ANTIBODIES WITH MODIFIED AFFINITY TO FcRn THAT PROMOTE ANTIGEN CLEARANCE

Abstract

To provide antigen binding molecules promoting antigen uptake into cells and promoting reduction of antigen concentration in plasma, and to provide pharmaceutical compositions comprising the antigen binding molecules, and production methods of the same.SOLUTION: There are provided antibodies whose antigen binding activity in the pH in early endosome is weaker in comparison to the antigen binding activity in the pH in plasma, and having human FcRn binding activity in the pH in plasma. There are provided antigen binding molecules comprising antigen binding domain and human FcRn binding domain, which have human FcRn binding activity in the pH neutral region, and whose human FcRn binding activity in the pH neutral region is stronger than KD 3.2 μM.EFFECT: The antibodies promote antigen uptake into cells, and increase the number of antigens to which a single molecule antibody can bind. Reduction of antigens in plasma is promoted by administering the antibodies, and pharmacokinetics of antibodies can be improved by using the antibodies.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for promoting intracellular uptake of antigens by an antigen-binding molecule, a method for increasing the number of antigens that can be bound by one antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administration of an antigen-binding molecule, a method for improving the pharmacokinetics of an antigen-binding molecule, a method for reducing the total or free antigen concentration in plasma, an antigen-binding molecule with improved intracellular uptake of antigens, an antigen-binding molecule with an increased number of antigens that can be bound, an antigen-binding molecule that can promote a decrease in plasma antigen concentration by administration of the molecule, an antigen-binding molecule with improved pharmacokinetics, a pharmaceutical composition comprising the antigen-binding molecule, a method for producing the above molecule, etc.

[0002] Priority The present invention claims priority to Japanese Patent Application No. 2010-079667, filed March 30, 2010, and Japanese Patent Application No. 2010-250830, filed November 9, 2010, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Document 1 and Non-Patent Document 2). On the other hand, various technologies applicable to second-generation antibody drugs have been developed, and technologies for improving effector function, antigen binding ability, pharmacokinetics, and stability, or reducing the risk of immunogenicity, etc. have been reported (Non-Patent Document 3). Since antibody drugs generally require a very high dose, problems include the difficulty of preparing a subcutaneous administration formulation and the high manufacturing cost. Methods for reducing the dose of antibody drugs include a method for improving the pharmacokinetics of the antibody and a method for improving the affinity between the antibody and the antigen.

[0004] Artificial amino acid substitution in the constant region has been reported as a method for improving the pharmacokinetics of antibodies (Non-Patent Documents 4 and 5). Affinity maturation technology (Non-Patent Document 6) has been reported as a technique for enhancing antigen-binding ability and antigen-neutralizing ability, and it is possible to enhance the binding activity to an antigen by introducing mutations into amino acids in the CDR region of the variable region, etc. By enhancing the antigen-binding ability, it is possible to improve the in vitro biological activity or reduce the dosage, and further to improve the drug efficacy in the body (Non-Patent Document 7).

[0005] On the other hand, the amount of antigen that can be neutralized by one antibody molecule depends on the affinity, and by strengthening the affinity, it is possible to neutralize the antigen with a small amount of antibody, and it is possible to strengthen the affinity of an antibody by various methods (Non-Patent Document 6). Furthermore, if it is possible to covalently bind to the antigen and make the affinity infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of bivalent antibodies) with one molecule of antibody. However, in the previous methods, the stoichiometric neutralization reaction of one molecule of antigen (two antigens in the case of bivalent antibodies) with one molecule of antibody was the limit, and it was impossible to completely neutralize the antigen with an antibody amount less than the amount of antigen. In other words, there was a limit to the effect of strengthening affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain its neutralizing effect for a certain period of time, it is necessary to administer an antibody amount equal to or greater than the amount of antigen produced in the body during that period, and there was a limit to reducing the required antibody dosage only by improving the pharmacokinetics of the antibody or affinity maturation technology described above. Therefore, in order to maintain the antigen neutralizing effect for a desired period of time with an antibody amount equal to or less than the antigen amount, it is necessary to neutralize multiple antigens with one antibody. As a new method for achieving this, an antibody that binds to an antigen in a pH-dependent manner has recently been reported (Patent Document 1). A pH-dependent antigen-binding antibody that strongly binds to an antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in endosomes can dissociate from the antigen in endosomes. A pH-dependent antigen-binding antibody can bind to an antigen again when the antibody is recycled into plasma by FcRn after dissociating from the antigen, so one pH-dependent antigen-binding antibody can repeatedly bind to multiple antigens.

[0006] In addition, the plasma retention of antigens is very short compared to antibodies that bind to FcRn and are recycled. When an antibody with such a long plasma retention binds to the antigen, the plasma retention of the antibody-antigen complex becomes as long as that of the antibody. Therefore, when an antigen binds to an antibody, the plasma retention of the antigen becomes longer, and the plasma antigen concentration increases.

[0007] IgG antibodies have a long plasma retention time by binding to FcRn. Binding between IgG and FcRn is observed only under acidic conditions (pH 6.0), and almost no binding is observed under neutral conditions (pH 7.4). IgG antibodies are nonspecifically taken up by cells, but return to the cell surface by binding to FcRn in endosomes under acidic conditions in endosomes, and dissociate from FcRn under neutral conditions in plasma. If a mutation is introduced into the Fc domain of IgG to lose the binding to FcRn under acidic conditions, the antibody will no longer be recycled from endosomes to plasma, and the plasma retention time of the antibody will be significantly impaired. A method for improving the plasma retention of IgG antibodies has been reported that improves the binding to FcRn under acidic conditions. By introducing amino acid substitutions into the Fc domain of IgG antibodies to improve the binding to FcRn under acidic conditions, the recycling efficiency from endosomes to plasma is increased, and as a result, the plasma retention time is improved. When introducing amino acid substitutions, it is important not to increase the binding to FcRn under neutral conditions. If an IgG antibody binds to FcRn under neutral conditions, even if it returns to the cell surface by binding to FcRn under the acidic conditions in the endosome, the IgG antibody will not be recycled into plasma unless it dissociates from FcRn in plasma under neutral conditions, and the plasma retention of the antibody will be impaired. For example, as described in J Immunol. 2002;169(9):5171-80., it has been reported that when an antibody that has been administered to mice and that has been shown to bind to mouse FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions into IgG1 is administered to mice, the plasma retention of the antibody is impaired.Furthermore, as described in J Immunol. 2009;182(12):7663-71., J Biol Chem. 2007 Jan 19;282(3):1709-17., and J Immunol. 2002 Nov 1;169(9):5171-80., it has been reported that the introduction of amino acid substitutions into IgG1 improves the binding to human FcRn under acidic conditions (pH 6.0), but at the same time, when an antibody that was shown to bind to human FcRn under neutral conditions (pH 7.4) was administered to cynomolgus monkeys, the plasma retention of the antibody did not improve and no change was observed in the plasma retention. Therefore, in antibody engineering techniques for improving antibody functions, efforts have been focused only on improving the plasma retention of antibodies by increasing the binding to human FcRn under acidic conditions without increasing the binding to human FcRn under neutral conditions (pH 7.4), and no benefits have been reported so far for increasing the binding to human FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions into the Fc domain of an IgG antibody. Even if the affinity of an antibody for an antigen is improved, it is not possible to promote the elimination of the antigen from plasma. It has been reported that the above-mentioned pH-dependent antigen-binding antibody is also effective as a method for promoting the elimination of antigens from plasma compared to normal antibodies (Patent Document 1).

[0008] Thus, a pH-dependent antigen-binding antibody binds to multiple antigens with a single antibody and can promote the elimination of antigens from plasma compared to a normal antibody, and therefore has effects that normal antibodies cannot achieve. However, no antibody engineering techniques have been reported to date that can further improve the effect of this pH-dependent antigen-binding antibody in repeatedly binding to antigens and in promoting the elimination of antigens from plasma.

[0009] The prior art documents of the present invention are listed below. [Prior art documents] [Patent documents]

[0010]

Patent Document 1

Non-licensed literature

[0011] [Non-licensed document 1] Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology 23, 1073 - 1078 (2005) [Non-licensed document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr;59(3):389-96. [Non-licensed document 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31;20(1):17-29. Review.

Non-licensed Document 4

Non-licensed Document 5

Outdoor Configuration6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

[0012] The present invention has been made in consideration of these circumstances, and an object of the present invention is to provide a method for promoting intracellular antigen uptake by an antigen-binding molecule, a method for increasing the number of antigens that can be bound by one antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administration of an antigen-binding molecule, a method for improving the pharmacokinetics of an antigen-binding molecule, an antigen-binding molecule with promoted intracellular antigen uptake, an antigen-binding molecule with an increased number of antigens that can be bound, an antigen-binding molecule that can promote a decrease in plasma antigen concentration by administration, an antigen-binding molecule with improved pharmacokinetics, a pharmaceutical composition comprising the antigen-binding molecule, and methods for producing them. [Means for solving the problem]

[0013] The present inventors have conducted intensive research into a method for promoting the uptake of an antigen into cells by an antigen-binding molecule (a molecule such as a polypeptide having antigen-binding ability), a method for an antigen-binding molecule to bind to an antigen multiple times, a method for promoting a decrease in the antigen concentration in plasma by administration of an antigen-binding molecule, and a method for improving the plasma retention of an antigen-binding molecule. As a result, the present inventors have found that an antigen-binding molecule that has the ability to bind to human FcRn at pH in early endosomes and has a higher binding activity to human FcRn at pH in plasma compared to intact human IgG immunoglobulin can promote the uptake of an antigen into cells, and further, that an antigen-binding molecule that has a weaker antigen-binding activity at pH in early endosomes compared to the antigen-binding activity at pH in plasma can further promote the uptake of an antigen into cells by the antigen-binding molecule, can increase the number of antigens that can be bound by one antigen-binding molecule, can promote a decrease in the antigen concentration in plasma by administration of the antigen-binding molecule, and can improve the pharmacokinetics of the antigen-binding molecule.

[0014] That is, the present invention relates to a method for promoting intracellular uptake of antigens by an antigen-binding molecule, a method for increasing the number of antigens that can be bound by one antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administration of an antigen-binding molecule, a method for improving the pharmacokinetics of an antigen-binding molecule, a method for reducing the total or free antigen concentration in plasma, an antigen-binding molecule with promoted intracellular uptake of antigens, an antigen-binding molecule with an increased number of antigens that can be bound, an antigen-binding molecule that can promote a decrease in plasma antigen concentration by administration of the molecule, an antigen-binding molecule with improved pharmacokinetics, a pharmaceutical composition comprising the antigen-binding molecule, and methods for producing them. More specifically, the present invention relates to [1] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in an acidic pH range and a neutral pH range, and the human FcRn-binding activity in a neutral pH range is greater than 3.2 μM. [2] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range, and the human FcRn-binding activity in a neutral pH range is 28-fold higher than that of intact human IgG. [3] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding molecule has human FcRn-binding activity in a neutral pH range, and the human FcRn-binding activity in a neutral pH range is stronger than 2.3 μM. [4] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding molecule has human FcRn-binding activity in a neutral pH range, and the human FcRn-binding activity in a neutral pH range is 38-fold higher than that of intact human IgG. [5] The antigen-binding molecule according to any one of [1] to [4], wherein the neutral pH range is pH 7.0 to 8.0. [6] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the total antigen concentration in plasma after administration of the antigen-binding molecule to a non-human animal is lower than the total antigen concentration in plasma after administration of a control antigen-binding molecule comprising the same antigen-binding domain and an intact human IgG Fc domain as the human FcRn-binding domain to a non-human animal. [7] An antigen-binding molecule, wherein the plasma antigen concentration after administration of the antigen-binding molecule to a non-human animal is lower than the total antigen concentration in plasma obtained from a non-human animal to which the antigen-binding molecule is not administered. [8] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, C=A / B The antigen / antigen-binding molecule molar ratio (C) of the antigen-binding molecule calculated as follows: C'=A' / B' is lower than the antigen / antigen-binding molecule molar ratio (C') of an antigen-binding molecule comprising the same antigen-binding domain and an intact human IgG Fc domain as the human FcRn-binding domain, calculated as follows: During the ceremony A is the total antigen concentration in plasma after administration of the antigen-binding molecule to a non-human animal; B is the plasma antigen-binding molecule concentration after administration of the antigen-binding molecule to a non-human animal; A' is the total antigen concentration in plasma after administration of a control antigen-binding molecule to a non-human animal; B' is the plasma antigen-binding molecule concentration after administration of a control antigen-binding molecule to a non-human animal; The antigen-binding molecule, [9] The antigen-binding molecule according to any one of [6] to [8], wherein the non-human animal is a human FcRn transgenic mouse.

[10] The antigen-binding molecule according to any one of [6] to [9], wherein the plasma antigen concentration is a long-term total plasma antigen concentration.

[11] The antigen-binding molecule according to any one of [6] to [9], wherein the plasma antigen concentration is a short-term total plasma antigen concentration.

[12] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding molecule has human FcRn-binding activity in an acidic pH range and a neutral pH range, and the human FcRn-binding activity in a neutral pH range is higher than that of an intact human IgG.

[13] The antigen-binding molecule according to any one of [1] to

[11] , wherein the antigen-binding activity of the antigen-binding domain in an acidic pH range is lower than that in a neutral pH range.

[14] The antigen-binding molecule of

[12] or

[13] , wherein the ratio of the antigen-binding activity in an acidic pH range to the antigen-binding activity in a neutral pH range is at least 2, as expressed by KD(acidic pH range) / KD(neutral pH range).

[15] The antigen-binding molecule according to any one of

[12] to

[14] , which comprises an amino acid mutation in the antigen-binding domain, comprising substitution of at least one amino acid in the antigen-binding domain with histidine or insertion of at least one histidine.

[16] The antigen-binding molecule according to any one of

[12] to

[14] , wherein the antigen-binding domain is obtained from an antigen-binding domain library.

[17] The antigen-binding molecule according to any one of [1] to

[16] , which comprises, as a human FcRn-binding domain, an Fc domain generated by substituting at least one amino acid in the Fc domain of a parent IgG with another amino acid.

[18] The human FcRn-binding domain is selected from the group consisting of: 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 370, 372, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 389, 390, 391, 392, 393, 394, 395, 396, 397, The antigen-binding molecule of any one of [1] to

[17] , which is a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from the amino acids at positions 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[19] EU Numbering Amino acid substitution at position 237, replacing Gly with Met; Amino acid substitution of Pro at position 238 to Ala; Amino acid substitution at position 239 replacing Ser with Lys; Amino acid substitution at position 248, replacing Lys with Ile; an amino acid substitution of Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; an amino acid substitution of Met at position 252 with Phe, Trp, or Tyr; Amino acid substitution at position 254 replacing Ser with Thr; Amino acid substitution at position 255, replacing Arg with Glu; an amino acid substitution of Thr at position 256 with Asp, Glu, or Gln; an amino acid substitution of Pro at position 257 with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; Amino acid substitution at position 258, replacing Glu with His; Amino acid substitution of Asp at position 265 to Ala; an amino acid substitution of Asp at position 270 with Phe; Amino acid substitution of Asn at position 286 with Ala or Glu; Amino acid substitution of Thr at position 289 to His; Amino acid substitution of Asn at position 297 to Ala; Amino acid substitution at position 298 replacing Ser with Gly; Amino acid substitution of Val at position 303 to Ala; Amino acid substitution of Val to Ala at position 305; an amino acid substitution replacing Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg; an amino acid substitution of Gln at position 311 with Ala, His, or Ile; an amino acid substitution of Asp at position 312 with Ala or His; Amino acid substitution of Leu at position 314 with Lys or Arg; an amino acid substitution of Asn at position 315 with Ala or His; Amino acid substitution of Lys at position 317 to Ala; an amino acid substitution of Asn at position 325 with Gly; an amino acid substitution at position 332 replacing Ile with Val; Amino acid substitution at position 334, replacing Lys with Leu; Amino acid substitution at position 360, replacing Lys with His; an amino acid substitution of Asp at position 376 with Ala; Amino acid substitution of Glu at position 380 to Ala; Amino acid substitution of Glu at position 382 to Ala; an amino acid substitution at position 384 replacing Asn or Ser with Ala; an amino acid substitution of Gly at position 385 with Asp or His; an amino acid substitution of Gln at position 386 with Pro; Amino acid substitution of Pro at position 387 to Glu; an amino acid substitution of Asn at position 389 with Ala or Ser; Amino acid substitution at position 424, replacing Ser with Ala; an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; Amino acid substitution at position 433, replacing His with Lys; an amino acid substitution replacing Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and Amino acid substitution at position 436 replacing Tyr or Phe with His Amino acid substitutions in the Fc domain of the parent IgG, comprising at least one amino acid substitution selected from Human FcRn-binding domain comprising The antigen-binding molecule according to any one of [1] to

[18] ,

[20] The human FcRn-binding domain is identical to the Fc domain of the parent IgG (EU numbering system). Met at amino acid 237; Ala at amino acid 238; Lys at amino acid 239; Ile at amino acid 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at amino acid position 250; Phe, Trp, or Tyr at amino acid 252; Thr at amino acid 254; Glu at amino acid 255, Asp, Glu, or Gln at amino acid 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257; His at amino acid 258, Ala at amino acid 265; Phe at amino acid 270, Ala or Glu at amino acid 286; His at amino acid 289; Ala at amino acid 297; Gly at amino acid 298; Ala at amino acid 303; Ala at amino acid 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid 307; Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid 308; Ala, Asp, Glu, Pro, or Arg at amino acid position 309; Ala, His, or Ile at amino acid 311; Ala or His at amino acid 312; Lys or Arg at amino acid 314; Ala or His at amino acid 315; Ala at amino acid 317; Gly at amino acid 325; Val at amino acid 332; Leu at amino acid 334, His at amino acid 360, Ala at amino acid 376; Ala at amino acid 380; Ala at amino acid 382; Ala at amino acid 384; Asp or His at amino acid 385; Pro at amino acid 386, Glu at amino acid 387, Ala or Ser at amino acid 389; Ala at amino acid 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid 428; Lys at amino acid 433; Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid 436 The antigen-binding molecule according to any one of [1] to

[18] , which comprises at least one amino acid selected from

[21] The antigen-binding molecule according to any one of

[18] to

[20] , wherein the parent IgG is selected from IgG obtained from a non-human animal.

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

[18] to

[20] , wherein the parent IgG is human IgG.

[23] The antigen-binding molecule according to any one of [1] to

[22] , which has antagonist activity.

[24] The antigen-binding molecule according to any one of [1] to

[23] , which binds to a membrane antigen or a soluble antigen.

[25] The antigen-binding molecule according to any one of [1] to

[24] , wherein the antigen-binding domain comprises an artificial ligand that binds to a receptor.

[26] The antigen-binding molecule according to any one of [1] to

[24] , wherein the antigen-binding domain comprises an artificial receptor that binds to a ligand.

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

[24] , which is an antibody.

[28] The antigen-binding molecule of

[27] , wherein the antibody is selected from a chimeric antibody, a humanized antibody, or a human antibody.

[29] A pharmaceutical composition comprising any one of the antigen-binding molecules according to [1] to

[28] .

[30] A method for promoting intracellular antigen uptake by an antigen-binding molecule, comprising: an antigen-binding domain and a human FcRn-binding domain, and having human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range;

[31] A method for promoting intracellular antigen uptake by an antigen-binding molecule, the method comprising: increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range, the antigen-binding activity of the antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain and having human FcRn-binding activity in an acidic pH range, and reducing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range.

[32] A method for increasing the number of antigens that can be bound to a single antigen-binding molecule by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range, the method comprising:

[33] A method for increasing the number of antigens that can be bound to a single antigen-binding molecule, the method comprising: increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range, the human FcRn-binding activity of the antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain and having human FcRn-binding activity in an acidic pH range, and decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range.

[34] A method for increasing the ability of an antigen-binding molecule to eliminate antigens from plasma, the antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain and having human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range.

[35] A method for increasing the plasma antigen elimination ability of an antigen-binding molecule, which comprises an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range and decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range.

[36] A method for improving the pharmacokinetics of an antigen-binding molecule, which comprises an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range.

[37] A method for improving the pharmacokinetics of an antigen-binding molecule, which comprises an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range and reducing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range.

[38] A method for promoting intracellular dissociation of an antigen bound to the antigen-binding molecule outside a cell from the antigen-binding molecule, comprising increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range and decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range, in an antigen-binding molecule that contains an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range.

[39] A method for promoting extracellular release of an antigen-binding molecule that has been taken up into a cell in an antigen-bound state, wherein the antigen-binding molecule comprises an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range and decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range;

[40] A method for reducing the total or free antigen concentration in plasma by increasing the human FcRn-binding activity of an antigen-binding molecule in a neutral pH range, the antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain and having human FcRn-binding activity in an acidic pH range.

[41] A method for reducing the total or free antigen concentration in plasma of an antigen-binding molecule that contains an antigen-binding domain and a human FcRn-binding domain and has human FcRn-binding activity in an acidic pH range, by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range and decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range.

[42] The method according to any one of

[30] to

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

[43] The method according to any one of

[30] to

[41] , wherein the increase in human FcRn-binding activity in a neutral pH range is achieved by substituting at least one amino acid in the parent IgG Fc domain of the human FcRn-binding domain with another amino acid.

[44] The increase in human FcRn-binding activity in the neutral pH range is due to the incorporation of the human FcRn-binding domain at the following positions (EU numbering): 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 350, 352, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 370, 372, 374, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, The method according to any one of

[30] to

[41] , wherein the increase is due to substitution of at least one amino acid selected from the amino acids at positions 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 with another amino acid;

[45] The method according to any one of

[31] ,

[33] ,

[35] ,

[37] to

[39] , and

[41] , wherein the antigen-binding activity of the antigen-binding molecule in an acidic pH range is reduced compared to the antigen-binding activity in a neutral pH range by substituting at least one amino acid of the antigen-binding molecule with histidine or inserting at least one histidine into the antigen-binding molecule.

[46] The method according to any one of

[31] ,

[33] ,

[35] ,

[37] to

[39] , and

[41] , wherein the antigen-binding domain is obtained from an antigen-binding domain library.

[47] The method according to any one of

[31] ,

[33] ,

[35] ,

[37] to

[39] , and

[41] , wherein the decrease in antigen-binding activity is indicated by an increase in the value of KD(acidic pH range) / KD(neutral pH range), which is the ratio of antigen-binding activity in an acidic pH range to antigen-binding activity in a neutral pH range, compared to before the histidine substitution or insertion.

[48] ​​A method for producing an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity of 3.2 μM or stronger in the neutral pH range, which is obtained by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule; (b) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) are linked; and (c) producing an antigen-binding molecule using the gene prepared in (b);

[49] A method for producing an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) modifying at least one amino acid in the antigen-binding domain of an antigen-binding molecule and selecting an antigen-binding molecule having higher antigen-binding activity in a neutral pH range than in an acidic pH range; (c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and (d) producing an antigen-binding molecule using the gene prepared in (c);

[50] A method for producing an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) selecting an antigen-binding molecule whose antigen-binding activity in a neutral pH range is higher than that in an acidic pH range; (c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and (d) producing an antigen-binding molecule using the gene prepared in (c);

[51] An antigen-binding molecule produced by the production method according to any one of

[48] to

[50] .

[52] A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity of 3.2 μM or stronger in the neutral pH range, which is obtained by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule; (b) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) are linked; and (c) producing an antigen-binding molecule using the gene prepared in (b);

[53] A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) modifying at least one amino acid in the antigen-binding domain of an antigen-binding molecule and selecting an antigen-binding molecule having higher antigen-binding activity in a neutral pH range than in an acidic pH range; (c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and (d) producing an antigen-binding molecule using the gene prepared in (c);

[54] A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) selecting an antigen-binding molecule whose antigen-binding activity in a neutral pH range is higher than that in an acidic pH range; (c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and (d) producing an antigen-binding molecule using the gene prepared in (c);

[55] The method according to any one of

[30] to

[54] , wherein the antigen-binding domain comprises an artificial ligand that binds to a receptor.

[56] The method according to any one of

[30] to

[54] , wherein the antigen-binding domain comprises an artificial receptor that binds to a ligand; and

[57] The method according to any one of

[30] to

[54] , wherein the antigen-binding molecule is an antibody. Regarding. Effect of the Invention

[0015] The present invention provides a method for promoting intracellular uptake of antigens by antigen-binding molecules, a method for increasing the number of antigens that can be bound by one antigen-binding molecule, and a method for promoting a decrease in plasma antigen concentration by administering the same. By promoting intracellular uptake of antigens by antigen-binding molecules, it becomes possible to promote a decrease in the antigen concentration in plasma by administering the antigen-binding molecule, and it becomes possible to improve the pharmacokinetics of the antigen-binding molecule and increase the number of antigens that can be bound by one antigen-binding molecule, thereby enabling the antigen-binding molecule to exert a more excellent effect in vivo than a normal antigen-binding molecule. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the time course of plasma soluble human IL-6 receptor concentration after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276) (steady-state infusion model) in which plasma soluble human IL-6 receptor (hsIL-6R) concentration is maintained at steady state. [Diagram 2] FIG. 1 is a schematic diagram showing that IgG antibody molecules dissociate from soluble antigens in endosomes, accelerating the disappearance of the antigens and allowing them to bind to new antigens again. [Diagram 3] FIG. 1 shows time courses of antibody plasma concentrations in human FcRn transgenic mice. [Figure 4] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in human FcRn transgenic mice. [Diagram 5] FIG. 1 shows time courses of antibody plasma concentrations in normal mice. [Figure 6] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in normal mice. [Figure 7] FIG. 1 shows the time course of plasma concentration of unbound soluble human IL-6 receptor in normal mice. [Figure 8] FIG. 1 shows time courses of soluble human IL-6 receptor plasma concentration in human FcRn transgenic mice. [Figure 9] FIG. 1 shows the time course of plasma soluble human IL-6 receptor concentration after administration of Fv4-IgG1-F14 at a low dose (0.01 mg / kg) or 1 mg / kg. [Figure 10] FIG. 1 shows the time course of plasma antibody concentration after administration of Fv4-IgG1-F14 at a low dose (0.01 mg / kg) or 1 mg / kg. [Figure 11] FIG. 1 shows the time course of plasma soluble human IL-6 receptor concentration after administration of anti-human IL-6 receptor antibody to normal mice in which the plasma soluble human IL-6 receptor concentration is maintained at a steady state. [Figure 12]FIG. 1 shows the time course of plasma antibody concentration after simultaneous injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 13] FIG. 1 shows the time course of plasma soluble human IL-6 receptor concentration after simultaneous injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 14] FIG. 1 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the plasma hsIL-6R concentration one day after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 15] FIG. 1 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the plasma antibody concentration one day after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 16] FIG. 1 shows the time course of the antigen / antibody molar ratio (C value) after simultaneous injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 17] FIG. 1 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the antigen / antibody molar ratio (C value) one day after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276). [Figure 18] FIG. 1 shows the time course of plasma hsIL-6R concentration after administration of a low dose (0.01 or 0.2 mg / kg) or 1 mg / kg of Fv4-IgG1-F14 to human FcRn transgenic mice (strain 276) in which plasma hsIL-6R concentration is maintained at a steady state (steady-state infusion model). [Figure 19]FIG. 1 shows the time course of plasma hsIL-6R concentration in human FcRn transgenic mice (lines 276 and 32) after co-injection of hsIL-6R and anti-human IL-6 receptor antibody. [Figure 20] FIG. 1 shows the time course of plasma antibody concentrations in human FcRn transgenic mice (lines 276 and 32) after co-injection of hsIL-6R and anti-human IL-6 receptor antibody. [Figure 21] FIG. 1 shows the time course of plasma hsIL-6R concentration after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentration is maintained at steady state. [Figure 22] FIG. 1 shows the time course of plasma antibody concentration after administration of an anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentration is maintained at a steady state. [Diagram 23] FIG. 1 shows the time course of the antigen / antibody molar ratio (C value) after administration of an anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which the plasma hsIL-6R concentration is maintained at a steady state. [Figure 24] FIG. 1 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the antigen / antibody molar ratio (C value) 1 day after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which the plasma hsIL-6R concentration is maintained at a steady state. [Diagram 25] FIG. 1 shows the time course of plasma antibody concentration after administration of anti-human IL-6 receptor antibodies having the Fc variants F11, F39, F48, and F264 to human FcRn transgenic mice (strain 32) in which plasma hsIL-6R concentration is maintained at a steady state (steady-state infusion model). [Figure 26] FIG. 1 shows the time course of plasma hsIL-6R concentration after administration of anti-human IL-6 receptor antibodies having the Fc variants F11, F39, F48, and F264 to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentration is maintained at a steady state. [Figure 27] FIG. 1 shows the time course of plasma antibody concentration after administration of anti-human IL-6 receptor antibodies having the Fc variants F157, F196, and F262 to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentration is maintained at a steady state. [Figure 28] FIG. 1 shows the time course of plasma hsIL-6R concentration after administration of anti-human IL-6 receptor antibodies having the Fc variants F157, F196, and F262 to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentration is maintained at a steady state. [Figure 29] FIG. 1 shows the pharmacokinetic models used for in silico testing of conventional and antigen-eliminating antibodies. [Diagram 30] FIG. 1 shows the time course of human IL-6 plasma concentration after human IL-6 and anti-human IL-6 antibody were simultaneously injected into normal mice. [Diagram 31] FIG. 1 shows the time course of plasma antibody concentration after human IL-6 and anti-human IL-6 antibody were simultaneously injected into normal mice. [Diagram 32] FIG. 1 shows sensorgrams of human IgA binding to CD89-Fc fusion protein at pH 7.4 and pH 6.0 using Biacore. [Diagram 33] FIG. 1 shows the time course of human IgA plasma concentration after simultaneous injection of human IgA and CD89-Fc fusion protein into normal mice. [Diagram 34] FIG. 1 shows the time course of plasma antibody concentrations after simultaneous injection of human IgA and CD89-Fc fusion protein into normal mice. [Diagram 35]FIG. 1 shows plasma concentrations of soluble human Plexin A1 7 hours after simultaneous injection of soluble human Plexin A1 and anti-human Plexin A1 antibody into normal mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule. More specifically, the present invention provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule having human FcRn-binding activity in an acidic pH range by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range. Furthermore, the present invention provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule having human FcRn-binding activity in an acidic pH range by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule.

[0018] Furthermore, the present invention relates to a human FcRn-binding domain comprising an Fc domain of a parent IgG, the human FcRn-binding domain comprising: The present invention provides a method for promoting intracellular antigen uptake by an antigen-binding molecule that has binding activity to human FcRn in an acidic pH range, by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from positions 0, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0019] The present invention further provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule, by lowering the antigen-binding activity (binding ability) of the antigen-binding molecule that promotes intracellular uptake of an antigen in an acidic pH range compared to its antigen-binding activity in a neutral pH range. The present invention further provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule, by modifying at least one amino acid in the antigen-binding domain in the antigen-binding molecule that promotes intracellular uptake of an antigen. The present invention further provides a method for promoting intracellular uptake of an antigen by an antigen-binding molecule, comprising substituting at least one amino acid in the antigen-binding domain in the antigen-binding molecule that promotes intracellular uptake of an antigen with histidine or inserting at least one histidine.

[0020] In the present invention, "uptake of antigen into cells" by an antigen-binding molecule means that the antigen is taken up into cells by endocytosis. In addition, in the present invention, "promoting uptake into cells" means that the rate at which an antigen-binding molecule bound to an antigen in plasma is taken up into cells is promoted and / or the amount of the taken-up antigen recycled into plasma is reduced. It is sufficient that the rate of uptake into cells is promoted compared to the antigen-binding molecule before increasing the binding activity of the antigen-binding molecule to human FcRn in a neutral pH range or before decreasing the antigen-binding activity (binding ability) of the antigen-binding molecule in an acidic pH range to that in a neutral pH range in addition to increasing the binding activity of the antigen-binding molecule to human FcRn, and it is preferable that the rate of uptake into cells is promoted more than that of intact human IgG, and it is particularly preferable that the rate of uptake is promoted more than that of intact human IgG. Therefore, in the present invention, whether or not the uptake of antigen into cells by an antigen-binding molecule has been promoted can be determined by whether or not the rate of uptake of antigen into cells has increased. The rate of antigen uptake into cells can be calculated, for example, by adding an antigen-binding molecule and an antigen to a culture medium containing human FcRn-expressing cells and measuring the decrease in the concentration of the antigen in the culture medium over time, or by measuring the amount of antigen taken up into human FcRn-expressing cells over time. By utilizing the method of promoting the rate of antigen uptake into cells by the antigen-binding molecule of the present invention, for example, by administering the antigen-binding molecule, the rate of antigen disappearance in plasma can be promoted. Therefore, whether or not the uptake of antigen into cells by the antigen-binding molecule has been promoted can also be confirmed by, for example, measuring whether or not the disappearance rate of antigen present in plasma has been accelerated, or whether or not the total antigen concentration in plasma has been reduced by administration of the antigen-binding molecule.

[0021] In the present invention, the term "total antigen concentration in plasma" refers to the sum of the antigen concentration bound to an antigen-binding molecule and the "free antigen concentration in plasma", which is the concentration of unbound antigen or unbound antigen. Various methods for measuring the "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described herein below.

[0022] In the present invention, "intact human IgG" refers to unmodified human IgG and is not limited to a specific class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as "intact human IgG" as long as it can bind to human FcRn in the acidic pH range. Preferably, the "intact human IgG" can be human IgG1.

[0023] The present invention also provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule. More specifically, the present invention provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule having binding activity to human FcRn in an acidic pH range by increasing the binding activity of the antigen-binding molecule to human FcRn in a neutral pH range. Furthermore, the present invention provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule having binding activity to human FcRn in an acidic pH range by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule.

[0024] Furthermore, the present invention relates to a human FcRn-binding domain comprising an Fc domain of a parent IgG, the human FcRn-binding domain comprising: The present invention provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule that has binding activity to human FcRn in an acidic pH range, by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from positions 0, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0025] In the present invention, "parent IgG" refers to an unmodified IgG that is subsequently modified to generate a variant, and the modified variant of the parent IgG is capable of binding to human FcRn in the acidic pH range (thus, the parent IgG does not necessarily need to have binding activity to human FcRn under acidic conditions). The parent IgG may be a naturally occurring IgG, or a variant or engineered version of a naturally occurring IgG. The parent IgG may refer to the polypeptide itself, a composition comprising the parent IgG, or an amino acid sequence that encodes the parent IgG. It should be noted that the "parent IgG" includes known commercially available IgGs produced by recombinant methods as outlined below. The source of the "parent IgG" may be obtained from any organism of a non-human animal or from a human, but is not limited thereto. Preferably, the organism is selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In another embodiment, the "parent IgG" may also be obtained from a cynomolgus monkey, a marmoset, a rhesus monkey, a chimpanzee, or a human. Preferably, the "parent IgG" is derived from human IgG1, but is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as the "parent IgG" as appropriate. Similarly, any class or subclass of IgG from any organism previously described herein can be preferably used as the "parent IgG". Examples of naturally occurring IgG variants or engineered forms are described, but are not limited to, in Curr Opin Biotechnol. 2009 Dec; 20(6): 685-91, Curr Opin Immunol. 2008 Aug; 20(4): 460-70, Protein Eng Des Sel. 2010 Apr; 23(4): 195-202, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338.

[0026] The present invention further provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule by reducing the antigen-binding activity (binding ability) in an acidic pH range of an antigen-binding molecule having an increased number of antigen-binding events compared to the antigen-binding activity in a neutral pH range. The present invention further provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule by modifying at least one amino acid in the antigen-binding domain in the antigen-binding molecule having an increased number of antigen-binding events. The present invention further provides a method for increasing the number of antigens that can be bound by one antigen-binding molecule, comprising substituting at least one amino acid in the antigen-binding domain in the antigen-binding molecule having an increased number of antigen-binding events with histidine or inserting at least one histidine.

[0027] In the present invention, the term "the number of antigens that one antigen-binding molecule can bind" refers to the number of antigens that can be bound to the antigen-binding molecule before it is decomposed and disappears. In the present invention, "increasing the number of antigens that one antigen-binding molecule can bind to" refers to increasing the number of cycles that can be completed before the antigen-binding molecule is decomposed and disappears, where one cycle is when an antigen binds to the antigen-binding molecule in plasma, the antigen-bound antigen-binding molecule is taken up into cells, dissociates the antigen in an endosome, and then the antigen-binding molecule returns to plasma. The number of cycles may be increased compared to the antigen-binding molecule before the antigen-binding molecule is decomposed and disappears, in addition to increasing the binding activity of the antigen-binding molecule to human FcRn in a neutral pH range, or the antigen-binding activity (binding ability) of the antigen-binding molecule in an acidic pH range is reduced to be lower than the antigen-binding activity in a neutral pH range in addition to increasing the binding activity of the antigen-binding molecule to human FcRn. Therefore, whether the number of cycles has increased can be determined by whether the "incorporation into cells has been promoted" as described above, or whether the "pharmacokinetics has been improved" as described below.

[0028] The present invention also provides a method for promoting intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule. More specifically, the present invention provides a method for promoting intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule by increasing the human FcRn-binding activity in a neutral pH range of an antigen-binding molecule that has human FcRn-binding activity in an acidic pH range and lowering the antigen-binding activity in an acidic pH range compared to the antigen-binding activity in a neutral pH range. The present invention further provides a method for promoting intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule by modifying at least one amino acid in the antigen-binding domain of the antigen-binding molecule and modifying at least one amino acid in the human FcRn-binding domain that has human FcRn-binding activity in an acidic pH range of the antigen-binding molecule. The present invention further provides a method for promoting intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule by substituting at least one amino acid in the antigen-binding domain of the antigen-binding molecule with histidine or inserting at least one histidine, and modifying the human FcRn-binding domain of a parent IgG. EU numbering in the Fc domain: 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332 The present invention provides a method for promoting dissociation of an antigen bound to an antigen-binding molecule outside a cell from the antigen-binding molecule within a cell, the method comprising: substituting at least one amino acid selected from among positions 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 with another amino acid.

[0029] In the present invention, the site where an antigen dissociates from an antigen-binding molecule may be any site within a cell, but is preferably within an early endosome. In the present invention, "dissociation of an antigen bound to an antigen-binding molecule outside a cell from the antigen-binding molecule within a cell" does not mean that all antigens bound to an antigen-binding molecule and taken up into a cell need to dissociate from the antigen-binding molecule within a cell, but it is sufficient that the antigen-binding activity of the antigen-binding molecule in an acidic pH range is lower than that in a neutral pH range, and the proportion of antigens dissociated from the antigen-binding molecule within a cell is higher than that before the human FcRn-binding activity in a neutral pH range is increased. In addition, a method for promoting dissociation of an antigen bound to an antigen-binding molecule outside a cell from an antigen-binding molecule within a cell can also be said to be a method for promoting uptake of an antigen-binding molecule bound to an antigen into a cell, and imparting to the antigen-binding molecule a property that makes it easier to promote dissociation of the antigen from the antigen-binding molecule within a cell.

[0030] The present invention also provides a method for promoting the extracellular release of an antigen-binding molecule that has been taken up into a cell in an antigen-bound state, when it is not bound to an antigen. More specifically, the present invention provides a method for promoting the extracellular release of an antigen-binding molecule that has been taken up into a cell in an antigen-bound state, when it is not bound to an antigen, by increasing the human FcRn-binding activity in a neutral pH range of an antigen-binding molecule that has human FcRn-binding activity in an acidic pH range and decreasing the antigen-binding activity in an acidic pH range to be lower than the antigen-binding activity in the neutral pH range. Furthermore, the present invention provides a method for promoting the extracellular release of an antigen-binding molecule that has been taken up into a cell in an antigen-bound state, when it is not bound to an antigen, by modifying at least one amino acid in the antigen-binding molecule and modifying at least one amino acid in the human FcRn-binding domain. Furthermore, the present invention provides a method for promoting the extracellular release of an antigen-binding molecule that has been taken up into a cell in an antigen-bound state, when it is not bound to an antigen, by substituting at least one amino acid in the antigen-binding molecule with histidine or inserting at least one histidine into the human FcRn-binding domain. EU numbering in the Fc domain: 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334 The present invention provides a method for promoting extracellular release, in a state where an antigen-binding molecule is not bound to an antigen, of an antigen-binding molecule that has been taken up into a cell in a state where it is bound to an antigen, wherein at least one amino acid selected from among positions 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0031] In the present invention, "extracellular release of antigen-binding molecules taken up into cells in a state bound to an antigen" does not necessarily mean that all antigen-binding molecules taken up into cells in a state bound to an antigen are released to the outside of cells in a state not bound to an antigen, but it is sufficient that the antigen-binding activity of the antigen-binding molecule in an acidic pH range is lower than that in a neutral pH range, and the proportion of antigen-binding molecules released to the outside of cells in a state not bound to an antigen is higher than that before the human FcRn-binding activity in the neutral pH range is increased. It is preferable that the antigen-binding molecule released to the outside of cells maintains its antigen-binding activity. In addition, a method for promoting the extracellular release of antigen-binding molecules taken up into cells in a state bound to an antigen in a state not bound to an antigen can also be said to be a method of promoting the intracellular uptake of antigen-binding molecules bound to antigens and imparting a property to the antigen-binding molecule that makes it easier to promote the extracellular release of the antigen-binding molecule in a state not bound to an antigen.

[0032] The present invention also provides a method for increasing the ability of an antigen to disappear in plasma by administration of an antigen-binding molecule. In the present invention, the term "method for increasing the ability of an antigen-binding molecule to disappear antigens from plasma" is synonymous with the term "method for increasing the ability of an antigen-binding molecule to disappear antigens from plasma." More specifically, the present invention provides a method for increasing the ability of an antigen-binding molecule having binding activity to human FcRn in an acidic pH range to disappear in plasma by increasing the binding activity of the antigen-binding molecule to human FcRn in a neutral pH range. Furthermore, the present invention provides a method for increasing the ability of an antigen-binding molecule having binding activity to human FcRn in an acidic pH range to disappear in plasma by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule.

[0033] Furthermore, the present invention relates to a human FcRn-binding domain comprising an Fc domain of a parent IgG, the human FcRn-binding domain comprising: The present invention provides a method for increasing the ability of an antigen-binding molecule that has binding activity to human FcRn in an acidic pH range to eliminate antigens in plasma, by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from positions 60, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0034] The present invention further provides a method for increasing the plasma antigen elimination ability of an antigen-binding molecule by reducing the antigen-binding activity in an acidic pH range of the antigen-binding molecule with increased plasma antigen elimination ability compared to the antigen-binding activity in a neutral pH range.The present invention further provides a method for increasing the plasma antigen elimination ability of an antigen-binding molecule by modifying at least one amino acid in the antigen-binding domain of the antigen-binding molecule with increased plasma antigen elimination ability.The present invention further provides a method for increasing the plasma antigen elimination ability by administering an antigen-binding molecule, characterized by substituting at least one amino acid in the antigen-binding domain of the antigen-binding molecule with increased plasma antigen elimination ability with histidine or inserting at least one histidine.

[0035] In the present invention, the term "plasma antigen elimination ability" refers to the ability of an antigen-binding molecule to eliminate antigens present in plasma from plasma when the antigen-binding molecule is administered to a living body or secreted in the living body. Therefore, in the present invention, "the antigen elimination ability of an antigen-binding molecule in plasma is increased" means that when the antigen-binding molecule is administered, the human FcRn-binding activity of the antigen-binding molecule in the neutral pH range is increased, or the antigen-binding activity in the acidic pH range is reduced to less than the antigen-binding activity in the neutral pH range in addition to the increase in the binding activity to human FcRn, and the rate at which the antigen is eliminated from plasma is increased compared to before the antigen-binding activity in the acidic pH range is reduced compared to the antigen-binding activity in the neutral pH range. Whether or not the antigen-binding molecule has increased in plasma antigen elimination ability can be determined, for example, by administering a soluble antigen and an antigen-binding molecule to a living body and measuring the plasma concentration of the soluble antigen after administration. When the concentration of soluble antigen in plasma after administration of a soluble antigen and an antigen-binding molecule is decreased by increasing the human FcRn-binding activity of the antigen-binding molecule in the neutral pH range, or by increasing the binding activity to human FcRn and decreasing the antigen-binding activity in the acidic pH range compared to the antigen-binding activity in the neutral pH range, it can be determined that the antigen-elimination ability of the antigen-binding molecule in plasma has increased. The soluble antigen may be an antigen-binding molecule-bound antigen or an antigen-binding molecule-unbound antigen, and the concentrations thereof can be determined as "plasma antigen-binding molecule-bound antigen concentration" and "plasma antigen-binding molecule-unbound antigen concentration", respectively (the latter is synonymous with "plasma free antigen concentration"). Since "plasma total antigen concentration" means the concentration obtained by adding up the antigen-binding molecule-bound antigen concentration and the antigen-binding molecule-unbound antigen concentration (or "plasma free antigen concentration"), the soluble antigen concentration can be determined as "plasma total antigen concentration". Various methods for measuring "total plasma antigen concentration" or "free plasma antigen concentration" are well known in the art, as described herein below.

[0036] The present invention further provides a method for improving the pharmacokinetics of an antigen-binding molecule. More specifically, the present invention provides a method for improving the pharmacokinetics of an antigen-binding molecule having human FcRn-binding activity in an acidic pH range by increasing the human FcRn-binding activity of the antigen-binding molecule in a neutral pH range. The present invention further provides a method for improving the pharmacokinetics of an antigen-binding molecule having human FcRn-binding activity in an acidic pH range by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule.

[0037] Furthermore, the present invention relates to a human FcRn-binding domain comprising an Fc domain of a parent IgG, which is a human FcRn-binding domain comprising: The present invention provides a method for improving the pharmacokinetics of an antigen-binding molecule that has binding activity to human FcRn in an acidic pH range by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from among positions 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0038] The present invention further provides a method for improving the pharmacokinetics of an antigen-binding molecule by reducing the antigen-binding activity of the antigen-binding molecule with improved pharmacokinetics in an acidic pH range compared to the antigen-binding activity in a neutral pH range.The present invention further provides a method for improving the pharmacokinetics of an antigen-binding molecule that has human FcRn-binding activity in an acidic pH range by modifying at least one amino acid in the antigen-binding domain of the antigen-binding molecule with improved pharmacokinetics.The present invention further provides a method for improving pharmacokinetics, comprising substituting at least one amino acid in the antigen-binding domain of the antigen-binding molecule with improved pharmacokinetics with histidine or inserting at least one histidine.

[0039] In the present invention, "improved pharmacokinetics", "improved pharmacokinetics", and "excellent pharmacokinetics" can be rephrased as "improved plasma (blood) retention", "improved plasma (blood) retention", "excellent plasma (blood) retention", and "prolonged plasma (blood) retention", and these terms are used interchangeably.

[0040] In the present invention, "improved pharmacokinetics" refers not only to an increase in the time from administration of an antigen-binding molecule to humans or non-human animals such as mice, rats, monkeys, rabbits, and dogs until the antigen-binding molecule disappears from plasma (e.g., until the antigen-binding molecule becomes unable to return to plasma due to intracellular degradation, etc.), but also to an increase in the time that the antigen-binding molecule remains in plasma in an antigen-binding state (e.g., in a state where the antigen-binding molecule is not bound to an antigen) from administration until it is decomposed and disappeared. Intact human IgG can bind to FcRn derived from non-human animals. For example, intact human IgG can bind more strongly to mouse FcRn than human FcRn (Int Immunol. 2001 Dec; 13(12): 1551-9), and therefore, for the purpose of confirming the properties of the antigen-binding molecule of the present invention, administration can be preferably performed using mice. As another example, mice in which the original FcRn gene has been disrupted and which have and express a transgene for the human FcRn gene (Methods Mol Biol. 2010; 602: 93-104) can also be used for administration to confirm the properties of the antigen-binding molecule of the present invention described below. Specifically, "improved pharmacokinetics" also includes the time until an antigen-binding molecule that is not bound to an antigen (antigen-unbound antigen-binding molecule) is decomposed and disappears being prolonged. Even if an antigen-binding molecule is present in plasma, if an antigen is already bound to the antigen-binding molecule, the antigen-binding molecule cannot bind to a new antigen. Therefore, if the time that an antigen-binding molecule is not bound to an antigen is prolonged, the time that it can bind to a new antigen is prolonged (there are more opportunities to bind to a new antigen), and the time that an antigen is not bound to an antigen-binding molecule in the body can be reduced, and the time that an antigen is bound to an antigen-binding molecule can be prolonged. If the elimination of an antigen from plasma can be accelerated by administration of an antigen-binding molecule, the plasma concentration of the antigen-unbound antigen-binding molecule will increase and the time that the antigen remains bound to the antigen-binding molecule will be extended.In other words, "improvement of the pharmacokinetics of an antigen-binding molecule" in the present invention includes improvement of any pharmacokinetic parameter of the antigen-free antigen-binding molecule (either an increase in plasma half-life, an increase in mean plasma residence time, or a decrease in plasma clearance), or an extension of the time during which the antigen is bound to the antigen-binding molecule after administration of the antigen-binding molecule, or an acceleration of the disappearance of the antigen from plasma by the antigen-binding molecule. It can be determined by measuring any parameter such as the plasma half-life, mean plasma residence time, or plasma clearance of the antigen-binding molecule or the antigen-free antigen-binding molecule (Understanding through Pharmacokinetics Exercises (Nanzando)). For example, when an antigen-binding molecule is administered to mice, rats, monkeys, rabbits, dogs, humans, etc., the plasma concentration of the antigen-binding molecule or the antigen-free antigen-binding molecule is measured, each parameter is calculated, and if the plasma half-life or mean plasma residence time is increased, the pharmacokinetics of the antigen-binding molecule can be said to be improved. These parameters can be measured by methods known to those skilled in the art, and can be appropriately evaluated, for example, by noncompartmental analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) in accordance with the attached instructions. The plasma concentration of antigen-binding molecules that are not bound to antigens can be measured by methods known to those skilled in the art, and can be, for example, the method described in Clin Pharmacol. 2008 Apr;48(4):406-17.

[0041] In the present invention, "improved pharmacokinetics" also includes the extension of the time during which an antigen is bound to an antigen-binding molecule after administration of the antigen-binding molecule. Whether or not the time during which an antigen is bound to an antigen-binding molecule after administration of the antigen-binding molecule is extended can be determined by measuring the plasma concentration of free antigen and measuring the time until the plasma concentration of free antigen or the ratio of the free antigen concentration to the total antigen concentration increases.

[0042] The plasma concentration of free antigen not bound to an antigen-binding molecule, or the ratio of the free antigen concentration to the total antigen concentration, can be measured by a method known to those skilled in the art, for example, the method measured in Pharm Res. 2006 Jan;23(1):95-103. In addition, when an antigen exhibits some function in vivo, whether the antigen is bound to an antigen-binding molecule (antagonist molecule) that neutralizes the antigen function can also be evaluated by whether the antigen function is neutralized. Whether the antigen function is neutralized can be evaluated by measuring some in vivo marker that reflects the antigen function. Whether the antigen is bound to an antigen-binding molecule (agonist molecule) that activates the antigen function can be evaluated by measuring some in vivo marker that reflects the antigen function.

[0043] Measurements such as the measurement of the plasma concentration of free antigen, the measurement of the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, and the measurement of markers in vivo are not particularly limited, but are preferably performed after a certain time has passed since the administration of an antigen-binding molecule. In the present invention, the period after a certain time has passed since the administration of an antigen-binding molecule is not particularly limited and can be determined by a person skilled in the art at an appropriate time depending on the properties of the administered antigen-binding molecule, etc., and examples thereof include 1 day after the administration of an antigen-binding molecule, 3 days after the administration of an antigen-binding molecule, 7 days after the administration of an antigen-binding molecule, 14 days after the administration of an antigen-binding molecule, and 28 days after the administration of an antigen-binding molecule. In the present invention, the "antigen concentration in plasma" means either the "total antigen concentration in plasma" which is the concentration of the antigen-binding molecule-bound antigen and the antigen-unbound antigen, or the "free antigen concentration in plasma" which is the concentration of the antigen-binding molecule-unbound antigen.

[0044] The total plasma antigen concentration can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more by administration of the antigen-binding molecule of the present invention, compared to administration of a control antigen-binding molecule comprising an intact human IgG Fc domain as the human FcRn-binding domain, or compared to administration of no antigen-binding domain molecule of the present invention.

[0045] The antigen / antigen-binding molecule molar ratio can be calculated as follows: A value = molar concentration of antigen at each time point B value = molar concentration of antigen-binding molecules at each time point C value = molar concentration of antigen per molar concentration of antigen-binding molecule at each time point (antigen / antigen-binding molecule molar ratio) C=A / B.

[0046] A smaller C value indicates a higher efficiency of antigen elimination per antigen-binding molecule, and a larger C value indicates a lower efficiency of antigen elimination per antigen-binding molecule.

[0047] The antigen / antigen-binding molecule molar ratio can be calculated as described above.

[0048] The antigen / antigen-binding molecule molar ratio can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more by administration of the antigen-binding molecule of the present invention, compared to administration of a control antigen-binding molecule comprising an intact human IgG Fc domain as the human FcRn-binding domain.

[0049] In the present invention, intact human IgG1, IgG2, IgG3, or IgG4 is preferably used as intact human IgG for use as a control intact human IgG to compare with an antigen-binding molecule in terms of human FcRn binding activity or in vivo activity. Preferably, a control antigen-binding molecule that contains the same antigen-binding domain as the antigen-binding molecule of interest and an intact human IgG Fc domain as the human FcRn binding domain can be appropriately used. More preferably, intact human IgG1 is used as a control intact human IgG to compare with an antigen-binding molecule in terms of human FcRn binding activity or in vivo activity.

[0050] The reduction in plasma total antigen concentration or antigen / antibody molar ratio can be evaluated as described in Examples 6, 8 and 13. More specifically, if the antigen-binding molecule does not cross-react with the mouse counterpart antigen, it can be evaluated by either the antigen-antibody co-injection model or the steady-state antigen infusion model using human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104). If the antigen-binding molecule cross-reacts with the mouse counterpart, it can be evaluated by simply injecting the antigen-binding molecule into human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories). In the co-injection model, a mixture of the antigen-binding molecule and the antigen is administered to the mouse. In the steady-state antigen infusion model, the mouse is implanted with an infusion pump filled with an antigen solution to achieve a constant plasma antigen concentration, and then the antigen-binding molecule is injected into the mouse. The test antigen-binding molecule is administered at the same dose. Total plasma antigen concentration, free plasma antigen concentration, and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.

[0051] The long-term effect of the present invention can be evaluated by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 2, 4, 7, 14, 28, 56, or 84 days after administration. In other words, for the purpose of evaluating the properties of the antigen-binding molecule of the present invention, the long-term plasma antigen concentration is determined by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule. Whether or not a reduction in the plasma antigen concentration or antigen / antigen-binding molecule molar ratio is achieved by the antigen-binding molecule described in the present invention can be determined by evaluating the reduction at any one or more time points described above.

[0052] The short-term effect of the present invention can be evaluated by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. In other words, for the purpose of evaluating the properties of the antigen-binding molecule of the present invention, the short-term plasma antigen concentration is determined by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration of the antigen-binding molecule.

[0053] The route of administration of the antigen-binding molecule of the present invention can be selected from intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, and intramuscular injection.

[0054] In the present invention, it is preferable that the pharmacokinetics in humans is improved. When it is difficult to measure plasma retention in humans, the plasma retention in humans can be predicted based on the plasma retention in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0055] In the present invention, the acidic pH range generally means pH 4.0 to pH 6.5. The acidic pH range is preferably a range indicated by any pH value within pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to 6.0, which is close to the pH in early endosomes in vivo. On the other hand, in the present invention, the neutral pH range generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value within pH 7.0 to pH 8.0, 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 particularly preferably pH 7.4, which is close to the pH in plasma (blood) in vivo. When the binding affinity between the human FcRn-binding domain and human FcRn is low at pH 7.4 and it is difficult to evaluate the binding affinity, pH 7.0 can be used instead of pH 7.4. Regarding the temperature used as the measurement condition, the binding affinity between the human FcRn-binding domain and human FcRn may 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 the human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 is an example of an embodiment of the present invention.

[0056] Therefore, in the present invention, "reducing the antigen-binding activity of an antigen-binding molecule in an acidic pH range compared to that in a neutral pH range" means making the antigen-binding activity of the antigen-binding molecule weaker at pH 4.0 to pH 6.5 than that at pH 6.7 to pH 10.0. Preferably, it means making the antigen-binding activity of the antigen-binding molecule weaker at pH 5.5 to pH 6.5 than that at pH 7.0 to pH 8.0, and particularly preferably, making the antigen-binding activity at the pH in early endosomes in vivo weaker than that at the pH in plasma, specifically, it means making the antigen-binding activity of the antigen-binding molecule weaker at pH 5.8 to pH 6.0 than that at pH 7.4.

[0057] Furthermore, in the present invention, the expression "reducing the antigen-binding activity of an antigen-binding molecule in an acidic pH range compared to that in a neutral pH range" can also be expressed as "reducing the antigen-binding activity of an antigen-binding molecule in a neutral pH range compared to that in an acidic pH range". That is, in the present invention, the ratio of the antigen-binding activity of an antigen-binding molecule in an acidic pH range to that in a neutral pH range may be increased. For example, as described below, an embodiment in which the value of KD(pH5.8) / KD(pH7.4) is increased. To increase the ratio of the antigen-binding activity of an antigen-binding molecule in an acidic pH range to that in a neutral pH range, for example, the antigen-binding activity in an acidic pH range may be decreased, the antigen-binding activity in a neutral pH range may be increased, or both may be performed.

[0058] In the present invention, "reducing the antigen-binding activity in an acidic pH range to be lower than that in a neutral pH range" may also be expressed as "making the antigen-binding ability in an acidic pH range weaker than that in a neutral pH range."

[0059] In the present invention, the binding activity to human FcRn in an acidic pH range means human FcRn-binding activity at pH 4.0 to pH 6.5, preferably at pH 5.5 to pH 6.5, and particularly preferably at pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Furthermore, in the present invention, the binding activity to human FcRn in a neutral pH range means human FcRn-binding activity at pH 6.7 to pH 10.0, preferably at pH 7.0 to pH 8.0, and particularly preferably at pH 7.4, which is close to the pH in plasma in vivo.

[0060] The antigen-binding molecule of the present invention has a human FcRn-binding domain. The human FcRn-binding domain is not particularly limited as long as the antigen-binding molecule has human FcRn-binding activity in the acidic pH range and neutral pH range, and may be a domain that has binding activity to human FcRn directly or indirectly. Examples of such domains include Fc domains of IgG immunoglobulins that have direct binding activity to human FcRn, albumin, albumin domain 3, anti-human FcRn antibodies, anti-human FcRn peptides, anti-human FcRn scaffold molecules, etc., or molecules that indirectly bind to IgG or albumin that have binding activity to human FcRn. In the present invention, a domain that has human FcRn-binding activity in the acidic pH range and neutral pH range is preferred. The domain may be used as it is as long as it already has human FcRn-binding activity in the acidic pH range and neutral pH range. When the domain has no or weak human FcRn-binding activity in the acidic and / or neutral pH range, the amino acids in the antigen-binding molecule may be modified to obtain human FcRn-binding activity, but it is preferable to modify the amino acids in the human FcRn-binding domain to obtain human FcRn-binding activity in the acidic and / or neutral pH range. Alternatively, the human FcRn-binding activity may be enhanced by modifying the amino acids in the domain that already has human FcRn-binding activity in the acidic and / or neutral pH range. The desired amino acid modification in the human FcRn-binding domain can be found by comparing the human FcRn-binding activity in the acidic and / or neutral pH range before and after the amino acid modification.

[0061] The human FcRn-binding domain is preferably a region that directly binds to human FcRn. A preferred example of a human FcRn-binding region is the Fc domain of an antibody. However, a region capable of binding to a polypeptide having binding activity to human FcRn, such as albumin or IgG, can indirectly bind to human FcRn via albumin, IgG, or the like. Thus, the human FcRn-binding region of the present invention may be a region that binds to a polypeptide having binding activity to human FcRn.

[0062] Furthermore, the antigen-binding molecule of the present invention is not particularly limited as long as it has an antigen-binding domain that has specific binding activity to a target antigen. A preferred example of the antigen-binding domain is a domain having an antibody antigen-binding region. Examples of the antibody antigen-binding region are CDR and variable region. When the antibody antigen-binding region is a CDR, it may contain all six CDRs contained in a full-length antibody, or may contain one or more CDRs. When the antibody binding region contains a CDR, the CDR may have amino acid deletion, substitution, addition and / or insertion, or may be a part of the CDR.

[0063] Furthermore, antigen-binding molecules targeted by the methods of the present invention include antigen-binding molecules with antagonist activity (antagonist antigen-binding molecules), antigen-binding molecules with agonist activity (agonist antigen-binding molecules), and molecules with cytotoxic activity. A preferred embodiment of the invention is an antagonist antigen-binding molecule, particularly an antagonist antigen-binding molecule that recognizes antigens such as receptors and cytokines.

[0064] The antigen-binding molecule of interest in the present invention is not particularly limited, and may be any antigen-binding molecule. The antigen-binding molecule used in the present invention preferably has an antigen-binding activity (antigen-binding domain) and a human FcRn-binding domain. In the present invention, it is particularly preferable that the antigen-binding molecule contains a binding domain to human FcRn. An example of an antigen-binding molecule having an antigen-binding domain and a human FcRn-binding domain can be an antibody. A preferred example of the antibody of the present invention can be an IgG antibody. When an IgG antibody is used as the antibody, the type is not limited, and IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. In addition, the antigen-binding molecule of the present invention may contain a constant region of the antibody, and amino acid mutations may be introduced into the constant region. Examples of amino acid mutations to be introduced include those that increase or decrease binding to Fcγ receptors (Proc Natl Acad Sci US A. 2006 Mar 14; 103(11): 4005-10.), but are not limited thereto. It is also possible to alter the pH-dependent binding by selecting an appropriate constant region, such as IgG2.

[0065] When the antigen-binding molecule of the present invention is an antibody, the antibody may be derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Furthermore, the antibody may be, for example, a chimeric antibody, particularly a modified antibody in which the amino acid sequence has been substituted, such as a humanized antibody. In addition, the antibody may be a bispecific antibody, an antibody modified with various molecules bound thereto, or a polypeptide containing an antibody fragment.

[0066] A "chimeric antibody" is an antibody produced by combining sequences derived from different animals. A specific example of a chimeric antibody is an antibody consisting of the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody.

[0067] A "humanized antibody", also called a reshaped human antibody, is an antibody derived from a mammal other than a human, for example, a mouse antibody, in which the complementarity determining region (CDR) of the antibody has been grafted onto the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General gene recombination techniques are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0068] A bispecific antibody is an antibody that has variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

[0069] Furthermore, examples of polypeptides containing antibody fragments include Fab fragments, F(ab')2 fragments, scFv (Nat Biotechnol. 2005 Sep;23(9):1126-36.), domain antibodies (dAbs) (WO2004 / 058821, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, Fc fusion proteins, and the like. For molecules containing an Fc domain, the Fc domain can be used as a human FcRn-binding domain. Furthermore, a human FcRn-binding domain may be fused to these molecules.

[0070] Furthermore, the antigen-binding molecule applicable to the present invention may be an antibody-like molecule. An antibody-like molecule (scaffold molecule, peptide molecule) is a molecule that exerts its function by binding to a target molecule (Current Opinion in Biotechnology 2006, 17:653-658, Current Opinion in Biotechnology 2007, 18:1-10, Current Opinion in Structural Biology 1997, 7:463-469, Protein Science 2006, 15:14-27), and examples thereof include DARPins (WO2002 / 020565), affibodies (WO1995 / 001937), avimers (WO2004 / 044011, WO2005 / 040229), and adnectins (WO2002 / 032925). Even these antibody-like molecules, as long as they can bind to a target molecule in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, can promote intracellular uptake of antigens by the antigen-binding molecule, promote a decrease in the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by one antigen-binding molecule.

[0071] The antigen-binding molecule may also be a protein in which a human FcRn-binding domain is fused to a receptor protein that binds to a target containing a ligand, such as TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60). Even if these receptor-human FcRn-binding domain fusion proteins can bind to a target molecule containing a ligand in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, they can promote the uptake of antigens into cells by the antigen-binding molecule, promote the reduction of the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by one antigen-binding molecule. The receptor protein is appropriately designed and modified to include a binding domain of the receptor protein to a target containing a ligand. As in the examples described above, including TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein, preferably, soluble receptor molecules are used in the present invention, which include the extracellular domain of the receptor protein required for binding to the target containing the ligand. These designed and modified receptor molecules are referred to as artificial receptors in the present invention. The methods used to design and modify receptor molecules to construct artificial receptor molecules are known in the art.

[0072] The antigen-binding molecule may also be a fusion protein of an artificial ligand protein that binds to a target but has a neutralizing effect and a human FcRn-binding domain, and an example of an artificial ligand protein is mutant IL-6 (EMBO J. 1994 Dec 15;13(24):5863-70.). If these artificial ligand fusion proteins can bind to a target molecule in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, it is possible to promote the uptake of antigens into cells by the antigen-binding molecule, promote the reduction of the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound to one antigen-binding molecule.

[0073] Furthermore, the antibody of the present invention may contain a modified sugar chain. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (WO99 / 54342, etc.), antibodies lacking fucose added to the sugar chain (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913, etc.), and antibodies with sugar chains containing bisecting GlcNAc (WO02 / 79255, etc.).

[0074] Conditions other than pH when measuring the binding activity to antigens or human FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009 / 125825, it is possible to measure the activity under conditions of MES buffer and 37°C. In another embodiment, as described in Example 4 or 5, it is possible to measure the activity using sodium phosphate buffer at 25°C. In addition, the antigen-binding activity and human FcRn-binding activity of antigen-binding molecules can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to soluble antigens can be evaluated by flowing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, it is possible to evaluate the binding activity to membrane antigens by flowing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized. The binding activity of an antigen-binding molecule to human FcRn can be measured by passing human FcRn or the antigen-binding molecule, respectively, as an analyte over a chip onto which the antigen-binding molecule or human FcRn has been immobilized.

[0075] In the present invention, as long as the antigen-binding activity in an acidic pH range is weaker than that in a neutral pH range, the ratio of the antigen-binding activity in an acidic pH range to that in a neutral pH range is not particularly limited, but preferably the ratio of the KD (Dissociation constant) for the antigen at pH 5.8 to the KD at pH 7.4, KD(pH5.8) / KD(pH7.4), is 2 or more, more preferably the KD(pH5.8) / KD(pH7.4) is 10 or more, and even more preferably the KD(pH5.8) / KD(pH7.4) is 40 or more. There is no particular upper limit to the KD(pH5.8) / KD(pH7.4) value, and it may be any value, such as 400, 1000, or 10000, as long as it can be produced by those skilled in the art.

[0076] When the antigen is a soluble antigen, the KD (dissociation constant) can be used as the value of the antigen-binding activity, whereas when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and the apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0077] In the present invention, another index showing the ratio of antigen-binding activity in the acidic pH range to that in the neutral pH range is, for example, the dissociation rate constant k d It is also possible to use k (Dissociation rate constant) instead of KD (dissociation constant) as an index of the binding activity ratio. d When using the dissociation rate constant, k d (dissociation rate constant) and k in the neutral pH range d (dissociation rate constant) ratio k d (pH acidic range) / k d The value of (neutral pH range) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. d (pH acidic range) / k d The upper limit of the value (neutral pH range) is not particularly limited, and may be any value, such as 50, 100, 200, etc., as long as it can be prepared within the technical common sense of a person skilled in the art.

[0078] If the antigen is a soluble antigen, the value of antigen binding activity is k d (dissociation rate constant) can be used, and when the antigen is a membrane antigen, the apparent k d It is possible to use the apparent dissociation rate constant k d (dissociation rate constant), and apparent k dThe (apparent dissociation rate constant) can be measured by a method known to those skilled in the art, for example, using Biacore (GE healthcare), a flow cytometer, or the like.

[0079] In the present invention, when the antigen-binding activity of an antigen-binding molecule is measured at different pH levels, it is preferable that the conditions other than pH be the same.

[0080] The method for reducing (weakening) the antigen-binding activity of an antigen-binding molecule in an acidic pH range compared to that in a neutral pH range (the method for imparting pH-dependent binding ability) is not particularly limited, and may be any method. Specifically, as described in WO2009 / 125825, for example, a method for reducing (weakening) the antigen-binding activity in an acidic pH range compared to that in a neutral pH range by substituting an amino acid in the antigen-binding molecule with histidine or inserting histidine into the antigen-binding molecule can be mentioned. It is already known that pH-dependent antigen-binding activity can be imparted to an antibody by substituting an amino acid in an antibody with histidine (FEBS Letter, 309(1), 85-88, (1992)). The position at which the histidine mutation (substitution) or insertion is introduced (performed) is not particularly limited, and any site may be substituted with histidine, or any site may be inserted with histidine. Preferred examples of the site of histidine mutation (substitution) or insertion include regions that affect the antigen-binding activity of the antigen-binding molecule, and include sites where the antigen-binding activity in an acidic pH range is lower (weaker) than that in a neutral pH range (the value of KD(acidic pH range) / KD(neutral pH range) is larger) compared to before the mutation or insertion. For example, in the present invention, when the antigen-binding molecule is an antibody, examples include the variable region and CDR of the antibody. The number of histidine mutations or insertions to be introduced (performed) can be appropriately determined by those skilled in the art, and only one site may be substituted with histidine, or only one site may be inserted with histidine, or two or more sites may be substituted with histidine, or two or more sites may be inserted with histidine. In addition, mutations other than histidine mutations (mutations to amino acids other than histidine (deletion, addition, insertion and / or substitution, etc.)) may be simultaneously introduced. Furthermore, histidine mutation and histidine insertion may be performed simultaneously. Substitution with or insertion of histidine may be performed randomly by methods known to those skilled in the art, such as histidine scanning, in which alanine is replaced with histidine.Then, antigen-binding molecules with a higher KD (acidic pH range) / KD (neutral pH range) value compared to before the mutation can be selected from the library of antigen-binding molecules into which histidine mutations or insertions have been randomly introduced.

[0081] When an amino acid of an antigen-binding molecule is substituted with histidine or histidine is inserted into an amino acid of an antigen-binding molecule, it is preferable, but not limited to, that the antigen-binding activity of the antigen-binding molecule after histidine substitution or insertion in a neutral pH range is equivalent to the antigen-binding activity of the antigen-binding molecule before histidine substitution or insertion in a neutral pH range. Here, "the antigen-binding activity of the antigen-binding molecule after histidine substitution or insertion in a neutral pH range is equivalent to the antigen-binding activity of the antigen-binding molecule before histidine substitution or insertion in a neutral pH range" means that the antigen-binding molecule after histidine substitution or insertion maintains 10% or more, preferably 50% or more, more preferably 80% or more, and more preferably 90% or more of the antigen-binding activity of the antigen-binding molecule before histidine substitution or insertion. When the antigen-binding activity of an antigen-binding molecule is reduced by histidine substitution or insertion, the antigen-binding activity may be made equivalent to the antigen-binding activity before the histidine substitution or insertion by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after such histidine substitution or insertion.

[0082] Another method for decreasing (weakening) the antigen-binding activity of an antigen-binding molecule in an acidic pH range compared to that in a neutral pH range is to substitute an amino acid in the antigen-binding molecule with a non-natural amino acid or to insert a non-natural amino acid into an amino acid in the antigen-binding molecule. It is known that non-natural amino acids can artificially control the pKa (Angew. Chem. Int. Ed. 2005, 44, 34; Chem Soc Rev. 2004 Sep 10; 33(7): 422-30.; Amino Acids. 1999; 16(3-4): 345-79.). Therefore, in the present invention, a non-natural amino acid can be used instead of the above-mentioned histidine. The position at which a non-natural amino acid is introduced is not particularly limited, and any site may be substituted with a non-natural amino acid or a non-natural amino acid may be inserted into any site. Preferred examples of sites at which a non-natural amino acid is substituted with or inserted into a non-natural amino acid include regions that affect the antigen-binding activity of an antigen-binding molecule. For example, when the antigen-binding molecule is an antibody, examples of the non-natural amino acid include the variable region and complementarity determining region (CDR) of the antibody. The number of non-natural amino acids introduced is not particularly limited, and only one position may be substituted with a non-natural amino acid, or only one position may be inserted with a non-natural amino acid. Alternatively, two or more positions may be substituted with a non-natural amino acid, or multiple positions may be inserted with a non-natural amino acid. In addition to the substitution or insertion of a non-natural amino acid, deletion, addition, insertion and / or substitution of other amino acids may be performed simultaneously. Furthermore, the above-mentioned histidine substitution and / or insertion and the substitution and / or insertion of a non-natural amino acid may be performed simultaneously. The non-natural amino acid used in the present invention may be any non-natural amino acid, and non-natural amino acids known to those skilled in the art may be used.

[0083] In the present invention, examples of sites for substitution with histidine or unnatural amino acids when the antigen-binding molecule is an antibody include antibody CDR sequences and sequences determining the CDR structure, and include, for example, the sites described in WO2009 / 125825. Amino acid positions are indicated by Kabat numbering (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH).

[0084] The Kabat numbering system is commonly used when referring to variable domain residues (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is commonly used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "Kabat EU index" refers to the numbering system for human IgG 1 The residue numbering refers to the EU antibody residue numbering. Unless otherwise specified herein, reference to the residue number in the variable domain of an antibody refers to the residue numbering according to Kabat numbering. Unless otherwise specified herein, reference to the residue number in the constant domain of an antibody refers to the residue numbering according to the EU numbering system (see, for example, WO2006073941).

[0085] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102 Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94

[0086] Among these alterations, H32, H61, L53, L90, and L94 are considered to be highly common alterations.

[0087] Furthermore, preferred modifications when the antigen is an IL-6 receptor (for example, human IL-6 receptor) include, but are not limited to, the following:

[0088] Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, and H102 Light chain: L24, L27, L28, L32, L53, L56, L90, L92, and L94

[0089] Specific preferred combinations of substitutions of multiple sites with histidine or unnatural amino acids include, for example, the combination of H27, H31, and H35, the combination of H27, H31, H32, H35, H58, H62, and H102, the combination of L32 and L53, the combination of L28, L32, and L53, etc. Furthermore, a preferred example of a combination of substitution sites between the heavy chain and the light chain is the combination of H27, H31, L32, and L53.

[0090] Only one of these positions may be substituted with histidine or a non-natural amino acid, or multiple positions may be substituted with histidine or a non-natural amino acid.

[0091] In addition, when the antigen-binding molecule is a substance containing an antibody constant region, another method for decreasing (weakening) the antigen-binding activity of the antigen-binding molecule in an acidic pH range compared to that in a neutral pH range can be a method of modifying amino acids in the antibody constant region. Specific examples of such antibody constant regions include a method of substituting with the antibody constant regions described in WO2009 / 125825 (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14). In addition, a method for modifying an antibody constant region can be, for example, a method of examining multiple constant region isotypes (IgG1, IgG2, IgG3, and IgG4) and selecting an isotype that has a decreased antigen-binding activity in an acidic pH range (has a faster dissociation rate in an acidic pH range). Another method can be a method of decreasing the antigen-binding activity in an acidic pH range (has a faster dissociation rate in an acidic pH range) by introducing amino acid substitutions into the amino acid sequence of a wild-type isotype (wild-type IgG1, IgG2, IgG3, or IgG4 amino acid sequence). The sequences of the hinge region of the antibody constant region vary greatly depending on the isotype (IgG1, IgG2, IgG3, and IgG4), and differences in the amino acid sequence of the hinge region greatly affect antigen-binding activity, so by selecting an appropriate isotype depending on the type of antigen or epitope, it is possible to select an isotype that has reduced antigen-binding activity in the acidic pH range (faster dissociation rate in the acidic pH range). In addition, since differences in the amino acid sequence of the hinge region greatly affect antigen-binding activity, the hinge region is considered to be a desirable site for amino acid substitution in the amino acid sequence of the wild-type isotype.

[0092] When the antigen-binding activity of an antigen-binding molecule in an acidic pH range is reduced (weakened) compared to the antigen-binding activity in a neutral pH range by the above-mentioned methods or the like (the value of KD(acidic pH range) / KD(neutral pH range) is increased), it is not particularly limited, but it is preferable that the value of KD(acidic pH range) / KD(neutral pH range) is usually 2-fold or more, preferably 5-fold or more, and more preferably 10-fold or more, compared to that of the original antibody.

[0093] An antigen-binding molecule whose antigen-binding activity in an acidic pH range is lower (weaker) than that in a neutral pH range (an antigen-binding molecule exhibiting pH-dependent binding) can be prepared by amino acid substitution or insertion from an antigen-binding molecule that does not have such properties using the above-mentioned methods, but another method includes a method of directly obtaining an antigen-binding molecule having such properties. For example, an antibody having the desired properties may be directly obtained by screening antibodies obtained by immunizing an animal (mouse, rat, hamster, rabbit, human immunoglobulin transgenic mouse, human immunoglobulin transgenic rat, human immunoglobulin transgenic rabbit, llama, camel, etc.) with an antigen using pH-dependent binding to the antigen as an index. The antibody may be produced by, but is not limited to, a hybridoma technique or a B cell cloning technique, which are methods known to those skilled in the art (Proc Natl Acad Sci US A. 1996 Jul 23; 93(15): 7843-8; J Immunol Methods. 2006 Oct 20; 316(1-2): 133-43; Journal of the Association for Laboratory Automation; WO2004 / 106377; WO2008 / 045140; and WO2009 / 113742). Alternatively, an antibody having a desired property may be directly selected by screening a library displaying an antigen-binding domain in vitro using pH-dependent antigen binding as an index. Such libraries include, but are not limited to, human naive libraries, immune libraries derived from non-human animals and humans, semi-synthetic libraries, and synthetic libraries, which are libraries known to those skilled in the art (Methods Mol Biol. 2002; 178: 87-100; J Immunol Methods. 2004 Jun; 289(1-2): 65-80; and Expert Opin Biol Ther. 2007 May; 7(5): 763-79). However, the methods are not particularly limited to these examples.

[0094] The present invention utilizes the difference in pH as an environmental difference between plasma and endosomes to obtain binding affinity of an antigen-binding molecule to an antigen that differs between plasma and endosomes (strong binding in plasma and weak binding in endosomes). Since the difference in the environment between plasma and endosomes is not limited to the difference in pH, the pH-dependent binding property that affects the binding of an antigen-binding molecule to an antigen can be replaced by using other factors whose concentrations differ between plasma and endosomes. Such factors can also be used to generate antibodies that bind to an antigen in plasma but dissociate from the antigen in endosomes. Thus, the present invention includes an antigen-binding molecule that includes an antigen-binding domain and a human FcRn-binding domain, has human FcRn-binding activity in an acidic pH range and a neutral pH range, has a lower antigen-binding activity in endosomes than in plasma, and has a higher human FcRn-binding activity in plasma than that of intact human IgG.

[0095] The method for enhancing the human FcRn-binding activity of the human FcRn-binding domain in the antigen-binding molecule of the present invention in a neutral pH range is not particularly limited, and any method may be used. Specifically, when the Fc domain of an IgG immunoglobulin is used as the human FcRn-binding domain, the human FcRn-binding activity in a neutral pH range can be enhanced by amino acid modification. Preferred examples of the Fc domain of an IgG immunoglobulin for modification include the Fc domain of a human parent IgG (IgG1, IgG2, IgG3, or IgG4, and engineered variants thereof). As for modification to other amino acids, amino acids at any positions may be modified as long as the human FcRn-binding activity in a neutral pH range is obtained or the human FcRn-binding activity can be enhanced. When the antigen-binding molecule contains the Fc domain of human IgG1 as the human FcRn-binding domain, it is preferable that the molecule contains a modification that enhances the binding to human FcRn in a neutral pH range compared to the parent human IgG1. Examples of amino acids that can be modified in this way include those at 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, and 324 (EU numbering). Examples of the amino acids at positions 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 can be mentioned. More specifically, examples of the amino acid modifications include those shown in Table 1. By using these modifications, the binding of the Fc domain of IgG immunoglobulin to human FcRn in the neutral pH range can be strengthened.

[0096] Furthermore, examples of modifications that can enhance the binding to human FcRn in the acidic pH range compared to the parent human IgG are shown in Table 2. Among these modifications, modifications that can enhance the binding to human FcRn even in the neutral pH range can be appropriately selected and used in the present invention. Furthermore, combinations of modifications that can enhance the binding to human FcRn of Fv4-IgG1 under acidic conditions are shown in Tables 6-1 and 6-2. Particularly preferred modified amino acids in the Fc domain of a parent human IgG include, for example, the amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering). By substituting at least one amino acid selected from these amino acids with another amino acid, the human FcRn-binding activity of an antigen-binding molecule in the neutral pH range can be increased.

[0097] Particularly preferred modifications include, for example, the EU numbering of the Fc domain of the parent IgG. Amino acid substitution at position 237, replacing Gly with Met; Amino acid substitution of Pro at position 238 to Ala; Amino acid substitution at position 239 replacing Ser with Lys; Amino acid substitution at position 248, replacing Lys with Ile; an amino acid substitution of Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; an amino acid substitution of Met at position 252 with Phe, Trp, or Tyr; Amino acid substitution at position 254 replacing Ser with Thr; Amino acid substitution at position 255, replacing Arg with Glu; an amino acid substitution of Thr at position 256 with Asp, Glu, or Gln; an amino acid substitution of Pro at position 257 with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; Amino acid substitution at position 258, replacing Glu with His; Amino acid substitution of Asp at position 265 to Ala; an amino acid substitution of Asp at position 270 with Phe; Amino acid substitution of Asn at position 286 with Ala or Glu; Amino acid substitution of Thr at position 289 to His; Amino acid substitution of Asn at position 297 to Ala; Amino acid substitution at position 298 replacing Ser with Gly; Amino acid substitution of Val at position 303 to Ala; Amino acid substitution of Val to Ala at position 305; an amino acid substitution replacing Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg; an amino acid substitution of Gln at position 311 with Ala, His, or Ile; an amino acid substitution of Asp at position 312 with Ala or His; Amino acid substitution of Leu at position 314 with Lys or Arg; an amino acid substitution of Asn at position 315 with Ala or His; Amino acid substitution of Lys at position 317 to Ala; an amino acid substitution of Asn at position 325 with Gly; an amino acid substitution at position 332 replacing Ile with Val; Amino acid substitution at position 334, replacing Lys with Leu; Amino acid substitution at position 360, replacing Lys with His; an amino acid substitution of Asp at position 376 with Ala; Amino acid substitution of Glu at position 380 to Ala; Amino acid substitution of Glu at position 382 to Ala; an amino acid substitution at position 384 replacing Asn or Ser with Ala; an amino acid substitution of Gly at position 385 with Asp or His; an amino acid substitution of Gln at position 386 with Pro; Amino acid substitution of Pro at position 387 to Glu; an amino acid substitution of Asn at position 389 with Ala or Ser; Amino acid substitution at position 424, replacing Ser with Ala; an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; Amino acid substitution at position 433, replacing His with Lys; an amino acid substitution replacing Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and Amino acid substitution at position 436 replacing Tyr or Phe with His Examples of the amino acid sequence that can be modified include the following. The number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of modifications at two or more amino acids include those shown in Table 3. Tables 4-1 to 4-5 show combinations of modifications that can strengthen the binding to human FcRn in the acidic pH range compared to the parent human IgG. Among these modifications, a combination of modifications that can strengthen the binding to human FcRn even in the neutral pH range can be appropriately selected and used in the present invention. Furthermore, combinations of modifications that can strengthen the binding to human FcRn for Fv4-IgG1 under neutral conditions are shown in Tables 6-1 and 6-2.

[0098] In the table, "^" means that an amino acid is inserted at the position following the corresponding number in the EU numbering system. For example, ^281S means that an S is inserted between the EU numbering positions 281 and 282.

[0099] [Table 1]

[0100] [Table 2]

[0101] [Table 3]

[0102] [Table 4-1]

[0103] Table 4-2 is a continuation of Table 4-1. [Table 4-2]

[0104] Table 4-3 is a continuation of Table 4-2.

Table 4-3

[0105] Table 4-4 is a continuation of Table 4-3.

Table 4-4

[0106] Table 4-5 is a continuation of Table 4-4.

Table 4-5

[0107] For these amino acid modifications, it is possible to appropriately implement them using known techniques. For example, modifications of the intact human IgG1 Fc domain have been performed in Drug Metab Dispos. 2007 Jan;35(1):86-94, Int Immunol. 2006 Dec;18(12):1759-69, J Biol Chem. 2001 Mar 2;276(9):6591-604, J Biol Chem. 2007;282(3):1709-17, J Immunol. 2002;169(9):5171-80, J Immunol. 2009;182(12):7663-71, Molecular Cell, Vol. 7, 867-877, April, 2001, Nat Biotechnol. 1997 Jul;15(7):637-40, Nat Biotechnol. 2005 Oct;23(10):1283-8, Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18709-14, EP2154157, US20070141052, WO2000 / 042072, WO2002 / 060919, WO2006 / 020114, WO2006 / 031370, WO2010 / 033279, WO2006 / 053301, WO2009 / 086320.

[0108] According to The Journal of Immunology, 2009 182: 7663-7671, the human FcRn-binding activity of intact human IgG1 in the acidic pH range (pH 6.0) is KD 1.7 μM, and the human FcRn-binding activity of intact human IgG1 in the neutral pH range is almost undetectable. Thus, preferred embodiments of the antigen-binding molecule used in the method of the present invention include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 20 μM or stronger and whose human FcRn-binding activity in the neutral pH range is the same as or stronger than that of intact human IgG. More preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 2.0 μM or stronger and whose human FcRn-binding activity in the neutral pH range is KD 40 μM or stronger. More preferred embodiments include antigen-binding molecules having human FcRn-binding activity in the acidic pH range of 0.5 μM or stronger (KD) and human FcRn-binding activity in the neutral pH range of 15 μM or stronger (KD). The KD values ​​shown here are values ​​measured by the method described in The Journal of Immunology, 2009, 182: 7663-7671 (in which the antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0109] Although it is possible to use KD (dissociation constant) as a value of human FcRn-binding activity, since the human FcRn-binding activity of intact human IgG is hardly observed in the neutral pH range (pH 7.4), it is difficult to calculate it as KD. One method for judging whether the human FcRn-binding activity at pH 7.4 is higher than that of intact human IgG is to judge the magnitude of the binding response when the analyte is applied at the same concentration in Biacore. That is, if the response when human FcRn is applied at pH 7.4 on a chip on which an antigen-binding molecule is immobilized is larger than the response when human FcRn is applied at pH 7.4 on a chip on which intact human IgG is immobilized, the human FcRn-binding activity of the antigen-binding molecule at pH 7.4 can be judged to be higher than that of intact human IgG.

[0110] pH 7.0 can also be used as the neutral pH range. By using pH 7.0 as the neutral pH, weak interaction between human FcRn and the FcRn-binding domain can be promoted. As the temperature used in the measurement conditions, the binding affinity may 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 the human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is similarly used to determine the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 is an example related to an embodiment of the present invention. In a preferred embodiment, the interaction between human FcRn and the FcRn-binding domain can be measured at pH 7.0 and 25° C. as described in Example 5. The binding affinity of an antigen-binding molecule to human FcRn can be measured by Biacore as described in Example 5.

[0111] In a more preferred embodiment, the antigen-binding molecule of the present invention has a higher human FcRn-binding activity than intact human IgG at pH 7.0 and 25° C. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25° C. is 28-fold higher than intact human IgG or a KD of 3.2 μM stronger. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25° C. is 38-fold higher than intact human IgG or a KD of 2.3 μM stronger.

[0112] Intact human IgG1, IgG2, IgG3, or IgG4 is preferably used as an intact human IgG for use as a control intact human IgG to compare with an antigen-binding molecule for its human FcRn-binding activity or in vivo activity. Preferably, a control antigen-binding molecule that contains the same antigen-binding domain as the antigen-binding molecule of interest and an intact human IgG Fc domain as the human FcRn-binding domain can be appropriately used. More preferably, intact human IgG1 is used as a control intact human IgG to compare with an antigen-binding molecule for its human FcRn-binding activity or in vivo activity.

[0113] More specifically, the antigen-binding molecule having a long-term effect on the antigen elimination activity in plasma described in the present invention has an FcRn binding activity in the range of 28-440 times higher than that of intact human IgG1 at pH 7.0 and 25° C., or has an FcRn binding activity with a KD in the range of 3.0 μM to 0.2 μM. For the purpose of evaluating the long-term effect of the antigen-binding molecule of the present invention on the antigen elimination activity in plasma, the long-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration and the antigen / antigen-binding molecule molar ratio in plasma 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration of the antigen-binding molecule. Whether or not a decrease in the plasma antigen concentration or the antigen / antigen-binding molecule molar ratio is achieved by the antigen-binding molecule described in the present invention can be determined by evaluating the decrease at any one or more of the time points described above.

[0114] More specifically, the antigen-binding molecule having a short-term effect on antigen elimination activity in plasma described in the present invention has a human FcRn-binding activity 440-fold higher than that of intact human IgG at pH 7.0 and 25° C., or has an FcRn-binding activity with a KD of 0.2 μM or stronger. For the purpose of evaluating the short-term effect of the antigen-binding molecule of the present invention on antigen elimination activity in plasma, the short-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration and the antigen / antigen-binding molecule molar ratio in plasma 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration of the antigen-binding molecule.

[0115] The methods of the present invention are applicable to any antigen-binding molecule regardless of the type of target antigen.

[0116] The antigens recognized by antigen-binding molecules such as antibodies targeted by the method of the present invention are not particularly limited, and any antigen-recognizing antibodies may be targeted. Examples of antibodies that improve pharmacokinetics by the method of the present invention include, for example, antibodies that recognize membrane antigens such as receptor proteins (membrane-bound receptors, soluble receptors) and cell surface markers, and antibodies that recognize soluble antigens such as cytokines. Specific examples of antigens recognized by antibodies that improve pharmacokinetics by the method of the present invention include, for example, 17-IA, 4-1 BB, 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, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, adiponectin, ADP-ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergens, α1-antichymotrypsin, α1-antitrypsin, α-synuclein, α-V / β-1 antagonist, aminin, amylin, amyloid β, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light Chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apoA1, apo serum amyloid A, apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracisanthracis) defense antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, β-2-microgloblin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, Blin glomerular stimulating factor (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMP, BOK, ボンベシン, bone-derived neurotrophic factor ( Complement 5a), CA125, CAD-8, カドヘリン-3, カルシトニン, cAMP, carbonic acid dehydratase-IX, carcinofetal antigen (CEA), cancer-associated antigen (carcinoma-associated antigen)antigen), カルジオトロフィン-1, カテプシンA, カテプシンB, カテプシンC / DP PI, カテプシンD, カテプシンE, カテプシンH, カテプシンL, カテプシンO, カテプシンS , カテプシンV, カテプシンX / Z / P, CBL, CCI, CCK2, CCL, CCL1 / I-309, CCL11 / エオタキシン, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-α, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / エオタキシン-2, CCL25 / TECK, CCL26 / エオタキシン-3, CCL27 / CTACK, CCL 28 / MEC, CCL3 / M1P-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-l-β, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP -2. CCL9 / 10 / MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, C D164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b , CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGR P acceptor, CINC, CKb8-1, Clostridium 18, CLC, Clostridiumbotulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, Complement factor 3 (C3), Complement factor D, Corticosteroid-binding globulin, Colony-stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / Fractalkine, CX3CR1, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL16, CXCL2 / Gro-β CXCL3 / Gro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, dipeptidyl peptidase IV, DK1, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF-like domain containing protein 7, elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, Endothelin receptor, Endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini, EpCAM, EphrinB2 / EphB4, Epha2 tyrosine kinase receptor, Epidermal growth factor receptor(EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, RSV F protein, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcαR, Fc epsilon RI, FcγIIb, FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF -2, FGF-2 receptor, FGF-3, FGF-8, acidic FGF, basic FGF, FGFR, FGFR-3, fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-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, GDNF, gelsolin, GFAP, GF-CSF, GFR-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein llb / llla (GP) llb / llla), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, growth hormone releasing factor, GRO-β, GRO-γ, H. pylori, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMVgB envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp120, Heparanase, Heparin cofactor II, Hepatic growth factor, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), Herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-16, IL-16 receptor, IL-17, IL-17 receptor, IL-18(IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, I L-3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7, IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF- beta receptor, INF-gamma, INF-gamma receptor, type I IFN, type I IFN receptor, influenza, inhibin, inhibin alpha, inhibin beta, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha-V / beta-3, integrin alpha-V / beta-6, integrin alpha4 / beta7, integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha5 / beta6, integrin alpha-delta (αV), integrin α-θ, integrin β1, integrin β2, integrin β3 (GPIIb-IIIa), IP-10, I-TAC, JE, kallikrein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (amylin, islet amyloid polypeptide), LAP (TGF-1), latency-associated peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (leutinizingLH, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotactin, lymphotoxin β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 aa), MDC (69 aa), megsin, Mer, MET tyrosine kinase receptor family, metalloprotease, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1 α, MIP-1 β, MIP-3 α, MIP-3 β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein, monocyte colony inhibitory factor factor), mouse gonadotropin-associated peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müllerian inhibitory factor, Mug, MuSK, myelin-associated glycoprotein, myeloid progenitor inhibitory factor-1 (MPIF-I), NAIP, nanobody, NAP, NAP-2, NCA90, NCAD, N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, neuroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, neurturin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural molecule derived from hepatitis C virus Protein type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteoinductive factor factor), osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, poly glycol chains of different sizes chain) (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, programmed cell death protein 1, proinsulin, prolactin, proprotein convertase PC9, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, sclerostin, SDF-1, SDF1 α, SDF1β, serine (SERINE), serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine monophosphate receptor 1, staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T cell receptor α / β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan specific, TGF-β RII, TGF-β RIIb, TGF-β RIII, TGF-β Rl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphoprotein receptor, thymic Ck-1, thyroid-stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, tissue factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-α, TNF-β, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2 / DR4), TNFRSF10B (TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B), TNFRSF10C (TRAIL R3 DcR1 / LIT / TRID), TNFRSF10D (TRAIL R4 DcR2 / TRUNDD), TNFRSF11A (RANK ODF R / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY CROWN / TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF Rl CD120a / p55-60), TNFRSF1B (TNF RII). CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII / TNFC). R) TNFRSF4 (OX40 ACT35 / TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1 / APT1 / CD95), TNFRSF6B (DcR3 M68 / TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 ligand / TL2), TNFSF11 (TRANCE / RANK diagram ODF / OPG diagram) TNFSF12 (TWEAK Apo-3 diagram / DR3 diagram) TNFSF13 (APRIL TALL2) TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM LINK / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR LINK AITR LINK / TL6), TNFSF1A (TNF-factor (Conectin) / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 factor gp34 / TXGP1), TNFSF5 (CD40 factor CD154 / gp39 / HIGM1 / IMD3 / TRAP) TNFSF6 (Fas factor Apo-1 factor / APT1 factor) TNFSF7 (CD27-factor CD70), TNFSF8 (CD30-factor CD153), TNFSF9 (4-1 BB-factor).CD137 ligand), TNF-α, TNF-β, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGFs) such as TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA 125, tumor-associated antigen exhibiting Lewis Y-related glycoprotein, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, viral antigen, vitamin B12 receptor, vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-1-β, XCL1 / lymphotactin, XCR1, XEDAR, XIAP, and XPD.

[0117] The antigen-binding molecules described herein can reduce the total plasma antigen concentration of the above antigens.The antigen-binding molecules described herein can also eliminate viruses, bacteria, and fungi in plasma by binding to the structural components of viruses, bacteria, and fungi.In particular, the F protein of RSV, lipoteichoic acid of Staphylococcus aureus, Clostridium difficile toxin, Shiga-like toxin II, Bacillus anthracis protective antigen, and Hepatitis C virus E2 glycoprotein can be used as structural components of viruses, bacteria, and fungi.

[0118] Although the methods of the present invention are not limited to a particular theory, for example, the relationship between making the antigen-binding ability of an antigen-binding molecule lower (weaker) in an acidic pH range than that in a neutral pH range and / or increasing (enhancing) the human FcRn-binding ability in a neutral pH range, promoting uptake of the antigen-binding molecule into cells and increasing the number of antigens that can be bound to one antigen-binding molecule, and promoting elimination of plasma antigen concentration can be explained as follows.

[0119] For example, in the case of an antibody whose antigen-binding molecule binds to a membrane antigen, the antibody administered into the body binds to the antigen, and then the antibody is taken up into an endosome in the cell by internalization together with the antigen while still bound to the antigen. The antibody then migrates to the lysosome while still bound to the antigen, and is degraded by the lysosome together with the antigen. The disappearance from the plasma via internalization is called antigen-dependent disappearance, and has been reported for many antibody molecules (Drug Discov Today. 2006 Jan;11(1-2):81-8). When one molecule of an IgG antibody binds to an antigen in a bivalent manner, one molecule of the antibody is internalized while bound to two molecules of the antigen, and is degraded in the lysosome as it is. Therefore, in the case of a normal antibody, one molecule of an IgG antibody cannot bind to three or more molecules of an antigen. For example, in the case of one molecule of an IgG antibody having neutralizing activity, three or more molecules of an antigen cannot be neutralized.

[0120] The relatively long plasma retention of IgG molecules (slow elimination) is due to the function of human FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to human FcRn expressed in endosomes under acidic conditions in the endosome. IgG molecules that cannot bind to human FcRn proceed to lysosomes, where they are degraded, but IgG molecules that bind to human FcRn migrate to the cell surface and dissociate from human FcRn under neutral conditions in plasma, returning to the plasma.

[0121] In addition, in the case of antibodies whose antigen-binding molecules bind to soluble antigens, the antibodies administered into the body bind to the antigen, and are then taken up into the cells while still bound to the antigen. Most of the antibodies taken up into the cells are released outside the cells by FcRn, but since they are released outside the cells while still bound to the antigen, they cannot bind to the antigen again. Therefore, as with antibodies that bind to membrane antigens, in the case of normal antibodies, one IgG antibody molecule cannot bind to three or more antigen molecules.

[0122] A pH-dependent antigen-binding antibody (an antibody that binds to an antigen under neutral conditions and dissociates under acidic conditions in an endosome) that strongly binds to an antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in an endosome can dissociate from the antigen in an endosome. A pH-dependent antigen-binding antibody can bind to an antigen again when the antibody is recycled into plasma by FcRn after dissociating from the antigen, so that one antibody can repeatedly bind to multiple antigens. In addition, since an antigen bound to an antigen-binding molecule is dissociated in an endosome and is not recycled into plasma, it is possible to promote the uptake of the antigen into cells by the antigen-binding molecule, and the administration of the antigen-binding molecule promotes the disappearance of the antigen, thereby making it possible to reduce the antigen concentration in plasma.

[0123] In an antibody that has pH-dependence on antigen binding (binding under neutral conditions and dissociating under acidic conditions), by imparting binding to human FcRn under neutral conditions (pH 7.4), it is possible to further promote uptake of the antigen into cells by the antigen-binding molecule, and administration of the antigen-binding molecule promotes antigen elimination, thereby lowering the antigen concentration in plasma. Usually, antibodies and antibody-antigen complexes are taken up into cells by nonspecific endocytosis, transported to the cell surface by binding to FcRn under acidic conditions in the endosome, and recycled into plasma by dissociating from FcRn under neutral conditions on the cell surface. Therefore, when an antibody that has sufficient pH-dependence on antigen binding (binding under neutral conditions and dissociating under acidic conditions) binds to an antigen in plasma and dissociates the bound antigen in the endosome, it is considered that the rate of antigen elimination is equal to the rate of uptake into cells by nonspecific endocytosis. When the pH dependency is insufficient, antigens that do not dissociate in endosomes are recycled into plasma, but when the pH dependency is sufficient, the rate of antigen elimination is limited by nonspecific endocytosis. Since FcRn transports antibodies from endosomes to the cell surface, it is thought that some FcRn is also present on the cell surface.

[0124] The present inventors considered that, while IgG immunoglobulin, which is one of antigen-binding molecules, generally has almost no binding ability to FcRn in the neutral pH range, IgG immunoglobulins having the ability to bind to FcRn in the neutral pH range can bind to FcRn present on the cell surface, and are taken up into cells in an FcRn-dependent manner by binding to FcRn present on the cell surface. The rate of uptake into cells via FcRn is faster than the rate of uptake into cells by nonspecific endocytosis. Therefore, by imparting the ability to bind to FcRn in the neutral pH range, it is possible to further increase the rate of disappearance of antigens. That is, antigen-binding molecules having the ability to bind to FcRn in the neutral pH range deliver antigens into cells more quickly than normal (intact human) IgG immunoglobulins, dissociate the antigen in the endosome, and recycle to the cell surface or plasma, where they bind to the antigen again and are taken up into cells via FcRn. By increasing the binding ability to FcRn in the neutral pH range, it is possible to increase the rotation rate of this cycle, thereby increasing the rate at which antigens are eliminated from plasma. Furthermore, by decreasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range, it is possible to further increase the efficiency. Furthermore, by increasing the number of rotations by one antigen-binding molecule, it is considered that the number of antigens that can be bound by one antigen-binding molecule also increases. The antigen-binding molecule of the present invention is composed of an antigen-binding domain and an FcRn-binding domain, and since the FcRn-binding domain does not affect the binding to antigen, and also in view of the above-mentioned mechanism, it is not dependent on the type of antigen, and it is considered that by decreasing the antigen-binding activity (binding ability) of the antigen-binding molecule in the acidic pH range compared to the antigen-binding activity in the neutral pH range and / or increasing the binding activity to FcRn at the pH in plasma, it is possible to promote the uptake of antigens into cells by the antigen-binding molecule and increase the rate at which antigens are eliminated.

[0125] <Substances that act as antigen-binding molecules> Furthermore, the present invention provides antigen-binding molecules that have human FcRn-binding activity in the acidic pH range and neutral pH range, and whose antigen-binding activity in the acidic pH range is lower than that in the neutral pH range. Specific examples include antigen-binding molecules that have human FcRn-binding activity at pH 5.8 to 6.0 and pH 7.4, which are considered to be the pH in early endosomes in vivo and the pH in plasma in vivo, respectively, and whose antigen-binding activity at pH 5.8 is lower than that at pH 7.4. An antigen-binding molecule whose antigen-binding activity at pH 5.8 is lower than that at pH 7.4 can also be said to be an antigen-binding molecule whose antigen-binding activity at pH 7.4 is higher than that at pH 5.8.

[0126] The antigen-binding molecule of the present invention having human FcRn-binding activity in an acidic pH range and a neutral pH range is preferably an antigen-binding molecule that has human FcRn-binding activity in an acidic pH range and has higher human FcRn-binding activity than that of intact human IgG in a neutral pH range, and the ratio of the binding activities is not limited as long as the human FcRn-binding activity at pH 7.4 is slightly higher.

[0127] According to The Journal of Immunology, 2009 182: 7663-7671, the human FcRn-binding activity of intact human IgG1 in the acidic pH range (pH 6.0) is KD 1.7 μM, and the human FcRn-binding activity of intact human IgG1 in the neutral pH range is almost undetectable. Thus, preferred embodiments of the antigen-binding molecules having human FcRn-binding activity in the acidic and neutral pH ranges of the present invention include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 20 μM or stronger and whose human FcRn-binding activity in the neutral pH range is equivalent to or stronger than that of intact human IgG, and more preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 2.0 μM or stronger and whose human FcRn-binding activity in the neutral pH range is KD 40 μM or stronger. More preferred embodiments include antigen-binding molecules having human FcRn-binding activity in the acidic pH range of 0.5 μM or stronger (KD) and human FcRn-binding activity in the neutral pH range of 15 μM or stronger (KD). The KD values ​​shown here are values ​​measured by the method described in The Journal of Immunology, 2009, 182: 7663-7671 (in which the antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0128] The present invention provides an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in an acidic pH range and a neutral pH range, and the human FcRn-binding activity and the antigen-binding activity in an acidic pH range lower than the neutral pH range are stronger than KD 3.2 μM. The present invention also provides an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range, and the human FcRn-binding activity in the neutral pH range is 28-fold higher than that of intact human IgG. The antigen-binding molecule of the present invention has a higher human FcRn-binding activity at pH 7.0 and 25° C. than that of intact human IgG. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25° C. is 28-fold higher than that of intact human IgG, or is stronger than KD 3.2 μM.

[0129] The present invention provides antigen-binding molecules comprising an antigen-binding domain and a human FcRn-binding domain, which have human FcRn-binding activity in a neutral pH range and which is stronger than 2.3 μM in the neutral pH range. The present invention also provides antigen-binding molecules comprising an antigen-binding domain and a human FcRn-binding domain, which have human FcRn-binding activity in a neutral pH range and which is 38-fold higher than the binding activity of intact human IgG.

[0130] In the present invention, the acidic pH range generally means pH 4.0 to pH 6.5. The acidic pH range is preferably a range indicated by any pH value within pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Meanwhile, in the present invention, the neutral pH range generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value within pH 7.0 to pH 8.0, 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 particularly preferably pH 7.4, which is close to the pH in plasma (blood) in vivo. When the binding affinity between the human FcRn-binding domain and human FcRn is low at pH 7.4 and it is difficult to evaluate the affinity, pH 7.0 can be used instead of pH 7.4. Regarding the temperature used as the measurement condition, the binding affinity between the human FcRn-binding domain and human FcRn may 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 the human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 is an example of an embodiment of the present invention.

[0131] In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25° C. is 38 times higher than intact human IgG, or KD 2.3 μM stronger. Intact human IgG1, IgG2, IgG3, or IgG4 is used as intact human IgG for the control intact human IgG application to compare with antigen-binding molecules for human FcRn binding activity. More preferably, intact human IgG1 is used for the control intact human IgG application to compare with antigen-binding molecules for human FcRn binding activity.

[0132] The present invention provides antigen-binding molecules that comprise an antigen-binding domain and a human FcRn-binding domain, and that give a lower total antigen concentration in plasma after administration of the antigen-binding molecule to a non-human animal than the total antigen concentration in plasma after administration of a control antigen-binding molecule to a non-human animal.

[0133] The present invention also provides an antigen-binding molecule, the plasma antigen concentration after administration of the antigen-binding molecule to a non-human animal is lower than the total antigen concentration in plasma obtained from a non-human animal not administered the antigen-binding molecule.

[0134] The total plasma antigen concentration can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, and 1000-fold or more by administration of an antigen-binding molecule of the present invention compared to administration of a control antigen-binding molecule comprising an intact human IgG Fc-binding domain as the human FcRn-binding domain, or compared to the case where the antigen-binding molecule of the present invention is not administered.

[0135] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: C=A / B The antigen / antigen-binding molecule molar ratio (C) of the antigen-binding molecule calculated as follows: C'=A' / B' and providing an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, the antigen / antigen-binding molecule molar ratio (C') of which is lower than that of an antigen-binding molecule comprising the same antigen-binding domain and an intact human IgG Fc domain as the human FcRn-binding domain, the C' being calculated as follows: During the ceremony A is the total antigen concentration in plasma after administration of the antigen-binding molecule to a non-human animal, B is the plasma antigen-binding molecule concentration after administration of the antigen-binding molecule to a non-human animal; A' is the total antigen concentration in plasma after administration of a control antigen-binding molecule to a non-human animal; B' is the plasma antigen-binding molecule concentration after administration of a control antigen-binding molecule to a non-human animal.

[0136] The antigen / antigen-binding molecule molar ratio can be reduced 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, and 1000-fold or more by administration of the antigen-binding molecule of the present invention compared to administration of an antigen-binding molecule comprising an intact human IgG Fc domain as a human FcRn-binding domain.

[0137] The reduction in plasma total antigen concentration or antigen / antibody molar ratio can be evaluated as described in Examples 6, 8 and 13. More specifically, if the antigen-binding molecule does not cross-react with the mouse counterpart antigen, it can be evaluated by either the antigen-antibody co-injection model or the steady-state antigen infusion model using human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories, Methods Mol Biol. (2010) 602: 93-104.). If the antigen-antibody molecule cross-reacts with the mouse counterpart, it can be evaluated by simply injecting the antigen-binding molecule into human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories). In the co-injection model, a mixture of the antigen-binding molecule and the antigen is administered to the mouse. In the steady-state antigen infusion model, the mouse is implanted with an infusion pump filled with an antigen solution to achieve a constant plasma antigen concentration, and then the antigen-binding molecule is injected into the mouse. The test antigen-binding molecule is administered at the same dose. Total plasma antigen concentration, free plasma antigen concentration, and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.

[0138] The route of administration of the antigen-binding molecule of the present invention can be selected from intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, and intramuscular injection.

[0139] More specifically, the antigen-binding molecule having a long-term effect on the antigen elimination activity in plasma described in the present invention has an FcRn binding activity in the range of 28-440 times higher than that of intact human IgG1 at pH 7.0 and 25° C., or has an FcRn binding activity with a KD in the range of 3.0 μM to 0.2 μM. For the purpose of evaluating the long-term effect of the antigen-binding molecule of the present invention on the antigen elimination activity in plasma, the long-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration and the antigen / antigen-binding molecule molar ratio in plasma 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration of the antigen-binding molecule. Whether or not a decrease in the plasma antigen concentration or the antigen / antigen-binding molecule molar ratio is achieved by the antigen-binding molecule described in the present invention can be determined by evaluating the decrease at any one or more of the time points described above.

[0140] More specifically, the antigen-binding molecule having a short-term effect on antigen elimination activity in plasma described in the present invention has a human FcRn-binding activity 440-fold higher than that of intact human IgG at pH 7.0 and 25° C., or has an FcRn-binding activity with a KD of 0.2 μM or stronger. For the purpose of evaluating the short-term effect of the antigen-binding molecule of the present invention on antigen elimination activity in plasma, the short-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration and the antigen / antigen-binding molecule molar ratio in plasma 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration of the antigen-binding molecule.

[0141] Furthermore, in the antigen-binding molecule of the present invention whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range, the ratio of binding activities is not limited as long as the antigen-binding activity in the acidic pH range is lower than that in the neutral pH range, and it is sufficient that the antigen-binding activity in the acidic pH range is slightly lower. A preferred embodiment includes an antigen-binding molecule whose antigen-binding activity at pH 7.4 is at least twice as high as that at pH 5.8, a more preferred embodiment includes an antigen-binding molecule whose antigen-binding activity at pH 7.4 is at least 10 times as high as that at pH 5.8, and a more preferred embodiment includes an antigen-binding molecule whose antigen-binding activity at pH 7.4 is at least 40 times as high as that at pH 5.8.

[0142] Specific examples include those described in WO2009 / 125825. More specifically, preferred embodiments of the antigen-binding molecule of the present invention having lower antigen-binding activity at pH 5.8 than at pH 7.4 have a ratio of KD(pH5.8) / KD(pH7.4) between KD at pH 5.8 and KD at pH 7.4 for the antigen of 2 or more, more preferably KD(pH5.8) / KD(pH7.4) of 10 or more, and even more preferably KD(pH5.8) / KD(pH7.4) of 40 or more. The upper limit of KD(pH5.8) / KD(pH7.4) is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be prepared by those skilled in the art.

[0143] In another preferred embodiment of the antigen-binding molecule of the present invention having lower antigen-binding activity at pH 5.8 than at pH 7.4, the k d and k at pH 7.4 d The ratio of k d (pH5.8) / k d (pH 7.4) is 2 or more, and more preferably k d (pH5.8) / k d (pH 7.4) is 5 or more, and more preferably k d (pH5.8) / k d(pH 7.4) is 10 or more, and more preferably k d (pH5.8) / k d (pH7.4) is 30 or higher. d (pH5.8) / k d The upper limit of the value (pH 7.4) is not particularly limited, and may be any value, such as 50, 100, 200, etc., as long as it can be produced by those skilled in the art.

[0144] Conditions other than pH when measuring the antigen-binding activity and human FcRn-binding activity can be appropriately selected by those skilled in the art and are not particularly limited, but for example, as described in the Examples, measurements can be performed in MES buffer at 37° C. In addition, the antigen-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, and for example, as described in the Examples, measurements can be performed using Biacore T100 (GE Healthcare) or the like.

[0145] The antigen-binding molecule of the present invention is considered to promote the uptake of antigens into cells, to easily dissociate from antigens in endosomes, and then bind to human FcRn and are released outside the cells, and to easily bind to antigens in plasma again. Therefore, for example, when the antigen-binding molecule is a neutralizing antigen-binding molecule, the antigen-binding molecule of the present invention can promote the reduction of antigen concentration in plasma by its administration. As a result, an antigen-binding molecule having human FcRn binding activity in an acidic pH range has an antigen-binding activity in an acidic pH range lower than that in a neutral pH range, and has human FcRn binding activity in a neutral pH range, and has excellent pharmacokinetics and is an antigen-binding molecule capable of binding to many antigens per antigen-binding molecule.

[0146] A preferred embodiment of the antigen-binding molecule having human FcRn-binding activity in the acidic pH range and neutral pH range is an antigen-binding molecule having a human FcRn-binding domain that has the ability to bind to human FcRn directly or indirectly. The domain may be used as it is if it already has human FcRn-binding activity in the acidic pH range and neutral pH range, or even if it has human FcRn-binding activity only in the acidic pH range and has no or weak human FcRn-binding activity in the neutral pH range, amino acids in the domain may be modified to obtain human FcRn-binding activity in the neutral pH range. Furthermore, amino acids in a domain that already has human FcRn-binding activity in the acidic pH range and neutral pH range may be modified to increase human FcRn-binding activity. For example, an antigen-binding molecule having the amino acid sequence of an IgG Fc domain in which at least one amino acid has been modified may be exemplified. The amino acid modification is not particularly limited, and any site may be modified as long as the human FcRn-binding activity in the neutral pH range is higher than that before modification.

[0147] Specific amino acid modifications for imparting human FcRn-binding activity in the acidic pH range and neutral pH range include modifications at, for example, 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, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, Examples of such modifications include amino acids at positions 27 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. More specifically, examples of such modifications include amino acid modifications at positions (EU numbering) shown in Table 1, Table 2, Table 6-1, 6-2, and Table 9. Preferred examples of the antigen-binding molecule include an amino acid sequence in which at least one amino acid selected from among positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) has been modified.

[0148] In a preferred embodiment, the EU numbering Amino acid substitution at position 237, replacing Gly with Met; Amino acid substitution of Pro at position 238 to Ala; Amino acid substitution at position 239 replacing Ser with Lys; Amino acid substitution at position 248, replacing Lys with Ile; an amino acid substitution of Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; an amino acid substitution of Met at position 252 with Phe, Trp, or Tyr; Amino acid substitution at position 254 replacing Ser with Thr; Amino acid substitution at position 255, replacing Arg with Glu; an amino acid substitution of Thr at position 256 with Asp, Glu, or Gln; an amino acid substitution of Pro at position 257 with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; Amino acid substitution at position 258, replacing Glu with His; Amino acid substitution of Asp at position 265 to Ala; an amino acid substitution of Asp at position 270 with Phe; Amino acid substitution of Asn at position 286 with Ala or Glu; Amino acid substitution of Thr at position 289 to His; Amino acid substitution of Asn at position 297 to Ala; Amino acid substitution at position 298 replacing Ser with Gly; Amino acid substitution of Val at position 303 to Ala; Amino acid substitution of Val to Ala at position 305; an amino acid substitution replacing Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg; an amino acid substitution of Gln at position 311 with Ala, His, or Ile; an amino acid substitution of Asp at position 312 with Ala or His; Amino acid substitution of Leu at position 314 with Lys or Arg; an amino acid substitution of Asn at position 315 with Ala or His; Amino acid substitution of Lys at position 317 to Ala; an amino acid substitution of Asn at position 325 with Gly; an amino acid substitution at position 332 replacing Ile with Val; Amino acid substitution at position 334, replacing Lys with Leu; Amino acid substitution at position 360, replacing Lys with His; an amino acid substitution of Asp at position 376 with Ala; Amino acid substitution of Glu at position 380 to Ala; Amino acid substitution of Glu at position 382 to Ala; an amino acid substitution at position 384 replacing Asn or Ser with Ala; an amino acid substitution of Gly at position 385 with Asp or His; an amino acid substitution of Gln at position 386 with Pro; Amino acid substitution of Pro at position 387 to Glu; an amino acid substitution of Asn at position 389 with Ala or Ser; Amino acid substitution at position 424, replacing Ser with Ala; an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; Amino acid substitution at position 433, replacing His with Lys; an amino acid substitution replacing Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and Amino acid substitution at position 436 replacing Tyr or Phe with His The following can be mentioned.

[0149] The number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of amino acid modifications at two or more amino acid positions include those shown in Table 3, Tables 4-1 to 4-5, Tables 6-1, 6-2, and Table 9.

[0150] In addition, examples of domains that have human FcRn-binding ability in the acidic and neutral pH ranges include the FcRn-binding domains of parent IgG, which are identified by EU numbering. The 237th amino acid is Met; Ala at amino acid 238; Lys at amino acid 239; Ile at amino acid 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at amino acid position 250; Phe, Trp, or Tyr at amino acid 252; Thr at amino acid 254; Glu at amino acid 255, Asp, Glu, or Gln at amino acid 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257; His at amino acid 258, Ala at amino acid 265; Phe at amino acid 270, Ala or Glu at amino acid 286; His at amino acid 289; Ala at amino acid 297; Gly at amino acid 298; Ala at amino acid 303; Ala at amino acid 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid 307; Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid 308; Ala, Asp, Glu, Pro, or Arg at amino acid position 309; Ala, His, or Ile at amino acid 311; Ala or His at amino acid 312; Lys or Arg at amino acid 314; Ala or His at amino acid 315; Ala at amino acid 317; Gly at amino acid 325; Val at amino acid 332; Leu at amino acid 334, His at amino acid 360, Ala at amino acid 376; Ala at amino acid 380; Ala at amino acid 382; Ala at amino acid 384; Asp or His at amino acid 385; Pro at amino acid 386, Glu at amino acid 387, Ala or Ser at amino acid 389; Ala at amino acid 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid 428; Lys at amino acid 433; Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid 436 The human FcRn-binding domain may comprise at least one amino acid selected from the group consisting of:

[0151] These amino acids may correspond to only one position, or may correspond to two or more positions. Examples of the combination of amino acids at two or more positions include the same amino acid combinations as those described in Table 3, Tables 4-1 to 4-5, Tables 6-1, 6-2, and Table 9.

[0152] Furthermore, preferred embodiments of antigen-binding molecules having lower antigen-binding activity in an acidic pH range than in a neutral pH range include antigen-binding molecules in which at least one amino acid in the antigen-binding molecule has been substituted with histidine or a non-natural amino acid, or in which at least one histidine or a non-natural amino acid has been inserted. The site at which the histidine or non-natural amino acid mutation is introduced is not particularly limited, and the antigen-binding activity in an acidic pH range is weaker than that in a neutral pH range compared to before the substitution (the value of KD(acidic pH range) / KD(neutral pH range) is increased, or k d (pH acidic range) / k dAny site may be used as long as the value of the neutral pH range increases). For example, when the antigen-binding molecule is an antibody, examples include the variable region and CDR of the antibody. The number of amino acids to be substituted with histidine or a non-natural amino acid or the number of amino acids to be inserted can be appropriately determined by those skilled in the art, and one amino acid may be substituted with histidine or a non-natural amino acid, one amino acid may be inserted, two or more amino acids may be substituted with histidine or a non-natural amino acid, or two or more amino acids may be inserted. In addition to the substitution with histidine or a non-natural amino acid or the insertion of histidine or a non-natural amino acid, deletion, addition, insertion and / or substitution of other amino acids may be performed at the same time. The substitution with histidine or a non-natural amino acid or the insertion of histidine or a non-natural amino acid may be performed randomly by a method such as histidine scanning in which alanine in alanine scanning is replaced with histidine, which is known to those skilled in the art. Among the antigen-binding molecules into which histidine or non-natural amino acid mutations were randomly introduced, the KD(pH5.8) / KD(pH7.4) or kD were compared with those before the mutation. d (pH5.8) / k d Antigen-binding molecules with a higher pH (pH 7.4) value can be selected.

[0153] A preferred example of an antigen-binding molecule in which a mutation to histidine or a non-natural amino acid has been performed and the antigen-binding activity in an acidic pH range is lower than that in a neutral pH range is, for example, an antigen-binding molecule whose antigen-binding activity at pH 7.4 after the mutation to histidine or a non-natural amino acid is equivalent to that at pH 7.4 before the mutation to histidine or a non-natural amino acid. In the present invention, the phrase "an antigen-binding molecule after the mutation to histidine or a non-natural amino acid has an antigen-binding activity equivalent to that of the antigen-binding molecule before the mutation to histidine or a non-natural amino acid" means that, when the antigen-binding activity of the antigen-binding molecule before the mutation to histidine or a non-natural amino acid is taken as 100%, the antigen-binding activity of the antigen-binding molecule after the mutation to histidine or a non-natural amino acid is at least 10%, preferably 50% or more, more preferably 80% or more, and more preferably 90% or more. The antigen-binding activity at pH 7.4 after the mutation to histidine or a non-natural amino acid may be higher than that at pH 7.4 before the mutation to histidine or a non-natural amino acid. When the antigen-binding activity of an antigen-binding molecule is reduced by substitution or insertion with histidine or a non-natural amino acid, the antigen-binding activity may be made equivalent to the antigen-binding activity before the histidine substitution or insertion by substitution, deletion, addition, and / or insertion of one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity is made equivalent by substitution, deletion, addition, and / or insertion of one or more amino acids after such histidine substitution or insertion.

[0154] Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, another preferred embodiment of the antigen-binding molecule having lower antigen-binding activity at pH 5.8 than at pH 7.4 includes a method in which the antibody constant region contained in the antigen-binding molecule is modified. Specific examples of the antibody constant region after modification include the antibody constant regions described in the Examples of WO2009 / 125825 (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14).

[0155] When the antigen-binding activity of an antigen-binding substance at pH 5.8 is weakened compared to the antigen-binding activity at pH 7.4 (the KD(pH 5.8) / KD(pH 7.4) value is increased) by the methods described above or the like, it is not particularly limited, but it is preferable that the KD(pH 5.8) / KD(pH 7.4) value is usually at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold that of the original antibody.

[0156] Furthermore, the present invention provides antigen-binding molecules in which at least one of the following amino acids has been substituted with histidine or a non-natural amino acid, where the amino acid positions are indicated by Kabat numbering (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH):

[0157] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102 Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94

[0158] Among these alterations, H32, H61, L53, L90, and L94 are considered to be highly common alterations.

[0159] Specific preferred combinations of substitutions of multiple sites with histidine or unnatural amino acids include, for example, the combination of H27, H31, and H35, the combination of H27, H31, H32, H35, H58, H62, and H102, the combination of L32 and L53, the combination of L28, L32, and L53, etc. Furthermore, a preferred example of a combination of substitution sites between the heavy chain and the light chain is the combination of H27, H31, L32, and L53.

[0160] The antigen-binding molecule of the present invention may have any other properties, for example, an agonist antigen-binding molecule or an antagonist antigen-binding molecule, as long as it has a lower antigen-binding activity in an acidic pH range than in a neutral pH range and has human FcRn-binding activity in both the acidic and neutral pH ranges. An example of a preferred antigen-binding molecule of the present invention is an antagonist antigen-binding molecule. An antagonist antigen-binding molecule is usually an antigen-binding molecule that inhibits the binding of a ligand (agonist) to a receptor and inhibits signal transduction into cells via the receptor.

[0161] Furthermore, the antigen recognized by the antigen-binding molecule of the present invention may be any antigen. Specific examples of the antigen recognized by the antigen-binding molecule of the present invention include the above-mentioned receptor proteins (membrane-bound receptors, soluble receptors), membrane antigens such as cell surface markers, and soluble antigens such as cytokines, for example, the antigens described above.

[0162] A preferred embodiment of the antigen-binding molecule of the present invention is an IgG immunoglobulin (IgG antibody) having an antigen-binding domain and a human FcRn-binding domain. When an IgG antibody is used as an antigen-binding molecule, the type is not limited, and IgG1, IgG2, IgG3, IgG4, etc. can be used.

[0163] The origin of the antigen-binding molecule of the present invention is not particularly limited, and the antigen-binding molecule may be of any origin, for example, a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, etc. Furthermore, the antigen-binding molecule may be, for example, the above-mentioned chimeric antibody, particularly a modified antibody in which the amino acid sequence has been substituted, such as a humanized antibody. In addition, the antigen-binding molecule may be the above-mentioned bispecific antibody, an antibody modified with various molecules bound thereto, a polypeptide containing an antibody fragment, an antibody with a modified sugar chain, etc.

[0164] A bispecific antibody refers to an antibody having variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody or a multispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

[0165] Furthermore, examples of polypeptides containing antibody fragments include Fab fragments, F(ab')2 fragments, scFv (Nat Biotechnol. 2005 Sep; 23(9): 1126-36), domain antibodies (dAbs) (WO2004 / 058821, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, and Fc fusion proteins. For molecules containing an Fc domain, the Fc domain can be used as a human FcRn-binding domain. Alternatively, an FcRn-binding domain may be fused to these molecules.

[0166] Furthermore, the antigen-binding molecule applicable to the present invention may be an antibody-like molecule. An antibody-like molecule (scaffold molecule, peptide molecule) is a molecule that exerts its function by binding to a target molecule (Current Opinion in Biotechnology 2006, 17:653-658, Current Opinion in Biotechnology 2007, 18:1-10, Current Opinion in Structural Biology 1997, 7:463-469, Protein Science 2006, 15:14-27), and examples thereof include DARPins (WO2002 / 020565), affibodies (WO1995 / 001937), avimers (WO2004 / 044011, WO2005 / 040229), and adnectins (WO2002 / 032925). Even these antibody-like molecules, as long as they can bind to a target molecule in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, can promote intracellular uptake of antigens by the antigen-binding molecule, promote a decrease in the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by one antigen-binding molecule.

[0167] The antigen-binding molecule may also be a protein in which a human FcRn-binding domain is fused to a receptor protein that binds to a target containing a ligand, such as TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60). Even if these receptor-human FcRn-binding domain fusion proteins can bind to a target molecule containing a ligand in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, they can promote the uptake of antigens into cells by the antigen-binding molecule, promote the reduction of the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by one antigen-binding molecule. The receptor protein is appropriately designed and modified to include a binding domain of the receptor protein to a target containing a ligand. As in the examples described above, including TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein, preferably, soluble receptor molecules are used in the present invention, which include the extracellular domain of the receptor protein required for binding to the target containing the ligand. These designed and modified receptor molecules are referred to as artificial receptors in the present invention. The methods used to design and modify receptor molecules to construct artificial receptor molecules are known in the art.

[0168] The antigen-binding molecule may also be a fusion protein of an artificial ligand protein that binds to a target but has a neutralizing effect and a human FcRn-binding domain, and an example of an artificial ligand protein is mutant IL-6 (EMBO J. 1994 Dec 15;13(24):5863-70.). If these artificial ligand fusion proteins can bind to a target molecule in a pH-dependent manner and / or have human FcRn-binding activity in the neutral pH range, it is possible to promote the uptake of antigens into cells by the antigen-binding molecule, promote the reduction of the antigen concentration in plasma by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound to one antigen-binding molecule.

[0169] Furthermore, the antibody of the present invention may contain a modified sugar chain. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (WO99 / 54342, etc.), antibodies lacking fucose added to the sugar chain (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913, etc.), and antibodies with sugar chains containing bisecting GlcNAc (WO02 / 79255, etc.).

[0170] Conditions other than pH when measuring the binding activity to antigens or human FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009 / 125825, it can be measured under conditions of MES buffer and 37°C. In addition, the antigen-binding activity and human FcRn-binding activity of antigen-binding molecules can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to soluble antigens can be evaluated by flowing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, the binding activity to membrane antigens can be evaluated by flowing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized. The binding activity of antigen-binding molecules to human FcRn can be evaluated by flowing human FcRn or antigen-binding molecules as analytes through a chip on which the antigen-binding molecule or human FcRn is immobilized.

[0171] Methods for producing chimeric antibodies are known. For example, in the case of a human-mouse chimeric antibody, a chimeric antibody can be obtained by linking DNA encoding the antibody V region and DNA encoding the human antibody C region, incorporating this into an expression vector, and introducing it into a host for production.

[0172] The term "humanized antibody" is also called a reshaped human antibody, and is an antibody derived from a mammal other than a human, for example, a mouse antibody, in which the complementarity determining region (CDR) of the mouse antibody is grafted onto the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General techniques for gene recombination are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576). Humanized antibodies can be produced by known methods, for example, by determining the CDRs of a mouse antibody, obtaining DNA encoding an antibody in which the CDRs are linked to the framework region (FR) of a human antibody, and producing the humanized antibody using a system using a conventional expression vector. Such DNA can be synthesized by PCR using as primers several oligonucleotides prepared so as to have overlapping portions at the terminal regions of both the CDRs and the FRs (see the method described in WO98 / 13388). The FRs of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. If necessary, the amino acids of the FRs in the variable regions of the antibody may be modified so that the CDRs of the reshaped human antibody form a suitable antigen-binding site (Sato et al., Cancer Res. (1993) 53: 10.01-6). Amino acid residues in the FR that can be modified include portions that bind directly to an antigen via non-covalent bonds (Amit et al., Science (1986) 233: 747-53), portions that affect or act on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and portions involved in VH-VL interactions (EP239400 Patent Publication).

[0173] When the antigen-binding molecule of the present invention is a chimeric antibody or a humanized antibody, the C region of these antibodies is preferably derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4, etc. can be used for the H chain, and Cκ, Cλ, etc. can be used for the L chain. In addition, amino acid mutations may be introduced into the human antibody C region as necessary to increase or decrease the binding to the Fcγ receptor, improve the stability of the antibody, or improve the production of the antibody. The chimeric antibody of the present invention preferably comprises a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody. On the other hand, the humanized antibody preferably comprises a CDR of an antibody derived from a mammal other than human, and FR and C regions derived from a human antibody. The constant region derived from a human antibody preferably contains a human FcRn binding region, and examples of such antibodies include IgG (IgG1, IgG2, IgG3, IgG4). The constant region used in the humanized antibody of the present invention may be the constant region of an antibody belonging to any isotype. Preferably, a constant region derived from human IgG1 is used, but is not limited thereto. Furthermore, the FRs derived from human antibodies used in humanized antibodies are not particularly limited, and may be those of antibodies belonging to any isotype.

[0174] The variable and constant regions of the chimeric and humanized antibodies of the present invention may be modified by deletion, substitution, insertion and / or addition, etc., so long as they exhibit the binding specificity of the original antibody.

[0175] Chimeric and humanized antibodies that utilize human-derived sequences have reduced immunogenicity in the human body and are therefore considered useful when administered to humans for therapeutic purposes.

[0176] The antigen-binding molecules of the present invention may be obtained by any method. For example, antigen-binding molecules that do not originally have human FcRn activity in the acidic pH range and neutral pH range, or antigen-binding molecules whose antigen-binding activity in the acidic pH range is higher than or similar to that in the neutral pH range, may be artificially modified into antigen-binding molecules having the desired activity by the amino acid modification or the like described above, or antibodies having the desired activity may be selected by screening from multiple antibodies obtained from the antibody libraries or hybridomas described below.

[0177] When modifying amino acids in an antigen-binding molecule, the amino acid sequence of the antigen-binding molecule before modification may be a known sequence, or the amino acid sequence of an antigen-binding molecule newly obtained by a method known to those skilled in the art may be used. For example, an antibody may be obtained from an antibody library, or may be obtained by cloning a gene encoding the antibody from a hybridoma that produces a monoclonal antibody.

[0178] Many antibody libraries are already known, and methods for constructing antibody libraries are also known, so that a person skilled in the art can obtain an appropriate antibody library. For example, for phage libraries, reference can be made to documents such as Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 324.0-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14, and Published Japanese Translation of PCT International Publication No. 20-504970. Other known methods such as a method using eukaryotic cells as a library (WO95 / 15393 pamphlet) and ribosome display method can be used. Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed on the surface of a phage as a single chain antibody (scFv) by phage display method, and a phage that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of a human antibody that binds to an antigen can be determined. Once the DNA sequence of the scFv that binds to an antigen is revealed, an appropriate expression vector can be prepared based on the sequence, and a human antibody can be obtained. These methods are already well known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388.

[0179] Methods for obtaining genes encoding antibodies from hybridomas basically involve using known techniques, using a desired antigen or cells expressing the desired antigen as a sensitizing antigen, immunizing with this according to conventional immunization methods, fusing the resulting immune cells with known parent cells by conventional cell fusion methods, screening for monoclonal antibody-producing cells (hybridomas) by conventional screening methods, synthesizing cDNA for the variable region (V region) of the antibody from the mRNA of the resulting hybridoma using reverse transcriptase, and linking this to DNA encoding the constant region (C region) of the desired antibody.

[0180] More specifically, although not limited to the following examples, for example, the sensitizing antigen for obtaining the antigen-binding molecule genes encoding the H and L chains can include both complete antigens having immunogenicity and incomplete antigens containing haptens that do not show immunogenicity. For example, a full-length protein or a partial peptide of a target protein can be used. In addition, it is known that substances composed of polysaccharides, nucleic acids, lipids, etc. can be antigens, and the antigen of the antigen-binding molecule of the present invention is not particularly limited. The antigen can be prepared by a method known to those skilled in the art, for example, in accordance with a method using baculovirus (e.g., WO98 / 46777, etc.). The hybridoma can be prepared, for example, in accordance with the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). When the antigen has low immunogenicity, it may be bound to a macromolecule having immunogenicity such as albumin and immunized. If necessary, the antigen can be made soluble by binding to other molecules. When a transmembrane molecule such as a membrane antigen (e.g., a receptor) is used as an antigen, the extracellular domain of the membrane antigen can be used as a fragment, or a cell expressing the transmembrane molecule on the cell surface can be used as an immunogen.

[0181] Antigen-binding molecule-producing cells can be obtained by immunizing an animal with a suitable sensitizing antigen as described above. Alternatively, lymphocytes capable of producing antigen-binding molecules can be immunized in vitro to obtain antigen-binding molecule-producing cells. Various mammals can be used as animals to be immunized, but rodents, lagomorphs, and primates are generally used. Examples of primates include rodents such as mice, rats, and hamsters, lagomorphs such as rabbits, and monkeys such as cynomolgus monkeys, rhesus monkeys, hamadryas baboons, and chimpanzees. In addition, transgenic animals having a repertoire of human antibody genes are also known, and human antibodies can also be obtained by using such animals (see WO96 / 34096; Mendez et al., Nat. Genet. 1997, 15: 146-56). Instead of using such transgenic animals, for example, a desired human antibody having binding activity to an antigen can be obtained by sensitizing human lymphocytes in vitro with a desired antigen or cells expressing the desired antigen, and fusing the sensitized lymphocytes with human myeloma cells, such as U266 (see JP-B-1-59878). Also, a desired human antibody can be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with a desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735).

[0182] Immunization of animals is carried out, for example, by appropriately diluting and suspending the sensitizing antigen in phosphate-buffered saline (PBS) or physiological saline, mixing with an adjuvant as necessary to emulsify the mixture, and then injecting the mixture intraperitoneally or subcutaneously into the animal. Thereafter, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. Antibody production can be confirmed by measuring the titer of the antibody of interest in the serum of the animal by a conventional method.

[0183] Hybridomas can be prepared by fusing antigen-binding molecule-producing cells obtained from an animal or lymphocytes immunized with a desired antigen with myeloma cells using a conventional fusing agent (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). If necessary, hybridoma cells are cultured and grown, and the binding specificity of the antigen-binding molecule produced by the hybridoma is measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Thereafter, if necessary, hybridomas producing the antigen-binding molecule whose specificity, affinity, or activity has been measured can be subcloned by techniques such as limiting dilution.

[0184] Subsequently, a gene encoding the selected antigen-binding molecule can be cloned from a hybridoma or an antigen-binding molecule-producing cell (such as a sensitized lymphocyte) using a probe capable of specifically binding to the antigen-binding molecule (for example, an oligonucleotide complementary to a sequence encoding an antibody constant region). Cloning from mRNA by RT-PCR is also possible. Immunoglobulins are classified into five different classes: IgA, IgD, IgE, IgG, and IgM. These classes are further divided into several subclasses (isotypes) (for example, IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2, etc.). The H chain and L chain used in the production of the antigen-binding molecule in the present invention may be derived from an antibody belonging to any of these classes and subclasses, and are not particularly limited, but IgG is particularly preferred.

[0185] Here, it is also possible to modify the genes encoding the H and L chains by genetic engineering techniques. For example, for antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, and camel antibodies, artificially modified genetically engineered antibodies, such as chimeric antibodies and humanized antibodies, can be appropriately produced for the purpose of reducing heterologous immunogenicity against humans. A chimeric antibody is an antibody consisting of the variable regions of the H and L chains of a mammal other than human, such as a mouse antibody, and the constant regions of the H and L chains of a human antibody, and can be obtained by linking DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host to produce it. A humanized antibody is also called a reshaped human antibody, and is synthesized by PCR from several oligonucleotides prepared to have overlapping portions at the ends of a DNA sequence designed to link the complementary determining region (CDR) of a mammal other than human, such as a mouse antibody. The resulting DNA is ligated to DNA encoding the human antibody constant region, then incorporated into an expression vector, which is then introduced into a host for production (see EP239400; WO96 / 02576). The FRs of the human antibody to be linked via the CDRs are selected from those whose complementarity determining regions form a good antigen-binding site. If necessary, amino acids in the framework regions of the antibody variable region may be substituted so that the complementarity determining regions of the reshaped human antibody form a suitable antigen-binding site (K. Sato et al., Cancer Res. 1993, 53: 10.01-10.06).

[0186] In addition to the above-mentioned humanization, it is also possible to carry out modifications to improve the biological properties of antibodies, such as their binding to antigens. The modifications in the present invention can be carried out by methods such as site-directed mutagenesis (see, for example, Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, cassette mutagenesis, and the like. In general, antibody mutants with improved biological properties have an amino acid sequence homology and / or similarity of 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 97%, 98%, 99%, etc.) to the amino acid sequence of the variable region of the original antibody. In the present specification, sequence homology and / or similarity is defined as the percentage of amino acid residues that are homologous (the same residues) or similar (amino acid residues classified into the same group based on the properties of general amino acid side chains) to the original antibody residues, after aligning the sequences and introducing gaps as necessary so that the sequence homology is maximized. Usually, natural amino acid residues are classified into the same group based on the properties of their side chains. (1) Hydrophobic: alanine, isoleucine, valine, methionine and leucine; (2) Neutral hydrophilic: asparagine, glutamine, cysteine, threonine and serine; (3) Acidic: aspartic acid and glutamic acid; (4) Basic: arginine, histidine and lysine; (5) residues that influence chain orientation: glycine and proline; and (6) Aromatic: tyrosine, tryptophan and phenylalanine They are classified into the following groups.

[0187] Usually, a total of six complementarity determining regions (hypervariable regions; CDRs) present in the variable regions of the H and L chains interact with each other to form the antigen-binding site of an antibody. It is known that even one of these variable regions has the ability to recognize and bind to an antigen, although with a lower affinity than one containing all of the binding sites. Therefore, the antibody genes encoding the H and L chains of the present invention may be sufficient as long as the polypeptides encoded by the genes maintain the binding ability to the desired antigen, and may encode fragments containing the antigen-binding sites of each of the H and L chains.

[0188] As described above, the heavy chain variable region is usually composed of three CDR regions and four FR regions. In a preferred embodiment of the present invention, the amino acid residues to be "modified" can be appropriately selected from, for example, amino acid residues located in the CDR region or FR region. In general, modification of amino acid residues in the CDR region may reduce the binding ability to an antigen. Therefore, the amino acid residues to be "modified" in the present invention are not particularly limited, but are preferably appropriately selected from amino acid residues located in the FR region. If it is confirmed that the binding ability is not reduced by modification even in the CDR, that site can be selected. In addition, a person skilled in the art can appropriately obtain a sequence that can be used as the FR of the variable region of an antibody in an organism such as a human or mouse by using a public database or the like.

[0189] Furthermore, the present invention provides genes encoding the antigen-binding molecules of the present invention. The genes encoding the antigen-binding molecules of the present invention may be any genes, including DNA, RNA, and other nucleic acid analogs.

[0190] The present invention further provides a host cell having the above gene. The host cell is not particularly limited, and examples thereof include Escherichia coli and various animal cells. The host cell can be used, for example, as a production system for producing and expressing the antibody of the present invention. Production systems for producing polypeptides include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0191] Examples of eukaryotic cells that can be used as host cells include animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108: 94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, and the like, amphibian cells such as Xenopus oocytes (Valle et al., Nature (1981) 291: 338-340), and insect cells such as Sf9, Sf21, and Tn5. In expressing the antibody of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. In animal cells, CHO cells are particularly preferred when large-scale expression is the objective. Vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE-dextran method, a method using the cationic ribosome DOTAP (manufactured by Boehringer Mannheim), electroporation, lipofection, or the like.

[0192] As for plant cells, for example, cells derived from Nicotiana tabacum and duckweed (Lemna minor) are known as protein production systems, and the antigen-binding molecules of the present invention can be produced by a method of callus culture of these cells. As for fungal cells, protein expression systems using yeasts, for example, cells of the genus Saccharomyces (Saccharomyces cerevisiae, Saccharomyces pombe, etc.), and filamentous fungi, for example, cells of the genus Aspergillus (Aspergillus niger, etc.), are known and can be used as hosts for producing the antigen-binding molecules of the present invention.

[0193] When prokaryotic cells are used, there are production systems using bacterial cells. In addition to the above-mentioned E. coli, production systems using Bacillus subtilis are known as bacterial cells, and these can be used to produce the antigen-binding molecules of the present invention.

[0194] <Screening method> The present invention provides a method for screening an antigen-binding molecule having human FcRn-binding activity in the acidic pH range and neutral pH range. Furthermore, the present invention provides a method for screening an antigen-binding molecule having human FcRn-binding activity in the acidic pH range and neutral pH range, and having lower antigen-binding activity in the acidic pH range than in the neutral pH range. The present invention also provides a method for screening an antigen-binding molecule capable of promoting intracellular uptake of an antigen. The present invention also provides a method for screening an antigen-binding molecule having an increased number of antigens capable of binding to one antigen molecule. The present invention also provides a method for screening an antigen-binding molecule capable of promoting antigen elimination. The present invention also provides a method for screening an antigen-binding molecule having improved pharmacokinetics. The present invention also provides a method for screening an antigen-binding molecule that promotes intracellular dissociation of an antigen bound to the antigen-binding molecule outside a cell. The present invention also provides a method for screening an antigen-binding molecule that is taken up into a cell in a state bound to an antigen, and then its release outside the cell in a state not bound to an antigen is promoted. Furthermore, the present invention provides a method for screening an antigen-binding molecule that is particularly useful when used as a pharmaceutical composition. These methods are particularly useful for screening antigen-binding molecules that have excellent plasma retention and plasma antigen elimination capabilities.

[0195] Specifically, the present invention provides a method for screening an antigen-binding molecule, comprising the following steps: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) modifying at least one amino acid in the antigen-binding domain of an antigen-binding molecule and selecting an antigen-binding molecule having higher antigen-binding activity in a neutral pH range than in an acidic pH range.

[0196] It should be noted that either step (a) or (b) may be performed first, and each step may be repeated two or more times. The number of times steps (a) and (b) are repeated is not particularly limited, but is usually within 10 times.

[0197] In the screening method of the present invention, the antigen-binding activity of the antigen-binding molecule in the neutral pH range is not particularly limited as long as it is an antigen-binding activity between pH 6.7 and pH 10.0, and examples thereof include the embodiments described in WO2009 / 125825. A preferred antigen-binding activity can be an antigen-binding activity between pH 7.0 and pH 8.0, and a more preferred antigen-binding activity can be an antigen-binding activity at pH 7.4. Furthermore, the antigen-binding activity of the antigen-binding molecule in the acidic pH range is not particularly limited as long as it is an antigen-binding activity between pH 4.0 and pH 6.5, and a preferred antigen-binding activity can be an antigen-binding activity between pH 5.5 and pH 6.5, and a more preferred antigen-binding activity can be an antigen-binding activity at pH 5.8 or pH 5.5.

[0198] The human FcRn-binding activity of an antigen-binding molecule in the neutral pH range is not particularly limited as long as it is a human FcRn-binding activity between pH 6.7 and pH 10.0; preferred examples of the human FcRn-binding activity include human FcRn-binding activity between pH 7.0 and pH 8.0, and more preferred examples of the human FcRn-binding activity include human FcRn-binding activity at pH 7.4.

[0199] The human FcRn-binding activity of an antigen-binding molecule in an acidic pH range is not particularly limited as long as it is a human FcRn-binding activity between pH 4.0 and pH 6.5; however, preferred examples of the human FcRn-binding activity include human FcRn-binding activity between pH 5.5 and pH 6.5, and more preferred examples of the human FcRn-binding activity include human FcRn-binding activity between pH 5.8 and pH 6.0.

[0200] In the present invention, the acidic pH range generally means pH 4.0 to pH 6.5. The acidic pH range is preferably a range indicated by any pH value within pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, and particularly preferably pH 5.8 to 6.0, which is close to the pH in early endosomes in vivo. On the other hand, in the present invention, the neutral pH range generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value within pH 7.0 to pH 8.0, 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 particularly preferably pH 7.4, which is close to the pH in plasma (blood) in vivo. When the binding affinity between the human FcRn-binding domain and human FcRn is low at pH 7.4 and it is difficult to evaluate the binding affinity, pH 7.0 can be used instead of pH 7.4. Regarding the temperature used as the measurement condition, the binding affinity between the human FcRn-binding domain and human FcRn may 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 the human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 is an example of an embodiment of the present invention.

[0201] The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than pH can be appropriately determined by those skilled in the art. The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be determined by KD (Dissociation constant), apparent KD (Apparent dissociation constant), and dissociation rate k d(Dissociation rate), or apparent k d These can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0202] According to The Journal of Immunology (2009) 182: 7663-7671, the human FcRn-binding activity of intact human IgG1 is KD 1.7 μM in the acidic pH range (pH 6.0), but the activity is barely detectable in the neutral pH range. Thus, in a preferred embodiment, antigen-binding molecules of the present invention having human FcRn-binding activity in the acidic pH range and in the neutral pH range can be screened, including antigen-binding molecules having human FcRn-binding activity in the acidic pH range of KD 20 μM or stronger and human FcRn-binding activity in the neutral pH range equivalent to or stronger than that of intact human IgG. In a more preferred embodiment, antigen-binding molecules of the present invention including antigen-binding molecules having human FcRn-binding activity in the acidic pH range of KD 2.0 μM or stronger and human FcRn-binding activity in the neutral pH range of KD 40 μM or stronger can be screened. In an even more preferred embodiment, antigen-binding molecules of the present invention can be screened, including antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 0.5 μM or stronger and whose human FcRn-binding activity in the neutral pH range is KD 15 μM or stronger. The above KD values ​​are determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (in which the antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0203] The present invention provides a method for screening an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having a human FcRn-binding activity of greater than 3.2 μM (KD) in the neutral pH range, which is obtained by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule; (b) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) are linked; and (c) producing an antigen-binding molecule using the gene prepared in (b).

[0204] In one embodiment, an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in the acidic pH range and neutral pH range, and whose human FcRn-binding activity and antigen-binding activity in the acidic pH range lower than the neutral pH range are stronger than KD 3.2 μM, can be screened according to the method used by those skilled in the art as described above in this specification. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25° C. is stronger than KD 3.2 μM.

[0205] The present invention provides a method for screening for an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range and whose human FcRn-binding activity in the neutral pH range is stronger than KD 2.3 μM. The present invention also provides a method for screening for an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range and whose human FcRn-binding activity in the neutral pH range is 38-fold higher than that of intact human IgG.

[0206] In the present invention, the antigen-binding molecule having human FcRn-binding activity in the neutral pH range is not particularly limited as long as it has human FcRn-binding activity at pH 6.7 to pH 10.0; however, preferably, the antigen-binding molecule has a higher human FcRn-binding activity at pH 6.7 to pH 10.0 than that of intact human IgG.

[0207] In the present invention, the antigen-binding molecule having human FcRn-binding activity in an acidic pH range is not particularly limited as long as it has human FcRn-binding activity at pH 4.0 to pH 6.5; however, an antigen-binding molecule whose human FcRn-binding activity at pH 5.5 to pH 6.5 is equal to or greater than that of intact human IgG is preferred.

[0208] Furthermore, in the present invention, the step of selecting antigen-binding molecules whose antigen-binding activity in a neutral pH range is higher than that in an acidic pH range is the same as the step of selecting antigen-binding molecules whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range.

[0209] As long as the antigen-binding activity in a neutral pH range is higher than that in an acidic pH range, the ratio of the antigen-binding activity in the neutral pH range to that in the acidic pH range is not particularly limited; however, the antigen-binding activity at pH 6.7 to pH 10.0 is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times, the antigen-binding activity at pH 4.0 to pH 6.5.

[0210] The screening method of the present invention may use a library such as a phage library.

[0211] In the method of the present invention, the antigen and the antigen-binding molecule may be bound in any state, and are not particularly limited. For example, the antigen may be bound to the antigen by contacting the antigen with an immobilized antigen-binding molecule, or the antigen may be bound to the antigen by contacting the antigen-binding molecule with an immobilized antigen. Alternatively, the antigen-binding molecule may be bound to the antigen by contacting the antigen in a solution.

[0212] Antigen-binding molecules to be screened by the screening methods of the present invention may be prepared in any manner, and examples of such antibodies that can be used include pre-existing antibodies, pre-existing antigen-binding domain libraries (such as phage libraries), antibodies or antigen-binding domain libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or antigen-binding domain libraries into which random amino acid modifications have been introduced, and antibodies or antigen-binding domain libraries into which histidine or non-natural amino acid mutations have been introduced (libraries with an increased histidine or non-natural amino acid content, antigen-binding domain libraries into which histidine or non-natural amino acid mutations have been introduced at specific sites, etc.).

[0213] The screening method of the present invention makes it possible to obtain antigen-binding molecules that bind to antigens multiple times and have excellent plasma retention properties. Thus, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules with excellent plasma retention properties.

[0214] Furthermore, the screening method of the present invention makes it possible to obtain antigen-binding molecules that can bind to an antigen two or more times when administered to animals such as humans, mice, monkeys, etc. Thus, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules that can bind to an antigen two or more times.

[0215] Furthermore, the screening method of the present invention makes it possible to obtain an antigen-binding molecule that, when administered to an animal such as a human, mouse, or monkey, is capable of binding to a greater number of antigens than the number of antigen-binding sites in the antigen-binding molecule. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that is capable of binding to a greater number of antigens than the number of antigen-binding sites in the antigen-binding molecule. For example, when the antibody is a neutralizing antibody, it can be used as a screening method for obtaining an antigen-binding molecule that is capable of neutralizing a greater number of antigens than the number of antigen-binding sites in the antigen-binding molecule.

[0216] Furthermore, the screening method of the present invention makes it possible to obtain antigen-binding molecules that are capable of intracellularly dissociating extracellularly bound antigens when administered to animals such as humans, mice, monkeys, etc. Thus, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules that intracellularly dissociate extracellularly bound antigens.

[0217] Furthermore, the screening method of the present invention makes it possible to obtain antigen-binding molecules that, when administered to animals such as humans, mice, monkeys, etc., are taken up into cells in an antigen-bound state and released outside the cells in an unantigen-bound state. Thus, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules that are taken up into cells in an antigen-bound state and released outside the cells in an unantigen-bound state.

[0218] Furthermore, the screening method of the present invention makes it possible to obtain antigen-binding molecules that can quickly eliminate antigens from plasma when administered to animals such as humans, mice, monkeys, etc. Thus, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules with increased (high) ability to eliminate antigens from plasma.

[0219] Furthermore, these antigen-binding molecules are considered to be particularly excellent as pharmaceuticals because they enable a reduction in the dosage and frequency of administration to patients, thereby enabling a reduction in the total dosage. Therefore, the screening method of the present invention can be used as a method for screening antigen-binding molecules for use as pharmaceutical compositions.

[0220] <Antigen-binding molecule production method> The present invention provides a method for producing an antigen-binding molecule that has human FcRn-binding activity at the pH in endosomes and at the pH in plasma, and whose antigen-binding activity at the pH in endosomes is lower than that at the pH in plasma.The present invention also provides a method for producing an antigen-binding molecule that has an excellent promoting effect on reducing the antigen concentration in plasma upon administration of the antigen-binding molecule and has excellent pharmacokinetics.Furthermore, the present invention provides a method for producing an antigen-binding molecule that is particularly useful when used as a pharmaceutical composition.

[0221] Specifically, the present invention provides a method for producing an antigen-binding molecule, comprising the steps of: (a) selecting an antigen-binding molecule having human FcRn-binding activity in a neutral pH range higher than that before modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule having human FcRn-binding activity in an acidic pH range; (b) modifying at least one amino acid in the antigen-binding domain of an antigen-binding molecule and selecting an antigen-binding molecule having higher antigen-binding activity in a neutral pH range than in an acidic pH range; (c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and (d) producing an antigen-binding molecule using the gene prepared in (c).

[0222] It should be noted that either step (a) or (b) may be performed first, and each step may be repeated two or more times. The number of times steps (a) and (b) are repeated is not particularly limited, but is usually within 10 times.

[0223] The linker functionally links the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b), but is not limited to any form. The human FcRn-binding domain and the antigen-binding domain may be linked by covalent or non-covalent forces. In particular, the linker may be a peptide linker or a chemical linker, or a binding pair such as a combination of biotin and streptavidin. Modification of a polypeptide comprising a human FcRn-binding domain and an antigen-binding domain is known in the art. In another embodiment, the human FcRn-binding domain and the antigen-binding domain of the present invention may be linked by forming a fusion protein of the human FcRn-binding domain and the antigen-binding domain. To construct a fusion protein of the human FcRn-binding domain and the antigen-binding domain, genes encoding the human FcRn-binding domain and the antigen-binding domain may be functionally linked to form a fusion polypeptide in frame. A linker comprising a peptide consisting of several amino acids may be appropriately inserted between the human FcRn-binding domain and the antigen-binding domain. A linker having the sequence (GGGGS) n A variety of flexible linkers are known in the art, such as a linker consisting of:

[0224] The antigen-binding molecules used in the production methods of the present invention may be prepared in any manner. For example, pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries in which amino acids have been randomly modified in these antibodies or libraries, antibodies or libraries in which histidine or unnatural amino acid mutations have been introduced (libraries with an increased histidine or unnatural amino acid content, libraries in which histidine or unnatural amino acid mutations have been introduced at specific sites, etc.), can be used.

[0225] In the above-mentioned production methods, the human FcRn-binding activity of the antigen-binding molecule in the neutral pH range is not particularly limited as long as it is a human FcRn-binding activity between pH 6.7 and pH 10.0; however, a preferred example of the human FcRn-binding activity is a human FcRn-binding activity between pH 7.0 and pH 8.0, and a more preferred example of the human FcRn-binding activity is a human FcRn-binding activity at pH 7.4.

[0226] The human FcRn-binding activity of an antigen-binding molecule in an acidic pH range is not particularly limited as long as it is a human FcRn-binding activity between pH 4.0 and pH 6.5; preferred examples of the human FcRn-binding activity include human FcRn-binding activity between pH 5.5 and pH 6.5, and more preferred examples of the human FcRn-binding activity include human FcRn-binding activity at pH 6.0.

[0227] In the present invention, the acidic pH range generally means pH 4.0 to pH 6.5. The acidic pH range is preferably a range indicated by any pH value within pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to 6.0, which is close to the pH in early endosomes in vivo. On the other hand, in the present invention, the neutral pH range generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value within pH 7.0 to pH 8.0, 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 particularly preferably pH 7.4, which is close to the pH in plasma (blood) in vivo. When the binding affinity between the human FcRn-binding domain and human FcRn is low at pH 7.4 and it is difficult to evaluate the binding affinity, pH 7.0 can be used instead of pH 7.4. Regarding the temperature used as the measurement condition, the binding affinity between the human FcRn-binding domain and human FcRn may 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 the human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 is an example of an embodiment of the present invention.

[0228] The present invention provides a method for producing an antigen-binding molecule, the method comprising the steps of: (a) selecting an antigen-binding molecule having a human FcRn-binding activity of greater than 3.2 μM (KD) in the neutral pH range, which is obtained by modifying at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule; (b) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) are linked; and (c) producing an antigen-binding molecule using the gene prepared in (b).

[0229] In a preferred embodiment, the antigen-binding molecules of the present invention having human FcRn-binding activity in the acidic pH range and the neutral pH range can be produced, including antigen-binding molecules having a human FcRn-binding activity in the acidic pH range of KD 20 μM or stronger and a human FcRn-binding activity in the neutral pH range equivalent to or stronger than that of intact human IgG. In a more preferred embodiment, antigen-binding molecules of the present invention including antigen-binding molecules having a human FcRn-binding activity in the acidic pH range of KD 2.0 μM or stronger and a human FcRn-binding activity in the neutral pH range of KD 40 μM or stronger can be similarly produced. In an even more preferred embodiment, antigen-binding molecules of the present invention including antigen-binding molecules having a human FcRn-binding activity in the acidic pH range of KD 0.5 μM or stronger and a human FcRn-binding activity in the neutral pH range of KD 15 μM or stronger can be preferably produced. The above KD value is determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (immobilizing the antigen-binding molecule on a chip and flowing human FcRn as an analyte). In one embodiment, an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in the acidic pH range and neutral pH range, and whose human FcRn-binding activity and antigen-binding activity in the acidic pH range lower than the neutral pH range are stronger than KD 3.2 μM, can be produced according to the method used by those skilled in the art as described herein above. In a more preferred embodiment, the human FcRn-binding activity of the antigen-binding molecule thus produced at pH 7.0 and 25°C is stronger than KD 3.2 μM.

[0230] The present invention provides a method for producing an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range and whose human FcRn-binding activity in the neutral pH range is stronger than KD 2.3 μM. The present invention also provides a method for producing an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has human FcRn-binding activity in a neutral pH range and whose human FcRn-binding activity in the neutral pH range is 38-fold higher than that of intact human IgG.

[0231] In the above-mentioned production method, the antigen-binding activity of the antigen-binding molecule in the neutral pH range is not particularly limited as long as it is an antigen-binding activity between pH 6.7 and pH 10.0, and examples thereof include the embodiments described in WO2009 / 125825. A preferred antigen-binding activity is an antigen-binding activity between pH 7.0 and pH 8.0, and a more preferred antigen-binding activity is an antigen-binding activity at pH 7.4. In addition, the antigen-binding activity of the antigen-binding molecule in the acidic pH range is not particularly limited as long as it is an antigen-binding activity between pH 4.0 and pH 6.5, and a preferred antigen-binding activity is an antigen-binding activity between pH 5.5 and pH 6.5, and a more preferred antigen-binding activity is an antigen-binding activity at pH 5.8 or pH 5.5.

[0232] The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than pH can be appropriately determined by those skilled in the art.

[0233] In the production methods of the present invention, the antigen-binding molecule having human FcRn-binding activity in the neutral pH range is not particularly limited as long as it has human FcRn-binding activity at pH 6.7 to pH 10.0; however, preferably, the antigen-binding molecule has human FcRn-binding activity at pH 6.7 to pH 10.0 higher than that of intact human IgG, more preferably, a human FcRn-binding activity with a KD of more than 40 μM, and even more preferably, a human FcRn-binding activity with a KD of more than 15 μM.

[0234] In the production methods of the present invention, the antigen-binding molecule having human FcRn-binding activity in the acidic pH range is not particularly limited as long as it has human FcRn-binding activity at pH 4.0 to pH 6.5; however, it is preferable that the human FcRn-binding activity at pH 5.5 to pH 6.5 is stronger than KD 20 μM, more preferably equal to or stronger than that of intact human IgG1 (KD stronger than 1.7 μM), and even more preferably stronger than KD 0.5 μM.

[0235] The KD values ​​shown here are values ​​measured by the method described in The Journal of Immunology, 2009 182: 7663-7671 (an antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0236] Furthermore, in the production method of the present invention, the step of selecting an antigen-binding molecule whose antigen-binding activity at pH 6.7 to pH 10.0 is higher than that at pH 4.0 to pH 6.5 means the same as the step of selecting an antigen-binding molecule whose antigen-binding activity at pH 4.0 to pH 6.5 is lower than that at pH 6.7 to pH 10.0.

[0237] As long as the antigen-binding activity in a neutral pH range is higher than that in an acidic pH range, the ratio of the antigen-binding activity in the neutral pH range to that in the acidic pH range is not particularly limited; however, the antigen-binding activity at pH 6.7 to pH 10.0 is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times, the antigen-binding activity at pH 4.0 to pH 6.5.

[0238] In the above-mentioned production method, the binding of the antigen to the antigen-binding molecule and the binding of the antigen to the human FcRn may be performed under any conditions, and is not particularly limited. For example, the antigen-binding molecule may be bound to the antigen-binding molecule by contacting the antigen or human FcRn with an immobilized antigen-binding molecule, or the antigen-binding molecule may be bound to the antigen or human FcRn by contacting the antigen-binding molecule with an immobilized antigen or human FcRn. Alternatively, the antigen-binding molecule may be bound to the antigen or human FcRn by contacting the antigen or human FcRn in a solution.

[0239] The antigen-binding molecule produced by the above-mentioned production method may be any antigen-binding molecule; preferred examples include antigen-binding molecules that have an antigen-binding domain and a human FcRn-binding domain, in which at least one amino acid in the human FcRn-binding domain has been modified, and in which an amino acid in the antigen-binding molecule has been substituted with histidine or at least one histidine has been inserted.

[0240] The modification of the human FcRn-binding domain is not particularly limited as long as it is an amino acid modification that enhances the human FcRn-binding activity in the neutral pH range. For example, the modifications may be performed at 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, 326, 327, 328, 329, 330, 332, 333 to 341, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 39 Examples of such modifications include amino acids at positions 27 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. More specifically, examples of such modifications include amino acids at positions (EU numbering) shown in Tables 1, 2, 6-1, and 6-2. Preferably EU numbering 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315 The human FcRn-binding activity in the neutral pH range can be increased by modifying at least one amino acid selected from the amino acids at positions 1 to 4, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436. The number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of modifications of two or more amino acids include those described in Table 3, Tables 4-1 to 4-5, and Tables 6-1 and 6-2.

[0241] In addition, the site where the histidine mutation is introduced is not particularly limited and may be introduced at any site, as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range. Furthermore, the histidine mutation may be introduced at one site or at two or more sites.

[0242] Therefore, the production method of the present invention may further include the above-mentioned amino acid modification step and the step of substituting or inserting histidine. Note that, in the production method of the present invention, a non-natural amino acid may be used instead of histidine. Therefore, the present invention can be understood by replacing the above-mentioned histidine with a non-natural amino acid.

[0243] Furthermore, another embodiment of the antigen-binding molecule produced by the above-mentioned production method includes, for example, an antigen-binding molecule comprising a modified antibody constant region. Thus, the production method of the present invention may further comprise a step of modifying amino acids in the antibody constant region.

[0244] The antigen-binding molecule produced by the production method of the present invention is an antigen-binding molecule that promotes a decrease in the antigen concentration in plasma when administered. Therefore, the production method of the present invention can be used as a method for producing an antigen-binding molecule that promotes a decrease in the antigen concentration in plasma when administered.

[0245] Furthermore, the antigen-binding molecule produced by the production method of the present invention is an antigen-binding molecule with improved pharmacokinetics. Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule with improved pharmacokinetics.

[0246] Furthermore, it is believed that the antigen-binding molecules produced by the production methods can increase the number of antigens that can be bound by one antigen-binding molecule when administered to animals such as humans, mice, monkeys, etc. Therefore, the production methods of the present invention can be used as methods for producing antigen-binding molecules that increase the number of antigens that can be bound by one antigen-binding molecule.

[0247] Furthermore, it is believed that the antigen-binding molecule produced by the production method of the present invention is capable of dissociating an antigen bound to the antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly when administered to animals such as humans, mice, monkeys, etc. Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule capable of dissociating an antigen bound to the antigen extracellularly intracellularly.

[0248] Furthermore, it is believed that the antigen-binding molecule produced by the production method of the present invention can release the antigen-binding molecule that has been taken up into cells in an antigen-bound state to the outside of the cells when administered to animals such as humans, mice, and monkeys. Therefore, the production method of the present invention can be used as a method for producing an antigen-binding molecule that is taken up into cells in an antigen-bound state and released to the outside of the cells in an antigen-unbound state.

[0249] Furthermore, these antigen-binding molecules are considered to be particularly excellent as pharmaceuticals because they have a higher effect of lowering the antigen concentration in plasma when administered than conventional antigen-binding molecules. Therefore, the production method of the present invention can be used as a method for producing antigen-binding molecules for use as pharmaceutical compositions.

[0250] The gene obtained in the production method of the present invention is usually carried (inserted) in an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, when E. coli is used as a host, a cloning vector such as pBluescript vector (Stratagene) is preferred, but various commercially available vectors can be used. When a vector is used for the purpose of producing the antigen-binding molecule of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it expresses the antigen-binding molecule in a test tube, in E. coli, in cultured cells, or in an individual organism. For example, the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for E. coli, the pME18S-FL3 vector (GenBank accession number AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms are preferred. The DNA of the present invention can be inserted into a vector by standard methods, for example, ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

[0251] The host cell is not particularly limited, and various host cells are used depending on the purpose. Examples of cells for expressing an antigen-binding molecule include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, and microinjection.

[0252] The host cells can be cultured according to known methods. For example, when animal cells are used as the host, for example, DMEM, MEM, RPMI1640, or IMDM can be used as the culture medium. In this case, serum supplements such as FBS and fetal calf serum (FCS) can be used in combination, or the cells can be cultured by serum-free culture. The pH during culture is preferably about 6 to 8. Culture is usually performed at 30 to 40°C for about 15 to 200 hours, and the medium is replaced, aerated, or stirred as necessary.

[0253] In order to secrete the antigen-binding molecule expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be incorporated into the polypeptide of interest. These signals may be endogenous or heterologous to the antigen-binding molecule of interest.

[0254] On the other hand, systems for producing polypeptides in vivo include, for example, production systems using animals and production systems using plants. A target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in the animal or plant body and then recovered. In the present invention, the "host" includes these animals and plants.

[0255] When animals are used, there are production systems using mammals and insects. Mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals can also be used.

[0256] For example, a polynucleotide encoding an antigen-binding molecule of the present invention is prepared as a fusion gene with a gene encoding a polypeptide that is specifically produced in milk, such as goat β-casein. Then, a polynucleotide fragment containing this fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The antigen-binding molecule of interest can be obtained from milk produced by a transgenic goat born from the goat that received the embryo or its progeny. To increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat, an appropriate hormone may be administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12: 699-702).

[0257] Furthermore, as an insect for producing the antigen-binding molecule of the present invention, for example, a silkworm can be used. When using a silkworm, the antigen-binding molecule of interest can be obtained from the body fluids of the silkworm by infecting the silkworm with a baculovirus into which a polynucleotide encoding the antigen-binding molecule of interest has been inserted.

[0258] Furthermore, when a plant is used to produce the antigen-binding molecule of the present invention, for example, tobacco can be used. When tobacco is used, a polynucleotide encoding the antigen-binding molecule of interest is inserted into a plant expression vector, for example, pMON 530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium is then infected into tobacco, for example, Nicotiana tabacum, and the desired antigen-binding molecule can be obtained from the tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-8). In addition, the same bacterium can be infected into duckweed (Lemna minor), and after cloning, the desired antigen-binding molecule can be obtained from the duckweed cells (Cox KM et al. Nat. Biotechnol. 2006 Dec;24(12):1591-1597).

[0259] The antigen-binding molecule thus obtained can be isolated from inside or outside the host cell (culture medium, milk, etc.) and purified as a substantially pure and homogeneous antigen-binding molecule. The separation and purification of the antigen-binding molecule can be performed using any separation and purification method that is used in the purification of ordinary polypeptides, and is not limited in any way. For example, the antigen-binding molecule can be separated and purified by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.

[0260] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be performed using liquid phase chromatography, such as HPLC and FPLC. Examples of columns used in affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0261] If necessary, before or after purification of the antigen-binding molecule, a suitable protein-modifying enzyme can be used to optionally modify the molecule or partially remove peptides. Examples of protein-modifying enzymes that can be used include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

[0262] <Pharmaceutical Composition> The present invention also relates to a pharmaceutical composition comprising the antigen-binding molecule of the present invention, an antigen-binding molecule isolated by the screening method of the present invention, or an antigen-binding molecule produced by the production method of the present invention. The antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention is useful as a pharmaceutical composition because it has a higher effect of lowering the antigen concentration in plasma when administered compared to a normal antigen-binding molecule. The pharmaceutical composition of the present invention can contain a pharma- ceutical acceptable carrier.

[0263] In the present invention, the pharmaceutical composition generally refers to an agent for treating or preventing a disease, or for testing or diagnosing a disease.

[0264] The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution or suspension injection with water or other pharma- ceutically acceptable liquid. For example, it can be formulated by appropriately combining with a pharma- ceutically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the indicated range is obtained.

[0265] Sterile compositions for injection can be formulated according to common pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, and other auxiliary agents (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80 (trademark), HCO-50, etc.) may be used in combination.

[0266] The oily liquid may be sesame oil or soybean oil, and may be used in combination with benzyl benzoate and / or benzyl alcohol as a solubilizing agent. In addition, it may be combined with a buffer (e.g., phosphate buffer and sodium acetate buffer), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled into a suitable ampule.

[0267] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, it can be an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0268] The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose of the pharmaceutical composition containing the antigen-binding molecule can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dose can be set to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these numerical values. The dose and administration method vary depending on the body weight, age, symptoms, etc. of the patient, but a person skilled in the art can set an appropriate dose and administration method taking these conditions into consideration.

[0269] In addition, the amino acids contained in the amino acid sequences described in the present invention may be modified after translation (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art), but even if an amino acid is modified after translation in this way, it is naturally included in the amino acid sequences described in the present invention.

[0270] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0271] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0272] [Example 1] Examination of improvement of the antigen elimination acceleration effect of antibodies Anti-IL-6 receptor antibody Preparation of anti-human IL-6 receptor antibodies with FcRn binding activity under neutral conditions H54 / L28-IgG1, which is described in WO2009 / 125825 and contains H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), is a humanized anti-IL-6 receptor antibody. A mutation was introduced into H54 (SEQ ID NO: 1) to increase FcRn binding under neutral pH conditions (pH 7.4). Specifically, H54-IgG1-F14 (SEQ ID NO: 3) was prepared by substituting Met at position 252 in the EU numbering system with Trp and Asn at position 434 with Trp. The amino acid substitutions were introduced according to the method known to those skilled in the art and described in Reference Example 1.

[0273] H54 / L28-IgG1, which contains H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), and H54 / L28-IgG1-F14, which contains H54-IgG1-F14 (SEQ ID NO: 3) and L28 (SEQ ID NO: 2), were expressed and purified by a method known to those skilled in the art and described in Reference Example 2.

[0274] In vivo testing of antibodies using the steady-state infusion model with human FcRn transgenic mouse strain 276 In vivo studies were performed using the steady-state infusion model with human FcRn transgenic mouse line 276 using H54 / L28-IgG1 and H54 / L28-IgG1-F14 prepared as described above. A model animal in which the plasma concentration of soluble human IL-6 receptor was maintained constant was created by implanting an infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) filled with soluble human IL-6 receptor subcutaneously in the dorsal region of a human FcRn transgenic mouse line 276 (B6.mFcRn- / -.hFcRn Tg line 276 + / + mouse (B6.mFcRn- / - hFCRN Tg276 B6.Cg-Fcgrt Tg(FCGRT)276Dcr (Jackson #4919)), Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104). Anti-human IL-6 receptor antibody was administered to the model animal, and the pharmacokinetics of soluble human IL-6 receptor after administration was evaluated. To suppress the production of neutralizing antibodies against soluble human IL-6 receptor, monoclonal anti-mouse CD4 antibody (R&D) was administered at 20 mg / kg before implanting the infusion pump and 14 days after administration of the antibody into the tail vein. Next, an infusion pump filled with 92.8 μg / ml of soluble human IL-6 receptor was implanted subcutaneously in the back of the mouse. Three days after implanting the infusion pump, anti-human IL-6 receptor antibodies (H54 / L28-IgG1 and H54 / L28-IgG1-F14) were administered at 1 mg / kg once into the tail vein. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration of the anti-human IL-6 receptor antibody. The collected blood was immediately centrifuged at 15,000 rpm at 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set at -20°C or lower until measurement.

[0275] Measurement of plasma hsIL-6R concentration by electrochemiluminescence The concentration of hsIL-6R (soluble human IL-6 receptor) in mouse plasma was measured by electrochemiluminescence. Standard samples of hsIL-6R adjusted to concentrations of 2,000, 1,000, 500, 250, 125, 62.5, and 31.25 pg / ml and mouse plasma samples diluted 50-fold or more were prepared. The samples were mixed with monoclonal anti-human IL-6R antibody (R&D) ruthenium-conjugated with SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and WT-IgG1 solution, and reacted at 37°C overnight. The final concentration of WT-IgG1 containing H(WT) (SEQ ID NO: 4) and L(WT) (SEQ ID NO: 5) as the anti-human IL-6 receptor antibody was 333 μg / ml, which is in excess of the anti-human IL-6 receptor antibody concentration contained in the sample, with the aim of making almost all of the hsIL-6R molecules in the sample bound to WT-IgG1. The sample was then dispensed onto an MA400 PR Streptavidin Plate (Meso Scale Discovery), reacted at room temperature for 1 hour, and washed. Read Buffer T (×4) (Meso Scale Discovery) was dispensed, and immediately measured using a Sector PR 400 Reader (Meso Scale Discovery). The hsIL-6R concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). Figure 1 shows the change in plasma hsIL-6R concentration after intravenous administration of H54 / L28-IgG1 and H54 / L28-IgG1-F14, measured by this method.

[0276] As shown in Figure 1, administration of H54 / L28-IgG1 caused a significant increase in plasma hsIL-6R concentrations compared to baseline hsIL-6R concentrations without antibody administration. Meanwhile, administration of H54 / L28-IgG1-F14 caused a decrease in the increase in plasma hsIL-6R concentrations compared to H54 / L28-IgG1. This decrease in increase was due to increased human FcRn binding at neutral pH in H54 / L28-IgG1-F14 compared to H54 / L28-IgG1. This indicates that increasing the binding affinity of an antibody to FcRn at neutral pH can increase antigen clearance, but the degree of increase in antigen clearance was smaller in H54 / L28-IgG1-F14 compared to H54 / L28-IgG1.

[0277] [Example 2] Examination of improvement of the antigen elimination acceleration effect of pH-dependent antigen-binding antibodies (preparation of antibodies) pH-dependent human IL-6 receptor-binding antibody H54 / L28-IgG1 comprising H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2) as described in WO 2009 / 125825 is a humanized anti-IL-6 receptor antibody, and Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) is a humanized anti-IL-6 receptor antibody in which the property of binding to soluble human IL-6 receptor in a pH-dependent manner (binding at pH 7.4 and dissociating at pH 5.8) has been imparted to H54 / L28-IgG1. In vivo testing in mice as described in WO 2009 / 125825 showed that the disappearance of soluble human IL-6 receptor was significantly accelerated in a group administered a mixture of Fv4-IgG1 and soluble human IL-6 receptor as an antigen, compared to a group administered a mixture of H54 / L28-IgG1 and soluble human IL-6 receptor as an antigen.

[0278] Soluble human IL-6 receptor bound to an antibody that binds to normal soluble human IL-6 receptor is recycled into plasma by FcRn together with the antibody, whereas an antibody that binds to soluble human IL-6 receptor in a pH-dependent manner dissociates the soluble human IL-6 receptor bound to the antibody under acidic conditions in the endosome. The dissociated soluble human IL-6 receptor is degraded by lysosomes, which allows the disappearance of soluble human IL-6 receptor to be significantly accelerated. Furthermore, antibodies that bind to soluble human IL-6 receptor in a pH-dependent manner are recycled into plasma by FcRn, and the recycled antibodies can bind to soluble human IL-6 receptor again. This process is repeated, allowing one antibody molecule to bind to soluble human IL-6 receptor multiple times (Figure 2).

[0279] Antibodies that bind to antigens in a pH-dependent manner are extremely useful because they accelerate the elimination of soluble antigens and each antibody molecule can bind to soluble antigens multiple times. To further improve this effect of accelerating antigen elimination, we investigated a method to enhance FcRn binding under neutral conditions (pH 7.4).

[0280] Preparation of pH-dependent human IL-6 receptor-binding antibody that binds to FcRn under neutral conditions Mutations were introduced into Fv4-IgG1, which contains VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7), to increase the binding to FcRn under neutral conditions (pH 7.4). Specifically, VH3-IgG1-v1 (SEQ ID NO: 8) was prepared by substituting Met at position 252 in the EU numbering system with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu in the EU numbering system of the heavy chain constant region of IgG1, and VH3-IgG1-v2 (SEQ ID NO: 9) was prepared by substituting Asn at position 434 in the EU numbering system with Trp in the heavy chain constant region of IgG1. The amino acid substitutions were introduced according to the method known to those skilled in the art described in Reference Example 1.

[0281] H54 / L28-IgG1 comprising H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7), Fv4-IgG1-v1 comprising VH3-IgG1-v1 (SEQ ID NO: 8) and VL3-CK (SEQ ID NO: 7), and Fv4-IgG1-v2 comprising VH3-IgG1-v2 (SEQ ID NO: 9) and VL3-CK (SEQ ID NO: 7) were expressed and purified by a method known to those skilled in the art and described in Reference Example 2.

[0282] [Example 3] Examination of improvement of the antigen elimination acceleration effect of pH-dependent antigen-binding antibodies (in vivo test) In vivo studies using human FcRn transgenic mice and normal mice The pharmacokinetics of hsIL-6R and anti-human IL-6 receptor antibody was evaluated after administration of hsIL-6R (soluble human IL-6 receptor: prepared in Reference Example 3) alone or simultaneous administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (B6.mFcRn- / -.hFcRn Tg strain 276 + / + mice, Jackson Laboratories, Methods Mol Biol. 2010;602:93-104.) and normal mice (C57BL / 6J mice, Charles River Japan). hsIL-6R solution (5 μg / mL) or a mixed solution of hsIL-6R and anti-human IL-6 receptor antibody (5 μg / mL, 0.1 mg / mL, respectively) was administered once to the tail vein at 10 mL / kg. At this time, the anti-human IL-6 receptor antibody was present in sufficient excess to the hsIL-6R, so it is believed that almost all of the hsIL-6R was bound to the antibody. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set at -20°C or lower until the measurement was performed. As the anti-human IL-6 receptor antibodies, the above-mentioned H54 / L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 were used for human FcRn transgenic mice, and the above-mentioned H54 / L28-IgG1, Fv4-IgG1, Fv4-IgG1-v1, and Fv4-IgG1-v2 were used for normal mice.

[0283] Measurement of plasma anti-human IL-6 receptor antibody concentrations by ELISA Anti-human IL-6 receptor antibody concentrations in mouse plasma were measured by ELISA. First, anti-human IgG (γ-chain specific) F(ab')2 antibody fragment (Sigma) was dispensed into Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International) and left to stand overnight at 4°C to prepare an anti-human IgG solid-phase plate. Standard curve samples with plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg / mL and mouse plasma measurement samples diluted 100-fold or more were prepared, and 200 μL of 20 ng / mL hsIL-6R was added to 100 μL of these standard curve samples and plasma measurement samples and left to stand at room temperature for 1 hour. Then, the mixture was dispensed into an anti-human IgG solid-phase plate and left to stand at room temperature for another 1 hour. Then, biotinylated anti-human IL-6 R antibody (R&D) was reacted at room temperature for 1 hour, and Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was reacted at room temperature for 1 hour. A color reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate, and the reaction was stopped with 1N sulfuric acid (Showa Chemical). The absorbance at 450 nm was measured using a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the standard curve using the analysis software SOFTmax PRO (Molecular Devices). The change in plasma antibody concentration in human FcRn transgenic mice after intravenous administration measured by this method is shown in Figure 3, and the change in plasma antibody concentration in normal mice is shown in Figure 5.

[0284] Measurement of plasma hsIL-6R concentration by electrochemiluminescence The concentration of hsIL-6R in mouse plasma was measured by electrochemiluminescence. Standard hsIL-6R curve samples adjusted to concentrations of 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg / mL and mouse plasma measurement samples diluted 50-fold or more were prepared. The samples were mixed with monoclonal anti-human IL-6R antibody (R&D) ruthenium-conjugated with SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and WT-IgG1 solution, and reacted overnight at 37°C. The final concentration of WT-IgG1 containing H(WT) (SEQ ID NO: 4) and L(WT) (SEQ ID NO: 5) as the anti-human IL-6 receptor antibody was 333 μg / mL, which is in excess of the anti-human IL-6 receptor antibody concentration contained in the sample, with the aim of making almost all of the hsIL-6R in the sample bound to WT-IgG1. The plate was then dispensed into an MA400 PR Streptavidin Plate (Meso Scale Discovery). After further reaction at room temperature for 1 hour and washing, Read Buffer T (×4) (Meso Scale Discovery) was dispensed and immediately measured using a SECTOR PR 400 reader (Meso Scale Discovery). The hsIL-6R concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma hsIL-6R concentration in human FcRn transgenic mice after intravenous administration measured by this method is shown in Figure 4, and the time course of plasma hsIL-6R concentration in normal mice is shown in Figure 6.

[0285] Measurement of plasma free hsIL-6R concentration by electrochemiluminescence To evaluate the extent to which soluble human IL-6 receptor is neutralized in plasma, the concentration of soluble human IL-6 receptor (free hsIL-6R concentration) not bound (neutralized) by anti-human IL-6 receptor antibody in mouse plasma was measured by electrochemiluminescence. hsIL-6R calibration curve samples adjusted to 10000, 5000, 2500, 1250, 625, 312.5, or 156.25 pg / mL and 12 μL of mouse plasma sample were added to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore) to adsorb all IgG type antibodies (mouse IgG, anti-human IL-6 receptor antibody, and anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex) present in the plasma to Protein A. The mixture was then spun down in a high-speed centrifuge and the pass-through solution was collected. Since the pass solution does not contain anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex bound to protein A, the free hsIL-6R concentration in plasma can be measured by measuring the hsIL-6R concentration in the pass solution. Next, the pass solution was mixed with monoclonal anti-human IL-6R antibody (R&D) ruthenium-conjugated with SULFO-TAG NHS Ester (Meso Scale Discovery) and biotinylated anti-human IL-6R antibody (R&D) and reacted at room temperature for 1 hour. The mixture was then dispensed into an MA400 PR Streptavidin Plate (Meso Scale Discovery). After further reaction at room temperature for 1 hour and washing, Read Buffer T (×4) (Meso Scale Discovery) was dispensed and immediately measured with a SECTOR PR 400 reader (Meso Scale Discovery). The hsIL-6R concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of free hsIL-6R concentrations in plasma in normal mice after intravenous administration, as measured by this method, is shown in Figure 7.

[0286] pH-dependent effect of human IL-6 receptor binding The results of in vivo tests of H54 / L28-IgG1 and Fv4-IgG1 having pH-dependent human IL-6 receptor binding were compared. As shown in Figures 3 and 5, the plasma retention of both antibodies was almost the same, but as shown in Figures 4 and 6, it was confirmed that hsIL-6R administered simultaneously with Fv4-IgG1 having pH-dependent human IL-6 receptor binding was eliminated more quickly than hsIL-6R administered simultaneously with H54 / L28-IgG1. This tendency was confirmed in both human FcRn transgenic mice and normal mice, and it was found that the plasma hsIL-6R concentration after 4 days could be reduced by approximately 17-fold and approximately 34-fold, respectively, by imparting pH-dependent human IL-6 receptor binding ability.

[0287] Effect of binding to FcRn under neutral conditions (pH 7.4) It has been reported that intact human IgG1 hardly binds to human FcRn (its affinity is extremely weak) under neutral conditions (pH 7.4). It has been reported that by substituting Asn to Trp at position 434 in the EU numbering system for intact human IgG1, the binding to human FcRn under neutral conditions (pH 7.4) is increased (J Immunol. 2009;182(12):7663-71.). The results of in vivo tests in human FcRn transgenic mice of Fv4-IgG1 and Fv4-IgG1-v2, in which this amino acid substitution was introduced into Fv4-IgG1, were compared. As shown in Figure 3, the plasma retention of both antibodies was almost the same, but as shown in Figure 4, it was confirmed that hsIL-6R disappeared more quickly when administered simultaneously with Fv4-IgG1-v2, which has increased binding to human FcRn under neutral conditions (pH 7.4), compared to hsIL-6R administered simultaneously with Fv4-IgG1. It was found that the plasma hsIL-6R concentration after 4 days could be reduced by about 4-fold by imparting binding to human FcRn under neutral conditions (pH 7.4).

[0288] Based on the homology between human FcRn and mouse FcRn, the substitution of Asn at position 434 (EU numbering) with Trp is thought to increase the binding to mouse FcRn under neutral conditions (pH 7.4). It has also been reported that the substitution of Met at position 252 (EU numbering) with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu increases the binding to mouse FcRn under neutral conditions (pH 7.4) (J Immunol. 2002;169(9):5171-80.). The results of in vivo tests in normal mice of Fv4-IgG1, and Fv4-IgG1-v1 and Fv4-IgG1-v2, in which these amino acid substitutions were introduced into Fv4-IgG1, respectively, were compared. As shown in Figure 5, compared with Fv4-IgG1, Fv4-IgG1-v1 and Fv4-IgG1-v2, which also had increased binding to mouse FcRn under neutral conditions (pH 7.4), had slightly decreased plasma retention (plasma antibody concentrations after 1 day were approximately 1.5-fold and 1.9-fold, respectively).

[0289] As shown in Figure 6, it was confirmed that hsIL-6R disappeared significantly faster when administered simultaneously with Fv4-IgG1-v1 or Fv4-IgG1-v2, which has increased binding to mouse FcRn under neutral conditions (pH 7.4), compared to hsIL-6R administered simultaneously with Fv4-IgG1. It was found that by imparting binding to mouse FcRn under neutral conditions (pH 7.4), Fv4-IgG1-v1 and Fv4-IgG1-v2 could reduce the plasma hsIL-6R concentration after 1 day by about 32-fold and about 80-fold, respectively. Although the plasma concentration of the antibody was slightly reduced as described above by imparting binding to mouse FcRn under neutral conditions (pH 7.4), it was found that the effect of reducing the plasma hsIL-6R concentration was significantly greater than that. Furthermore, it was found that hsIL-6R administered simultaneously with Fv4-IgG1-v1 or Fv4-IgG1-v2 eliminated hsIL-6R more quickly than hsIL-6R alone. As shown in FIG. 6, it was found that the plasma hsIL-6R concentration after 1 day could be reduced by about 4-fold and about 11-fold by administering hsIL-6R simultaneously with Fv4-IgG1-v1 or Fv4-IgG1-v2, respectively, compared to hsIL-6R alone. This means that the antibody that binds to soluble IL-6 receptor in a pH-dependent manner and further binds to mouse FcRn under neutral conditions (pH 7.4) could accelerate the elimination of soluble IL-6 receptor. In other words, by administering such an antibody to the living body, it becomes possible to reduce the antigen concentration in plasma in the living body.

[0290] As shown in Figure 7, free hsIL-6R concentrations were detected up to 7 days after administration of H54 / L28-IgG1, whereas free hsIL-6R was not detected 1 day or later after administration of Fv4-IgG1, and free hsIL-6R was not detected 7 hours or later after administration of Fv4-IgG1-v1 or Fv4-IgG1-v2. Specifically, Fv4-IgG1, which has pH-dependent binding to hsIL-6R, showed a lower free hsIL-6R concentration compared to H54 / L28-IgG1, indicating that conferring pH-dependent binding to hsIL-6R exerts a greater hsIL-6R neutralizing effect. Furthermore, Fv4-IgG1-v1 and Fv4-IgG1-v2, which have increased FcRn binding at pH 7.4 compared to Fv4-IgG1, showed an even lower free hsIL-6R concentration, confirming that increased FcRn binding at pH 7.4 can exert an even greater hsIL-6R neutralizing effect.

[0291] A normal neutralizing antibody such as H54 / L28-IgG1 reduces the clearance of the antigen to which it is bound when the antibody is administered, and the antigen remains in plasma longer. It is not desirable for the antigen, whose action is to be neutralized, to remain in plasma longer by administration of the antibody. By imparting pH dependency to the binding to the antigen (binding under neutral conditions and dissociating under acidic conditions), the plasma retention of the antigen can be shortened. In this study, the plasma retention of the antigen could be further shortened by imparting binding to human FcRn under neutral conditions (pH 7.4). Furthermore, it was shown that the clearance can be increased to a level higher than that of the antigen alone by administering an antibody that binds to the antigen in a pH-dependent manner and is imparted with binding to FcRn under neutral conditions (pH 7.4). Until now, no method has been known to increase clearance by administration of an antibody to a level higher than that of the antigen alone, and the method discovered in this study is extremely useful as a method for eliminating antigens from plasma by administration of an antibody. This study was the first to reveal the advantage of increasing FcRn binding under neutral conditions (pH 7.4).Furthermore, similar effects were observed in both Fv4-IgG1-v1 and Fv4-IgG1-v2, which have different amino acid substitutions that increase FcRn binding under neutral conditions (pH 7.4).As such, it is believed that any amino acid substitution that increases human FcRn binding under neutral conditions (pH 7.4) has the effect of accelerating antigen elimination, regardless of the type of amino acid substitution.That is, the amino acid substitution of Pro at position 257 (EU numbering) with Ile reported in J Biol Chem. 2007;282(3):1709-17, the amino acid substitution of Gln at position 311 (EU numbering) with Ile reported in J Immunol. 2009;182(12):7663-71., an amino acid substitution replacing Asn at position 434 (EU numbering) with Ala, Tyr, or Trp, an amino acid substitution replacing Met at position 252 (EU numbering) with Tyr, an amino acid substitution replacing Thr at position 307 (EU numbering) with Gln, an amino acid substitution replacing Val at position 308 (EU numbering) with Pro, an amino acid substitution replacing Thr at position 250 (EU numbering) with Gln, an amino acid substitution replacing Met at position 428 (EU numbering) with Leu, an amino acid substitution replacing Glu at position 380 (EU numbering) with Ala, an amino acid substitution replacing Ala at position 378 (EU numbering) with Val, an amino acid substitution replacing Tyr at position 436 (EU numbering) with Ile, J Biol Chem. 2006 Aug It is believed that antibody molecules that eliminate antigens from plasma upon administration can be produced by using amino acid substitutions such as the amino acid substitution reported in Nat Biotechnol. 18;281(33):23514-24 of replacing Met at position 252 (EU numbering) with Tyr, the amino acid substitution reported in Nat Biotechnol. 2005 Oct;23(10):1283-8 of replacing Ser at position 254 (EU numbering) with Thr, and the amino acid substitution reported in Nat Biotechnol. 2005 Oct;23(10):1283-8 of replacing His at position 433 with Lys, the amino acid substitution reported in Nat Biotechnol. 2005 Oct;23(10):1283-8 of replacing Asn at position 434 with Phe, and the amino acid substitution reported in Nat Biotechnol. 2005 Oct;23(10):1283-8 of replacing Tyr at position 436 with His, as well as combinations of these amino acid substitutions.

[0292] [Example 4] Evaluation of binding activity to human FcRn As a measurement system for evaluating the interaction between antibodies and FcRn using Biacore, a system in which antibodies are immobilized on a sensor chip and human FcRn is used as an analyte, as described in J Immunol. 2009;182(12):7663-71., has been reported. For this purpose, human FcRn was prepared as described in Reference Example 4. Using this system, the binding activity (dissociation constant KD) of Fv4-IgG1, Fv4-IgG1-v1, and Fv4-IgG1-v2 to human FcRn at pH 6.0 and pH 7.4 was evaluated. The antibody, which is the test substance, was directly immobilized on a Series S sensor chip CM5 and subjected to the test. The antibody was immobilized on the sensor chip using 50 mmol / L sodium phosphate / 150 mmol / L NaCl, 0.05% (v / v%) Surfactant P20 pH 6.0 as the running buffer, and the amine coupling kit was used according to the manufacturer's manual, aiming for an immobilization amount of 500 RU.

[0293] Measurements were performed using the sensor chips prepared using 50 mmol / L sodium phosphate / 150 mmol / L NaCl, 0.05% Surfactant P20 pH 6.0 or 50 mmol / L sodium phosphate / 150 mmol / L NaCl, 0.05% Surfactant P20 pH 7.4 as the running buffer. All measurements were performed at 25°C. Human FcRn dilutions and running buffer (as a control solution) were injected at a flow rate of 5 μL / min for 10 minutes to allow human FcRn to interact with the antibody on the sensor chip. After that, the running buffer was run at a flow rate of 5 μL / min for 1 minute to observe the dissociation of FcRn, and then 20 mmol / L Tris-HCl / 150 mmol / L NaCl, pH 8.1 was injected twice at a flow rate of 30 μL / min for 15 seconds to regenerate the sensor chip.

[0294] The measurement results were analyzed using Biacore T100 Evaluation Software (Ver. 2.0.1). The dissociation constants (KD) were calculated from the measurement results of at least six different concentrations of FcRn by analysis using the steady-state affinity method. The results of the binding activity (dissociation constant KD) of Fv4-IgG1, Fv4-IgG1-v1, and Fv4-IgG1-v2 to human FcRn at pH 6.0 and pH 7.4 are shown in Table 5 below.

[0295] [Table 5]

[0296] The binding of Fv4-IgG1 to human FcRn at pH 7.4 was very weak, and the KD value could not be calculated (NA). In contrast, Fv4-IgG1-v1 and Fv4-IgG1-v2 were found to bind to human FcRn at pH 7.4, and the KD values ​​were calculated to be 36.55 μM and 11.03 μM, respectively. In addition, the KD values ​​for human FcRn at pH 6.0 were calculated to be 1.99 μM, 0.32 μM, and 0.11 μM, respectively. As shown in FIG. 3, in human FcRn transgenic mice, the disappearance of hsIL-6R was accelerated by Fv4-IgG1-v2 compared to Fv4-IgG1, so it is considered that antigen disappearance can be accelerated by modifying human IgG1 to make the binding to human FcRn at pH 7.4 stronger than 11.03 μM at least. Furthermore, as shown in J Immunol. 2002;169(9):5171-80, human IgG1 binds to mouse FcRn about 10 times stronger than human FcRn, and therefore, it is expected that the binding of Fv4-IgG1-v1 and Fv4-IgG1-v2 to mouse FcRn at pH 7.4 is also about 10 times stronger than the binding to human FcR. The accelerated elimination of hsIL-6R for Fv4-IgG1-v1 and Fv4-IgG1-v2 in normal mice shown in Figure 6 is greater than the accelerated elimination of hsIL-6R for Fv4-IgG1-v2 in human FcRn transgenic mice shown in Figure 4, and therefore, it is considered that the acceleration of elimination of hsIL-6R increases depending on the strength of binding to FcRn at pH 7.4.

[0297] [Example 5] Preparation of pH-dependent human IL-6 receptor-binding antibody with enhanced binding to human FcRn under neutral conditions In order to further enhance the antigen elimination effect of the pH-dependent human IL-6 receptor-binding antibody in human FcRn transgenic mice, various modifications were introduced into Fv4-IgG1 to enhance binding to human FcRn under neutral conditions. Specifically, the amino acid modifications shown in Tables 6-1 and 6-2 were introduced into the heavy chain constant region of Fv4-IgG1 to produce various mutants (amino acid numbers at the mutation sites are based on EU numbering). The introduction of amino acid substitutions was performed according to the method known to those skilled in the art and described in Reference Example 1.

[0298] [Table 6-1]

[0299] Table 6-2 is a continuation of Table 6-1. [Table 6-2]

[0300] The prepared variant containing the heavy chain and L(WT) (SEQ ID NO: 5) was expressed and purified by a method known to those skilled in the art, as described in Reference Example 2.

[0301] Binding evaluation to human FcRn Kinetic analysis of human FcRn and antibodies was performed using Biacore T100 (GE Healthcare). For this purpose, human FcRn was prepared as described in Reference Example 4. An appropriate amount of Protein L (ACTIGEN) was immobilized on a sensor chip CM4 (GE Healthcare) by the amine coupling method, and the target antibody was captured thereon. Next, the FcRn dilution solution and running buffer (as a control solution) were injected, and human FcRn was allowed to interact with the antibody captured on the sensor chip. The running buffer used was 50 mmol / L sodium phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, pH 7.0, and each buffer was also used for diluting FcRn. The chip was regenerated with 10 mmol / L glycine-HCl, pH 1.5. All measurements were performed at 25°C. From the sensorgrams obtained in the measurements, the kinetic parameters of the binding rate constant ka (1 / Ms) and the dissociation rate constant k d The KD (M) of each antibody for human FcRn was calculated based on the calculated KD (1 / s) using Biacore T100 Evaluation Software (GE Healthcare).

[0302] The results of the evaluation of binding to human FcRn under neutral conditions (pH 7.0) by Biacore are shown in Tables 6-1 and 6-2. Here, the binding of intact IgG1 was very weak and the KD could not be calculated, so it is indicated as ND in Table 6-1.

[0303] [Example 6] In vivo test of pH-dependent human IL-6 receptor-binding antibody with enhanced binding to human FcRn under neutral conditions Using the heavy chains conferred with the ability to bind to human FcRn under neutral conditions prepared in Example 4, pH-dependent human IL-6 receptor-binding antibodies having the ability to bind to human FcRn under neutral conditions were produced, and the antigen elimination effect in vivo was examined. Specifically, Fv4-IgG1 comprising VH3-IgG1 and VL3-CK; Fv4-IgG1-v2, comprising VH3-IgG1-v2 and VL3-CK; Fv4-IgG1-F14, comprising VH3-IgG1-F14 and VL3-CK; Fv4-IgG1-F20, which comprises VH3-IgG1-F20 and VL3-CK; Fv4-IgG1-F21, comprising VH3-IgG1-F21 and VL3-CK; Fv4-IgG1-F25, which comprises VH3-IgG1-F25 and VL3-CK; Fv4-IgG1-F29, comprising VH3-IgG1-F29 and VL3-CK; Fv4-IgG1-F35, which comprises VH3-IgG1-F35 and VL3-CK; Fv4-IgG1-F48, which comprises VH3-IgG1-F48 and VL3-CK; Fv4-IgG1-F93, which comprises VH3-IgG1-F93 and VL3-CK; Fv4-IgG1-F94 containing VH3-IgG1-F94 and VL3-CK was expressed and purified by a method known to those skilled in the art and described in Reference Example 2.

[0304] For the prepared pH-dependent human IL-6 receptor-binding antibodies, in vivo testing was performed using human FcRn transgenic mice (B6.mFcRn- / -.hFcRn Tg lineage 276+ / + mice, Jackson Laboratories, Methods Mol Biol. 2010;602:93-104.) in the same manner as in Example 3.

[0305] The time course of the plasma soluble human IL-6 receptor concentration in the human FcRn transgenic mice after intravenous administration is shown in FIG. 8. The test results showed that all pH-dependent human IL-6 receptor-binding antibodies with enhanced binding to human FcRn under neutral conditions showed lower plasma soluble human IL-6 receptor concentrations compared to Fv4-IgG1, which has almost no binding ability to human FcRn under neutral conditions. Among them, an example that showed a particularly remarkable effect was shown to be that the plasma concentration of soluble human IL-6 receptor administered simultaneously with Fv4-IgG1-F14 one day later was approximately 54-fold lower than that administered simultaneously with Fv4-IgG1. In addition, the plasma concentration of soluble human IL-6 receptor administered simultaneously with Fv4-IgG1-F21 7 hours later was approximately 24-fold lower than...

Claims

[Claim 1] The invention described herein.

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