311; Pro for the amino acid of position 309; Ala, Asp, or Pro for the amino acid of position 312; Ala or Leu for the amino acid at position 314; Lys for the amino acid of position 316; Pro for the amino acid of position 317; Asn or Thr for the amino acid at position 318; Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid of position 332; Asn, Thr, or Trp for the amino acid of position 339; Pro for the amino acid of position 341; Glu, His, Lys, Gln, Arg, Thr, or Tyr for the amino acid of position 343; Arg for the amino acid at position 375; Gly, lie, Met, Pro, Thr, Val or for the amino acid of position 376; Lys for the amino acid of position 377; Asp, Asn, or Val for the amino acid of position 378; Ala, Asn, Ser, or Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid of position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid of position 386; Ala, Arg, His, Pro, Ser, or Thr for the amino acid of position 387; Asn, Pro, or Ser for the amino acid of position 389; Asn for the amino acid of position 423; Asn for the amino acid of position 427; Leu, Met, Phe, Ser, or Thr for the amino acid of position 428; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid of position 430; His or Asn for the amino acid of position 431; Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid of position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid of position 436; Lys, Leu, Thr, or Trp for the amino acid of position 438;
Lys for the amino acid of position 440; and Lys for the amino acid at position 442; Ile, Pro, or Thr for the amino acid of position 308; as indicated by the EU numbering following amino acids of the Fc region represented by any of SEQ ID NOs: 13, 14, 15, and 16.
[42]
42. Method according to any of claims 26 to 37, characterized in that the Fc region is an Fc region with an enhanced FcRn-binding activity under a condition of neutral pH range in relation to that of the represented Fc region by any of SEQ ID Nos: 13, 14, 15 and 16.
[43]
43. Method according to claim 42, characterized by the fact that the Fc region is an Fc region with a substitution of at least one or more amino acids selected from the group consisting of positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the amino acid sequence of the Fc region represented by any of SEQ ID Nos: 13, 14, 15 and 16.
[44]
44. Method according to claim 43, characterized by the fact that the Fc region includes at least one or more amino acids selected from the group consisting of: Met for the amino acid at position 237; Ile for the amino acid of position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr for the amino acid of position 250; PHE, Trp or Tyr for the amino acid of position 252; Thr for the amino acid at position 254; Glu for the amino acid of position 255; ASP, Asn, Glu or Gln for the amino acid at position 256;
Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val for the amino acid of position 257; His for the amino acid of position 258; Wing for the amino acid of position 265; Ala or Glu for the amino acid of position 286; His for the amino acid of position 289; Wing for the amino acid of position 297; Wing for the amino acid of position 303; Wing for the amino acid of position 305; Ala, Asp, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the amino acid of position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr for the amino acid of position 308; Ala, Asp, Glu, Pro or Arg for the amino acid of position 309; Ala, His, or Ile for the amino acid at position 311; Ala or His for the amino acid at position 312; Lys or Arg for the amino acid of position 314; Ala, Asp or His for the amino acid at position 315; Wing for the amino acid of position 317; Val for the amino acid of position 332; Read for the amino acid of position 334; His for the amino acid at position 360; Wing for the amino acid of position 376; Wing for amino acid at position 380; Wing for the amino acid of position 382; Wing for the amino acid of position 384; Asp or His for the amino acid of position 385; Pro for the amino acid of position 386; Glu for the amino acid of position 387;
Ala or Ser for the amino acid of position 389; Wing for the amino acid of position 424; Ala, Asp, Phe Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid position 428; Lys for the amino acid of position 433; Ala, Phe, His, Ser, Trp or Tyr for the amino acid of position 434; and His, Ile, Leu, Phe, Thr or Val for the amino acid at position 436; as indicated by the EU numbering in the amino acid sequence of the Fc region represented by any of SEQ ID Nos: 13, 14, 15 and 16.
[45]
45. Method according to any one of claims 26 to 41, characterized by the fact that the Fc region includes an Fc region that has greater Fcg receptor binding activity than the Fc region of a native human IgG.
[46]
46. Method according to claim 45, characterized by the fact that the Fc region comprises in its amino acid sequence at least one or more amino acids that are different from the amino acids of the Fc region of native human IgG selected from the group consisting of the 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 (EU numbering).
[47]
47. The method of claim 46, characterized
due to the fact that the Fc region comprises in its amino acid sequence at least one amino acid selected from the group consisting of: Lys or Tyr for the amino acid of position 221; Phe, Trp, Glu or Tyr for the amino acid of position 222; Phe, Trp, Glu or Lys for the amino acid of position 223; Phe, Trp, Glu or Tyr for the amino acid of position 224; Glu, Lys or Trp for the amino acid of position 225; Glu, Gly, Lys or Tyr for the amino acid of position 227; Glu, Gly, Lys or Tyr for the amino acid of position 228; Ala, Glu, Gly or Tyr for the amino acid of position 230; Glu, Gly, Lys, Pro or Tyr for the amino acid of position 231; Glu, Gly, Lys or Tyr for the amino acid of position 232; Ala, Asp, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for 234 theamino acid; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 236; ASP, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 237; Asp, Glu, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid at position 238; Asp, Glu, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 239; Ala, Ile, Met or Thr for the amino acid at position 240; Asp, Glu, Leu, Arg, Trp or Tyr for the amino acid of the
tion 241; Leu, Glu, Leu, Gln, Arg, Trp or Tyr for the amino acid of position 243; His for the amino acid at position 244; Wing for the amino acid of position 245; ASP, Glu, His, or Tyr for the amino acid at position 246; Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid at position 247; Glu, His, Gln or Tyr for the amino acid at position 249; Glu or Gln for the amino acid of position 250; Phe for the amino acid of position 251; Phe, Met or Tyr for the amino acid of position 254; Glu, Leu or Tyr for the amino acid at position 255; Ala, Met or Pro for the amino acid at position 256; Asp, Glu, His, Ser or Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for the amino acid at position 260; Ala, Glu, Phe, Ile or Thr for the amino acid of position 262; Ala, Ile, Met or Thr for the amino acid at position 263; Asp, Glu, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 264; Ala, Gly, Leu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 265; Ala, Ile, Met or Thr for the amino acid at position 266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 267; Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp for the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 269;
Phe, glu, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr for the amino acid of position 270; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 271; Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 272; Phe or Ile for the amino acid of position 273; Asp, Glu, Phe Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 274; Leu or Trp for the amino acid at position 275; Asp, Glu, Phe Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid at position 278; Wing for the amino acid of position 279; Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr for the amino acid of position 280; Asp, Lys, Pro or Tyr for the amino acid of position 281; Glu, Gly, Lys, Pro or Tyr for the amino acid of position 282; Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr for the amino acid of heading 283; Asp, Glu, Leu, Asn, Thr or Tyr for the amino acid of position 284; Asp, Glu, Lys, Gln, Trp or Tyr for the amino acid of position 285; Glu, Gly, Pro or Tyr for the amino acid of position 286; Asn, Asp, Glu or Tyr for the amino acid of position 288; Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr for the 290 position amino acid;
Asp, Glu, Gly, His, Ile, Gln or Thr for the amino acid in position 291; Ala, Asp, Glu, Pro, Thr or Tyr for the amino acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 293; Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 294; Asp, Glu, Phe Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acidof 295 position; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val for the amino acid of position 296; Asp, Glu, Phe Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 297; Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr for the 299 amino acid; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp for the amino acid at position 300; Asp, Glu, His, or Tyr for the amino acid of position 301; Ile for the amino acid of position 302; Asp, Gly or Tyr for the amino acid of position 303; Asp, His, Leu, Asn or Thr for the amino acid at position 304; Glu, Ile, Thr or Tyr for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr for the amino acid of position 311; Phe for the amino acid at position 313; Read for the amino acid at position 315;
Glu or Gln for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid at position 320; Ala, Asp, Phe Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr for the amino acid of position 322; Ile for the amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 324; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 325; Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr for the amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 328; ASP, Glu, Phe Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 329; Cys, Phe, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid at position 330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr for the amino acid at position 331; Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr for the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr for the 333 amino acid; Ala, Glu, Phe, Ile, Leu, Pro or Thr for the amino acid of position 334;
ASP, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr for the amino acid of position 335; Glu, Lys or Tyr for the amino acid of position 336; Glu, His, or Asn for the amino acid at position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr for the amino acid of position 339; Ala or Val for the amino acid of position 376; Gly or Lys for the amino acid of position 377; Asp for the amino acid of position 378; Asn for the amino acid of position 379; Ala, Asn or Ser for the amino acid of position 380; Ala or Ile for the amino acid of position 382; Glu for the amino acid of position 385; Thr for the amino acid at position 392; Read for the amino acid of position 396; Lys for the amino acid of position 421; Asn for the amino acid of position 427; Phe or Leu for the amino acid of position 428; Met for the amino acid of position 429; Trp for the amino acid of position 434; Ile for the amino acid of position 436; and Gly, His, Ile, Leu or Tyr for the amino acid at position 440; as indicated by the EU numbering.
[48]
48. Method according to any one of claims 26 to 44, characterized by the fact that the Fc region has greater binding activity towards an Fcγ-inhibiting receptor than towards an Fcγ-activating receptor.
[49]
49. Method according to claim 48, characterized by the fact that the Fcγ inhibitory receptor is human FcγRIIb.
[50]
50. The method of claim 48 or 49, characterized
characterized by the fact that the Fcγ activation receptor is human FcγRII, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) or human FcγRIIIa (F).
[51]
51. Method according to any of claims 48 to 50, characterized in that the amino acid at position 238 or 328 (EU numbering) in the Fc region is different from the amino acid in the Fc region of native human IgG.
[52]
52. Method according to claim 51, characterized by the fact that the amino acid at position 238 of the Fc region is Asp or the amino acid at position 328 of the Fc region is Glu, as indicated by the numbering EU.
[53]
53. Method according to claim 51 or 52, characterized in that the amino acid sequence of the Fc region is composed of at least one or more amino acids selected from the group consisting of: Asp for the amino acid of position 233; Trp or Tyr for the amino acid of position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp or Tyr for the amino acid of position 237; Asp for the amino acid at position 239; Ala, Gln or Val for the amino acid at position 267; Asn, Asp or Glu for the amino acid of position 268; Gly for the amino acid at position 271; Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser or Thr for the amino acid of position 326; Arg, Lys or Met for the amino acid at position 330; Ile, Leu or Met for the amino acid of position 323; and Asp for the amino acid at position 296; as indicated by the EU numbering.
[54]
54. Invention, characterized by the fact that it is in any form of its embodiments (for example, product, method, use, kit, composition, etc.) or in any applicable category of claim, encompassed by the matter initially described, revealed , or illustrated in the patent application as a whole, including illustrative examples thereof.

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法律状态:
2020-11-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-03| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-05-04| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-05-18| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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JP2012123773|2012-05-30|

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JP2012-123773|2012-05-30|

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