An antigen-binding molecule that repeatedly binds to multiple antigen molecules

Abstract

To provide: an antigen binding molecule with enhanced uptake of antigen into cells; an antigen binding molecule with an increased number of bonds to an antigen; an antigen binding molecule which can promote the decrease of antigen concentration in blood plasma when administered; and an antigen binding molecule with improved retention in blood plasma.SOLUTION: An antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain, in which the antigen binding activity under two different calcium concentration conditions is different, the antigen binding activity of the antigen binding molecule under low calcium concentration conditions being lower than antigen binding activity under high calcium concentration conditions, and which has binding activity to human FcRn under neutral pH conditions.SELECTED DRAWING: None

Description

[Background technology]

[0001] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Many IgG-type antibody drugs are currently on the market, and numerous antibody drugs are currently being developed (Non-Patent Document 1, Non-Patent Document 2). Meanwhile, various technologies applicable to second-generation antibody drugs have been developed, including those that improve effector function, antigen-binding ability, pharmacokinetics, and stability, or reduce the risk of immunogenicity (Non-Patent Document 3). Because antibody drugs generally require very high dosages, challenges include the difficulty of preparing subcutaneous formulations and high manufacturing costs. Potential methods for reducing the dosage of antibody drugs include improving the pharmacokinetics of antibodies and improving the affinity between antibodies and antigens.

[0002] 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 antigen-binding activity by introducing mutations into amino acids in the CDR region of the variable region, etc. Enhanced antigen-binding ability can improve in vitro biological activity or reduce dosage, and can also improve in vivo efficacy (Non-Patent Document 7).

[0003] On the other hand, the amount of antigen that can be neutralized per antibody molecule depends on affinity. By increasing affinity, it is possible to neutralize the antigen with a small amount of antibody. Various methods are possible to increase antibody affinity (Non-Patent Document 6). Furthermore, if an antibody can be covalently bound to an antigen and affinity can be increased infinitely, it would be possible to neutralize one antigen molecule with one antibody molecule (two antigens in the case of a bivalent antibody). However, previous methods were limited to a stoichiometric neutralization reaction of one antigen molecule with one antibody molecule (two antigens in the case of a bivalent antibody), making it impossible to completely neutralize an antigen with an antibody amount less than the antigen amount. In other words, there was a limit to the effectiveness of increasing 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, an antibody amount greater than the amount of antigen produced in the body during that period must be administered. Therefore, there was a limit to reducing the required antibody dosage by simply improving the pharmacokinetics of the antibody or affinity maturation technology as described above. Therefore, in order to maintain the antigen neutralizing effect for a desired period with an antibody amount equal to or less than the antigen amount, it is necessary for one antibody to neutralize multiple antigens.

[0004] 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). pH-dependent antigen-binding antibodies bind strongly to antigens under the neutral conditions of plasma and dissociate from the antigen under the acidic conditions of endosomes, and are capable of dissociating from the antigen in endosomes. After dissociating from the antigen, pH-dependent antigen-binding antibodies are recycled into plasma by FcRn and can bind to the antigen again, allowing a single antibody to repeatedly bind to multiple antigens.

[0005] Furthermore, the plasma retention of antigens is very short compared to antibodies that bind to FcRn and are recycled. When an antibody with long plasma retention binds to such an antigen with short plasma retention, the plasma retention of the antibody-antigen complex becomes as long as that of the antibody. Therefore, binding of the antigen to the antibody actually increases the plasma retention, and the plasma antigen concentration increases. In such cases, improving the affinity of the antibody for the antigen does not 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 conventional antibodies (Patent Document 1).

[0006] Thus, pH-dependent antigen-binding antibodies bind to multiple antigens with a single antibody and can accelerate antigen elimination from plasma compared to conventional antibodies, thereby exhibiting effects not possible with conventional antibodies. However, the only known method to achieve the effects of pH-dependent antigen-binding antibodies, which can repeatedly bind to antigens and accelerate antigen elimination from plasma, is to impart pH dependency to the antigen-antibody reaction by utilizing the pH difference between plasma and endosomes.

[0007] Prior art documents relating to the present invention are listed below. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 2009 / 125825, ANTIGEN-BINDING MOLECULE CAPABLE OF BINDING TO TWO OR MORE ANTIGEN MOLECULES REPEATEDLY [Non-patent literature]

[0009] [Non-Patent 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

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

[0010] The present invention has been made in light of these circumstances, and aims to provide a method for promoting intracellular antigen uptake using an antigen-binding molecule, a method for increasing the number of antigen bindings using a single antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, a method for improving plasma retention of an antigen-binding molecule, an antigen-binding molecule with promoted intracellular antigen uptake, an antigen-binding molecule with an increased number of antigen bindings, an antigen-binding molecule whose administration can promote a decrease in plasma antigen concentration, an antigen-binding molecule with improved plasma retention, a pharmaceutical composition comprising the antigen-binding molecule, and methods for producing the same. [Means for solving the problem]

[0011] The present inventors have conducted extensive research into methods for promoting the intracellular uptake of antigens by antigen-binding molecules (molecules such as polypeptides capable of binding to antigens), increasing the number of times a single antigen-binding molecule binds to an antigen, promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, and improving the plasma retention of antigen-binding molecules. As a result, the present inventors focused on the difference in calcium concentrations between plasma and early endosomes and found that the use of antigen-binding molecules that have a calcium-dependent antigen-antibody reaction can promote the intracellular uptake of antigens by antigen-binding molecules, increase the number of times a single antigen-binding molecule binds to an antigen by binding multiple times to an antigen, and promote a decrease in plasma antigen concentration by administering an antigen-binding molecule, and improve the plasma retention of antigen-binding molecules.

[0012] Specifically, the present invention relates to a method for promoting intracellular antigen uptake using an antigen-binding molecule having a calcium-dependent antigen-antibody reaction, a method for increasing the number of antigen bindings using a single antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, a method for improving plasma retention of an antigen-binding molecule, antigen-binding molecules with promoted intracellular antigen uptake, antigen-binding molecules with an increased number of antigen bindings, antigen-binding molecules whose administration can promote a decrease in plasma antigen concentration, antigen-binding molecules with improved plasma retention, pharmaceutical compositions containing the antigen-binding molecules, and methods for producing them. More specifically, the present invention relates to the following: [1] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions differs, the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition, and the antigen-binding molecule has human FcRn-binding activity under a neutral pH condition. [2] The antigen-binding molecule of [1], wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM. [3] The antigen-binding molecule of [1], wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM. [4] the antigen-binding molecule of [1] or [2], wherein the low calcium concentration is an ionized calcium concentration in an endosome; [5] the antigen-binding molecule of [1] or [3], wherein the high calcium concentration is ionized calcium concentration in plasma; [6] The antigen-binding molecule of any of [1] to [5], wherein the FcRn-binding domain is an Fc region. [7] The antigen-binding molecule of any of [1] to [6], further comprising an antigen-binding activity under acidic pH conditions that is lower than that under neutral pH conditions. [8] the antigen-binding molecule of [7], wherein at least one amino acid is substituted with histidine or at least one histidine is inserted; [9] The antigen-binding molecule of any one of [1] to [8], which binds to a membrane antigen or a soluble antigen.

[10] The antigen-binding molecule of any of [1] to [9], wherein the antigen is selected from the group consisting of IL-6R, IL-6, IgA, human glypican 3, and IgE.

[11] An antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions differs, the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition, and the light chain or heavy chain contained in the antigen-binding domain contains a calcium-binding motif derived from a human antibody.

[12] the antigen-binding molecule of

[11] , wherein the calcium-binding motif is contained in the light chain CDR1, CDR2, and / or CDR3 of the antigen-binding domain;

[13] the antigen-binding molecule of

[12] , wherein the calcium-binding motif is contained at positions 30, 31, and / or 32 according to the Kabat numbering system in the light chain CDR1;

[14] the antigen-binding molecule of

[12] or

[13] , wherein the calcium-binding motif is contained at position 50 according to the Kabat numbering system in the light chain CDR2;

[15] The antigen-binding molecule of any of

[12] to

[14] , wherein the calcium-binding motif is contained at position 92 according to the Kabat numbering system in the light chain CDR3.

[16] the antigen-binding molecule of any of

[12] to

[15] , wherein the antigen to which the antigen-binding molecule binds is either IgA or glypican 3.

[17] the antigen-binding molecule of

[11] , wherein the calcium-binding motif is contained in heavy chain CDR1, CDR2, and / or CDR3 of the antigen-binding domain;

[18] the antigen-binding molecule of

[16] , wherein the calcium-binding motif is contained at positions 95, 96, 100a, and / or 101 according to the Kabat numbering system in the heavy chain CDR3;

[19] the antigen-binding molecule of

[17] or

[18] , wherein the antigen to which the antigen-binding molecule binds is either IL-6R or IL-6;

[20] The antigen-binding molecule of any of

[11] to

[19] , which comprises an FcRn-binding domain that has FcRn-binding activity under a neutral pH condition.

[21] the antigen-binding molecule of

[20] , wherein the FcRn-binding domain is an Fc region.

[22] the antigen-binding molecule of any one of [1] to

[10] ,

[20] , or

[21] , wherein the amino acid sequence of the Fc region differs from that of a native Fc region by any one or more of the following amino acids: 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering);

[23] Amino acids represented by EU numbering in the Fc region: The amino acid at position 237 is Met; The amino acid at position 248 is Ile; The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; Amino acid at position 252 is Phe, Trp, or Tyr; The amino acid at position 254 is Thr; The amino acid at position 255 is Glu; The amino acid at position 256 is Asp, Glu, or Gln; The amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His; The amino acid at position 265 is Ala, The amino acid at position 286 is Ala or Glu, The amino acid at position 289 is His; The amino acid at position 297 is Ala, The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, the amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg; The amino acid at position 311 is Ala, His, or Ile; The amino acid at position 312 is Ala or His, The amino acid at position 314 is Lys or Arg, The amino acid at position 315 is Ala, Asp, or His; The amino acid at position 317 is Ala, The amino acid at position 332 is Val; The amino acid at position 334 is Leu, The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is Asp or His, The amino acid at position 386 is Pro, The amino acid at position 387 is Glu; The amino acid at position 389 is Ala or Ser; The amino acid at position 424 is Ala, The amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys; The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, or The amino acid at position 436 is His, Ile, Leu, Val, The antigen-binding molecule of

[22] , which is a combination of any one or more of the following:

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

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

[25] A method for producing an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, the method comprising the following steps (a) to (e): (a) determining the antigen-binding activity of an antigen-binding molecule under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding molecule under a high calcium concentration condition; (c) selecting antigen-binding molecules whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition; (d) obtaining a gene encoding the antigen-binding molecule selected in step (c); (e) producing an antigen-binding molecule using the gene obtained in step (d).

[26] A method for producing an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, the method comprising the following steps (a) to (e): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) placing the antigen-binding molecule bound to the antigen in step (a) under a low calcium concentration condition; (c) obtaining the antigen-binding molecules dissociated in step (b); (d) obtaining a gene encoding the antigen-binding molecule obtained in step (c); (e) producing an antigen-binding molecule using the gene obtained in step (d).

[27] A method for producing an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, the method comprising the following steps (a) to (f): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under low calcium concentration conditions; (b) selecting antigen-binding molecules that do not bind to the antigen in step (a); (c) contacting the antigen-binding molecule selected in step (b) with an antigen under high calcium concentration conditions; (d) obtaining the antigen-binding molecule bound to the antigen in step (c); (e) obtaining a gene encoding the antigen-binding molecule obtained in step (d); (f) producing an antigen-binding molecule using the gene obtained in step (e).

[28] The production method of any of

[25] to

[27] , further comprising the step of modifying amino acids in the antigen-binding molecule to confer or enhance binding activity to human FcRn under neutral pH conditions.

[29] The production method of any of

[25] to

[27] , further comprising the step of modifying amino acids in the antigen-binding molecule to reduce the antigen-binding activity under acidic pH conditions compared to the antigen-binding activity under neutral pH conditions.

[30] The method according to any one of

[25] to

[27] , wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM.

[31] The method according to any one of

[25] to

[27] , wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM.

[32] The production method according to any one of

[25] to

[27] , wherein the low calcium concentration is an ionized calcium concentration in an endosome.

[33] The method according to any one of

[25] to

[27] , wherein the high calcium concentration is an ionized calcium concentration in plasma.

[34] the production method of

[29] , wherein the amino acid modification in the antigen-binding molecule is a modification in which at least one or more amino acids in the antigen-binding molecule are substituted with histidine or at least one histidine is inserted;

[35] The production method according to any one of

[25] to

[34] , wherein the antigen to which the antigen-binding molecule binds is an antigen selected from the group consisting of IL-6R, IL-6, IgA, human glypican 3, and IgE.

[36] The production method according to any one of

[25] to

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

[37] A pharmaceutical composition comprising the antigen-binding molecule of any of [1] to

[24] or the antigen-binding molecule produced by the production method of any of

[25] to

[36] , and a pharmaceutically acceptable carrier.

[38] The pharmaceutical composition according to

[37] , which is used to promote the uptake of an antigen into cells.

[39] The pharmaceutical composition according to

[37] , which is used to promote a decrease in antigen concentration in plasma.

[40] a pharmaceutical composition used to promote intracellular antigen uptake or a decrease in plasma antigen concentration, the pharmaceutical composition comprising an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions differs, and the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition;

[41] The pharmaceutical composition according to

[40] , wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM.

[42] The pharmaceutical composition according to

[40] , wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM.

[43] The pharmaceutical composition according to

[40] or

[41] , wherein the low calcium concentration is an ionized calcium concentration in an endosome.

[44] The pharmaceutical composition according to

[40] or

[42] , wherein the high calcium concentration is an ionized calcium concentration in plasma.

[45] The pharmaceutical composition according to any one of

[40] to

[44] , wherein the FcRn-binding domain contained in the antigen-binding molecule is an Fc region.

[46] The pharmaceutical composition of any of

[40] to

[45] , wherein the antigen-binding activity of the antigen-binding molecule under acidic pH conditions is lower than that under neutral pH conditions.

[47] the pharmaceutical composition of

[46] , wherein at least one amino acid in the antigen-binding molecule is substituted with histidine or at least one histidine is inserted;

[48] ​​The pharmaceutical composition of any of

[40] to

[47] , wherein the antigen to which the antigen-binding molecule binds is an antigen selected from the group consisting of IL-6R, IL-6, IgA, human glypican 3, and IgE.

[49] A method for screening for an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding molecule under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding molecule under a high calcium concentration condition; (c) selecting antigen-binding molecules whose antigen-binding activity under low calcium concentration conditions is lower than that under high calcium concentration conditions.

[50] A method for screening for an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, comprising the following steps (a) to (c): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) placing the antigen-binding molecule bound to the antigen in step (a) under a low calcium concentration condition; (c) recovering the antigen-binding molecules dissociated in step (b).

[51] A method for screening for an antigen-binding molecule having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, comprising the following steps (a) to (d): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under low calcium concentration conditions; (b) selecting antigen-binding molecules that do not bind to the antigen in step (a); (c) allowing the antigen-binding molecule selected in step (b) to bind to an antigen under high calcium concentration conditions; (d) a step of obtaining the antigen-binding molecule bound to the antigen in step (c).

[52] The screening method according to any one of

[49] to

[51] , wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM.

[53] The screening method according to any one of

[49] to

[51] , wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM.

[54] The screening method according to any one of

[49] to

[52] , wherein the low calcium concentration is an ionized calcium concentration in an endosome.

[55] The screening method according to any one of

[49] to

[51] or

[53] , wherein the high calcium concentration is the ionized calcium concentration in plasma.

[56] The screening method of any of

[49] to

[55] , wherein the antigen to which the antigen-binding molecule binds is an antigen selected from the group consisting of IL-6R, IL-6, IgA, human glypican 3, and IgE.

[57] The screening method according to any one of

[49] to

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

[58] A method for promoting intracellular uptake of an antigen by an antigen-binding molecule, comprising administering the antigen-binding molecule of any of [1] to

[24] or the antigen-binding molecule produced by the production method of any of

[25] to

[36] .

[59] A method for promoting a decrease in plasma antigen concentration by administering the antigen-binding molecule of any of [1] to

[24] or the antigen-binding molecule produced by the production method of any of

[25] to

[36] .

[60] A method for increasing the number of antigen bindings per antigen-binding molecule, using the antigen-binding molecule of any of [1] to

[24] or the antigen-binding molecule produced by the production method of any of

[25] to

[36] .

[61] A method for improving the plasma retention of an antigen-binding molecule, using the antigen-binding molecule of any of [1] to

[24] or the antigen-binding molecule produced by the production method of any of

[25] to

[36] .

[62] A method for promoting intracellular antigen uptake by an antigen-binding molecule by administering an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions is different, and the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition.

[63] A method for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions is different, and the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition.

[64] A method for increasing the number of antigen binding events per antigen-binding molecule, using an antigen-binding molecule that contains an antigen-binding domain and a human FcRn-binding domain, and whose antigen-binding activity differs under two different calcium concentration conditions, and whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[65] A method for improving the plasma retention of an antigen-binding molecule by using an antigen-binding molecule that comprises an antigen-binding domain and a human FcRn-binding domain, wherein the antigen-binding activity under two different calcium concentration conditions differs, and the antigen-binding activity of the antigen-binding molecule under a low calcium concentration condition is lower than the antigen-binding activity under a high calcium concentration condition.

[66] The method according to any one of

[62] to

[65] , wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM.

[67] The method according to any one of

[62] to

[66] , wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM.

[68] The method according to any one of

[62] to

[67] , wherein the low calcium concentration is an ionized calcium concentration in an endosome.

[69] The method according to any one of

[62] to

[68] , wherein the high calcium concentration is the ionized calcium concentration in plasma.

[70] The method of any of

[62] to

[69] , wherein the FcRn-binding domain contained in the antigen-binding molecule is an Fc region.

[71] The method of any of

[62] to

[70] , wherein the antigen-binding activity of the antigen-binding molecule under acidic pH conditions is lower than that under neutral pH conditions.

[72] the method of

[71] , wherein at least one amino acid of the antigen-binding molecule is substituted with histidine or at least one histidine is inserted;

[73] The method of any of

[62] to

[72] , wherein the antigen to which the antigen-binding molecule binds is an antigen selected from the group consisting of IL-6R, IL-6, IgA, human glypican 3, and IgE.

[74] The method of any of

[62] to

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

[0013] The present invention also relates to kits for use in the methods of the present invention, comprising the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention. The present invention also relates to agents that promote antigen uptake into cells by antigen-binding molecules, promote a decrease in plasma antigen concentration, increase the number of antigen bindings by a single antigen-binding molecule, or improve the plasma retention of antigen-binding molecules, which contain as an active ingredient the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention. The present invention also relates to use of the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention in the production of agents that promote antigen uptake into cells by antigen-binding molecules, promote a decrease in plasma antigen concentration, increase the number of antigen bindings by a single antigen-binding molecule, or improve the plasma retention of antigen-binding molecules. The present invention also relates to the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention for use in the methods of the present invention. [Effects of the Invention]

[0014] The present invention provides a method for promoting intracellular antigen uptake by an antigen-binding molecule, a method for increasing the number of antigen bindings by a single antigen-binding molecule, a method for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, and a method for improving plasma retention of an antigen-binding molecule. Promotion of intracellular antigen uptake by an antigen-binding molecule enables the administration of an antigen-binding molecule to promote a decrease in plasma antigen concentration and improve the plasma retention of the antigen-binding molecule, thereby increasing the number of antigen bindings by a single antigen-binding molecule and enabling the antigen-binding molecule to exert a more effective effect in vivo than conventional antigen-binding molecules. [Brief explanation of the drawings]

[0015] [Figure 1]This figure shows that a pH-dependent binding antibody repeatedly binds to a soluble antigen. (i) The antibody binds to a soluble antigen, (ii) it is nonspecifically taken up into cells by pinocytosis, (iii) the antibody binds to FcRn in the endosome, and the soluble antigen dissociates from the antibody, (iv) the soluble antigen is transported to the lysosome and degraded, (v) the antibody from which the soluble antigen has dissociated is recycled into plasma by FcRn, and (vi) the recycled antibody becomes able to bind to the soluble antigen again. [Figure 2] This figure shows that a pH-dependent binding antibody repeatedly binds to a membrane antigen. (i) The antibody binds to the membrane antigen, (ii) the antibody-membrane antigen complex is internalized into the cell, (iii) the antibody dissociates from the membrane antigen in the endosome, (iv) the membrane antigen is translocated to the lysosome and degraded, (v) the antibody dissociated from the membrane antigen is recycled into plasma, and (vi) the recycled antibody becomes able to bind to the membrane antigen again. [Figure 3] FIG. 1 shows the mode of interaction of a pH-dependent binding antibody with an antigen in plasma (pH 7.4) and in endosomes (pH 6.0). [Figure 4] FIG. 1 shows the mode of interaction of a calcium-dependent binding antibody with an antigen in plasma (Ca2+ 2 mM) and in endosomes (Ca2+ 3 μM). [Figure 5] FIG. 1 shows the mode of interaction of a pH- and calcium-dependent binding antibody with an antigen in plasma (pH 7.4, Ca2+ 2 mM) and in endosomes (pH 6.0, Ca2+ 3 μM). [Figure 6] FIG. 1 shows sensorgrams using Biacore showing the interaction of anti-human IL-6 receptor antibodies with soluble human IL-6 receptor at 2 mM Ca 2+ and 3 μM Ca 2+ . [Figure 7] FIG. 10 shows Biacore sensorgrams showing the interaction of H54 / L28-IgG1 with soluble human IL-6 receptor at 2 mM Ca2+ and 3 μM Ca2+. [Figure 8]FIG. 10 shows Biacore sensorgrams showing the interaction of FH4-IgG1 with soluble human IL-6 receptor at 2 mM Ca2+ and 3 μM Ca2+. [Figure 9] FIG. 10 shows Biacore sensorgrams showing the interaction of 6RL#9-IgG1 with soluble human IL-6 receptor at 2 mM Ca 2+ and 3 μM Ca 2+ . [Figure 10] FIG. 1 shows the time course of antibody concentrations in plasma of H54 / L28-IgG1, FH4-IgG1, and 6RL#9-IgG1 in normal mice. [Figure 11] FIG. 1 shows the time course of soluble human IL-6 receptor (hsIL-6R) concentration in plasma of normal mice treated with H54 / L28-IgG1, FH4-IgG1, and 6RL#9-IgG1. [Figure 12] FIG. 1 shows the time course of antibody concentration in plasma in normal mice for H54 / L28-N434W, FH4-N434W, and 6RL#9-N434W. [Figure 13] FIG. 1 shows the time course of soluble human IL-6 receptor (hsIL-6R) concentration in plasma of normal mice treated with H54 / L28-N434W, FH4-N434W, and 6RL#9-N434W. [Figure 14] FIG. 1 shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody, determined by X-ray crystal structure analysis. [Figure 15] FIG. 1 shows sensorgrams using Biacore showing the interaction of anti-human IL-6 antibodies with human IL-6 at Ca2+ 1.2 mM and Ca2+ 3 μM. [Figure 16]1 shows ion-exchange chromatograms of an antibody comprising the human Vk5-2 sequence and an antibody comprising the hVk5-2_L65 sequence, in which the glycosylation sequence in the human Vk5-2 sequence has been altered. The solid line represents the chromatogram of the antibody comprising the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 48, and light chain: hVk5-2, a fusion molecule of SEQ ID NOs: 41 and 28), and the dashed line represents the chromatogram of the antibody having the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 48), light chain: hVk5-2_L65 (SEQ ID NO: 47)). [Figure 17] Figure 1 shows ion exchange chromatograms of an antibody containing the LfVk1_Ca sequence (heavy chain: GC_H, SEQ ID NO: 102 and light chain: LfVk1_Ca, SEQ ID NO: 61) and an antibody containing a sequence in which the Asp (D) residue in the LfVk1_Ca sequence has been modified to an Ala (A) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is designated as the main peak, and the y-axis is normalized to the main peak. [Figure 18] Figure 1 shows ion exchange chromatograms of an antibody containing the LfVk1_Ca sequence (heavy chain: GC_H, SEQ ID NO: 102 and light chain: LfVk1_Ca, SEQ ID NO: 61) and an antibody containing the LfVk1_Ca6 sequence (heavy chain: GC_H, SEQ ID NO: 102 and light chain: LfVk1_Ca6, SEQ ID NO: 75) in which the Asp (D) residue at position 30 (Kabat numbering) in the LfVk1_Ca sequence has been altered to a Ser (S) residue, after storage at 5°C (solid line) or 50°C (dotted line). The highest peak in each ion exchange chromatogram after storage at 5°C is designated as the main peak, and the y-axis is normalized to the main peak. [Figure 19] FIG. 1 shows sensorgrams using Biacore showing the interaction of anti-human CD4 antibodies with soluble human CD4 at Ca 2+ 1.2 mM and Ca 2+ 3 μM. [Figure 20] FIG. 1 shows time courses of anti-human CD4 antibody concentrations in plasma of normal mice. [Figure 21]FIG. 1 shows the time course of soluble human CD4 concentration in plasma of normal mice in the soluble human CD4 alone administration group, the TNX355-IgG1 antibody administration group, the Q425 antibody administration group, and the Q425L9 antibody administration group. [Figure 22] FIG. 1 shows sensorgrams using Biacore showing the interaction of anti-human IgA antibody with human IgA at Ca 2+ 1.2 mM and Ca 2+ 3 μM. [Figure 23] FIG. 1 shows the time course of antibody concentrations in plasma of normal mice in the GA1-IgG1 antibody-administered group, the GA2-IgG1 antibody-administered group, and the GA3-IgG1 and GA2-N434W antibody-administered groups. [Figure 24] FIG. 1 shows the time course of human IgA concentration in plasma of normal mice in the human IgA only administration group, the GA1-IgG1 antibody administration group, the GA2-IgG1 antibody administration group, the GA3-IgG1 antibody administration group, and the GA2-N434W antibody administration group. [Figure 25] FIG. 1 shows the time course of unbound human IgA concentration in plasma from normal mice in the GA1-IgG1 antibody-administered group, the GA2-IgG1 antibody-administered group, the GA3-IgG1 antibody-administered group, and the GA2-N434W antibody-administered group. [Figure 26] FIG. 1 is a graph illustrating the efficiency of a conventional antibody, which forms a large immune complex against a multimeric antigen, in eliminating an antigen per antibody molecule. [Figure 27] FIG. 10 is a graph illustrating the efficiency of antigen elimination per antibody molecule of a pH / Ca-dependent antibody containing the constant region of natural IgG1, which forms a large immune complex against a multimeric antigen. [Figure 28] FIG. 1 illustrates the efficiency of antigen elimination per antibody molecule of a multispecific pH / Ca-dependent antibody that recognizes two or more epitopes present in a monomeric antigen and is suitable for forming large immune complexes. [Figure 29] FIG. 1 shows the interaction of anti-human glypican 3 antibodies with recombinant human glypican 3 at Ca 2+ 1.2 mM and Ca 2+ 3 μM, as determined by ELISA. [Figure 30]FIG. 1 shows the interaction of anti-human IgE antibodies with recombinant human IgE at 2 mM Ca2+ and 3 μM Ca2+ using ELISA. [Figure 31] FIG. 1 shows the time course of antibody plasma concentration in human FcRn transgenic mice. [Figure 32] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in human FcRn transgenic mice. [Figure 33] FIG. 1 shows time courses of antibody plasma concentrations in normal mice. [Figure 34] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in normal mice. [Figure 35] FIG. 1 shows the time course of plasma concentration of unbound soluble human IL-6 receptor in normal mice. [Figure 36] FIG. 1 shows time courses of plasma soluble human IL-6 receptor concentrations in human FcRn transgenic mice. [Figure 37] 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 38] 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 39] FIG. 1 shows the time course of plasma soluble human IL-6 receptor concentrations after administration of anti-human IL-6 receptor antibody to normal mice in which plasma soluble human IL-6 receptor concentrations maintained steady state. [Figure 40] FIG. 1 shows the time course of plasma antibody concentrations after simultaneous administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276). [Figure 41] FIG. 1 shows the time course of soluble human IL-6 receptor plasma concentrations after simultaneous administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 276). [Figure 42] 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-administration of hsIL-6R and an anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276). [Figure 43] 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-administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276). [Figure 44] Fig. 1 shows the time course of the antigen / antibody molar ratio (C value) after simultaneous administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276). [Figure 45] Fig. 14 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-administration of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276). [Figure 46] 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 concentrations are maintained at a steady state (steady-state infusion model). [Figure 47] FIG. 1 shows the time course of plasma hsIL-6R concentration in human FcRn transgenic mice (lines 276 and 32) after co-administration of hsIL-6R and anti-human IL-6 receptor antibody. [Figure 48] FIG. 1 shows the time course of plasma antibody concentrations in human FcRn transgenic mice (lines 276 and 32) after simultaneous administration of hsIL-6R and anti-human IL-6 receptor antibody. [Figure 49] 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 a steady state. [Figure 50] Fig. 1 shows the time course of plasma antibody concentrations after administration of anti-human IL-6 receptor antibodies to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentrations are maintained at a steady state. [Figure 51] Fig. 1 shows the time course of the antigen / antibody molar ratio (C value) 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 concentrations maintained a steady state. [Figure 52] Fig. 14 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 remains steady. [Figure 53] FIG. 1 shows the time course of plasma antibody concentrations after administration of anti-human IL-6 receptor antibodies containing the Fc variants F11, F39, F48, and F264 to human FcRn transgenic mice (strain 32) (steady-state infusion model) in which plasma hsIL-6R concentrations are maintained at a steady state. [Figure 54] 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 55]FIG. 1 shows the time course of plasma antibody concentrations after administration of anti-human IL-6 receptor antibodies having the Fc variants F157, F196, and F262 to human FcRn transgenic mice (strain 32) in which plasma hsIL-6R concentrations are maintained at a steady state (steady-state infusion model). [Figure 56] 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. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides methods for promoting intracellular antigen uptake by antigen-binding molecules, methods for increasing the number of antigen binding events per antigen-binding molecule, methods for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, and methods for improving plasma retention of an antigen-binding molecule. Specifically, the present invention provides methods for promoting intracellular antigen uptake by antigen-binding molecules, methods for increasing the number of antigen binding events per antigen-binding molecule, methods for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, and methods for improving plasma retention of an antigen-binding molecule, all of which are achieved by using antigen-binding molecules whose antigen-binding activity (sometimes referred to as "binding activity" in the present invention) under low calcium concentrations is lower than that under high calcium concentrations.

[0017] amino acid As used herein, amino acids are represented by one-letter or three-letter codes, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V.

[0018] antigen As used herein, the structure of an "antigen" is not limited to a specific structure, as long as it contains an epitope to which an antigen-binding domain binds. In another sense, an antigen can be inorganic or organic, and can be exogenous or endogenous to the organism to which the present invention is administered. Suitable examples of antigens to which the antigen-binding domain contained in an antigen-binding molecule whose pharmacokinetics are improved by the methods of the present invention bind include membrane antigens such as receptor proteins (membrane-bound receptors and soluble receptors) and cell surface markers, soluble antigens such as cytokines, and antigens containing epitopes present only in exogenous organisms. Antigens include the following molecules: 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 anthracis(Anthracis) defensive antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BL-S (ByS), 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, carbonate 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, eotaxin, EpCAM, ephrin B2 / 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 (FGF-acidic), basic FGF (FGF-basic), 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- β receptor, INF-γ, INF-γ receptor, type I IFN, type I IFN receptor, influenza, inhibin, inhibin α, inhibin β, 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 α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin α-δ (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 (leutinizinghormone) (LH), 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, NCA 90, 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, nonstructural protein type 3 from hepatitis C virus (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, polyglycol 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 metabolites, 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-l-β, XCLl / lymphotactin, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98 DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b,Examples of such molecules include C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones and growth factors, which exist in a soluble form in biological fluids without being anchored to cells.

[0019] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on an antigen to which an antigen-binding domain in an antigen-binding molecule disclosed herein binds. Thus, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of an antigen-binding molecule that recognizes the epitope to the antigen. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute the epitope. Furthermore, when the epitope is a sugar chain, the epitope can also be identified by a specific sugar chain structure.

[0020] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope, typically comprising at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0021] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids comprising the epitope is not the single, defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may encompass an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0022] Binding activity Methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for IL-6R are exemplified below; however, methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for an antigen other than IL-6R can also be appropriately carried out in accordance with the examples below.

[0023] For example, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region of IL-6R cDNA encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to the test antigen-binding molecule containing the IL-6R antigen-binding domain is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide of binding of the antigen-binding molecule to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

[0024] Furthermore, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a conformational epitope can be confirmed as follows. For this purpose, IL-6R-expressing cells are prepared. Examples of such confirmation include when a test antigen-binding molecule containing an IL-6R antigen-binding domain binds strongly to IL-6R-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence forming the extracellular domain of immobilized IL-6R. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity toward human IL-6R-expressing cells.

[0025] Methods for measuring the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) using IL-6R-expressing cells as antigens.

[0026] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody-binding titer toward IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule toward IL-6R-expressing cells to be compared.

[0027] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0028] For example, one suitable method for measuring the antigen-binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain is as follows: First, the test antigen-binding molecule is reacted with cells expressing IL-6R and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the desired concentration. For example, the antigen-binding molecule can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, the Geometric Mean value allows the binding activity of the test antigen-binding molecule, represented by the amount of binding of the test antigen-binding molecule, to be measured.

[0029] Whether a test antigen-binding molecule containing an IL-6R antigen-binding domain shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0030] Specifically, in a cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to wells coated with IL-6R protein.

[0031] The amount of test antigen-binding molecules bound to the wells via the IL-6R protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0032] If a competitor antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of a candidate competitor antigen-binding molecule, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competitor antigen-binding molecule or competes for binding to the same epitope.

[0033] When the structure of the epitope to which a test antigen-binding molecule containing an IL-6R antigen-binding domain binds has been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0034] For example, such binding activity can be measured by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. Alternatively, binding activity to the mutant peptide bound to a column can be measured by flowing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0035] Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with a suitable buffer to the desired concentration and used. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured.

[0036] In this method, "substantially no binding to mutant IL-6R-expressing cells" can be determined, for example, by the following method. First, test and control antigen-binding molecules bound to mutant IL-6R-expressing cells are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the polypeptide complex using the following formula:

[0037] ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0038] The Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant IL-6R-expressing cells, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. In this case, it is particularly preferred that the test antigen-binding molecules used to determine the ΔGeo-Mean comparison values ​​for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. An antigen-binding molecule previously confirmed to recognize an epitope in IL-6R is used as a control antigen-binding molecule.

[0039] A test antigen-binding molecule is deemed to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding molecule for the mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding molecule for the IL-6R-expressing cells. The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be determined to be identical.

[0040] antigen-binding domain As used herein, the term "antigen-binding domain" may refer to any domain of any structure as long as it binds to a target antigen. Examples of such domains include the variable regions of the heavy and light chains of an antibody, a module called an A domain of approximately 35 amino acids contained in Avimer, a cell membrane protein present in vivo (WO 2004 / 044011, WO 2005 / 040229), Adnectin (WO 2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on the cell membrane, Affibody (WO 1995 / 001937) using an IgG-binding domain consisting of a 58-amino acid, three-helix bundle of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeats), which are regions exposed on the molecular surface of ankyrin repeats (AR) with a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Preferred examples of the antigen-binding domain of the present invention include an anticalin molecule (WO 2002 / 020565), which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish, are repeatedly stacked (WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present invention include antigen-binding domains comprising the variable regions of the heavy and light chains of antibodies.Suitable examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0041] The antigen-binding domains in the antigen-binding molecules of the present invention can bind to the same epitope. Here, the same epitope can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 15. Alternatively, the same epitope can be present in a protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in SEQ ID NO: 15. Alternatively, the antigen-binding domains in the antigen-binding molecules of the present invention can bind to different epitopes. Here, the different epitopes can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 15. Alternatively, the different epitopes can be present in a protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in SEQ ID NO: 15.

[0042] calcium-binding motif The antigen-binding domain of the antigen-binding molecule of the present invention comprises a calcium-binding motif. The calcium-binding motif can be contained anywhere in the antigen-binding domain, as long as the antigen-binding activity under low calcium concentration conditions is lower than that under high calcium concentration conditions. When the antigen-binding domain is an antibody variable region, the calcium-binding motif can be contained in either the heavy chain or the light chain of the variable region. Alternatively, the calcium-binding motif can be contained in both the heavy and light chains. In another non-limiting embodiment, the calcium-binding motif can be contained in either the framework sequence of the variable region or the CDR sequence of the variable region. Alternatively, the calcium-binding motif can be contained in both the framework sequence and the CDR sequence.

[0043] In a non-limiting embodiment of the present invention, the calcium-binding motif comprises an amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration. Suitable examples of such amino acid residues include amino acids with metal chelating activity. Suitable examples of amino acids with metal chelating activity include serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), glutamic acid (Glu(E)), histidine (His(H)), and tyrosine (Tyr(Y)). A calcium-binding motif present in an existing antigen-binding domain that exhibits lower antigen-binding activity under low calcium concentrations than under high calcium concentrations can be used as the calcium-binding motif of the present invention. A non-limiting example of such an existing antigen-binding domain is a calcium-binding motif contained in the variable region of an antibody whose antigen-binding activity is lower under low calcium concentrations than under high calcium concentrations. Non-limiting examples of such antibodies include the IL-6 receptor antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2, and the IL-6 antibody comprising SEQ ID NO: 25 and SEQ ID NO: 26. Troponin C, calmodulin, parvalbumin, myosin light chain, and other antibodies are known to have multiple calcium ion-binding sites and are thought to have originated from a common origin in molecular evolution, and their binding motifs can also be used as calcium-binding motifs in the present invention.

[0044] When the antigen-binding domain of the present invention is an antibody variable region, the calcium-binding motif may be contained in either the heavy chain or the light chain of the variable region. Alternatively, the calcium-binding motif may be contained in both the heavy and light chains. In another non-limiting embodiment, the calcium-binding motif may be contained in either the framework sequence of the variable region or the CDR sequence of the variable region. Alternatively, the calcium-binding motif may be contained in both the framework sequence and the CDR sequence. It is also possible to design the CDR1, CDR2, and / or CDR3 of the heavy or light chain to contain these calcium-binding motifs. For example, in a non-limiting embodiment of the present invention, the calcium-binding motif contained in the light-chain variable region of the human antibody represented by SEQ ID NO: 41, SEQ ID NO: 63, or SEQ ID NO: 64 can be designed to be contained in the light-chain variable region of an antigen-binding molecule of the present invention. Examples of such calcium-binding motifs include calcium-binding motifs in which one or more amino acids at positions 30, 31, 32, 50, and / or 92 according to the Kabat numbering system contain an amino acid with metal chelating activity. A non-limiting embodiment of such calcium-binding motifs is a calcium-binding motif in which one to four amino acids identical to one or more of the five amino acids at positions 30, 31, 32, 50, and / or 92, as determined by Kabat numbering, in the light-chain variable region of the human antibody represented by SEQ ID NO: 41, SEQ ID NO: 63, or SEQ ID NO: 64 are contained in the corresponding amino acid positions according to Kabat numbering. In this case, among the five amino acid positions at positions 30, 31, 32, 50, and / or 92, as determined by Kabat numbering, in the light-chain variable region, an amino acid position that is not identical to an amino acid at the corresponding amino acid position in the light-chain variable region of the human antibody represented by SEQ ID NO: 41, SEQ ID NO: 63, or SEQ ID NO: 64 preferably contains an amino acid having metal chelating activity. Furthermore, for example, in another non-limiting embodiment of the present invention, the calcium-binding motif contained in the heavy-chain variable region represented by SEQ ID NO: 1 can also be designed to be contained in the heavy-chain variable region of an antigen-binding molecule of the present invention.Such calcium-binding motifs include those in which the amino acids at positions 95, 96, and / or 100a according to the Kabat numbering contain an amino acid with metal chelating activity. For example, in another non-limiting embodiment of the present invention, the calcium-binding motif contained in the heavy chain variable region of SEQ ID NO: 25 can also be designed to be contained in the heavy chain variable region of an antigen-binding molecule of the present invention. Such calcium-binding motifs include those in which the amino acids at positions 95 and / or 101 according to the Kabat numbering contain an amino acid with metal chelating activity. Examples of amino acids with metal chelating activity include serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), glutamic acid (Glu(E), histidine (His(H)), and tyrosine (Tyr(Y)). Furthermore, the carbonyl group of the main chain of the amino acid at these positions may be involved in calcium ion binding. As described in the Examples below, by grafting amino acids contained in a calcium-binding motif into a desired antigen-binding domain, it was surprisingly possible to confer calcium ion-binding activity to the antigen-binding domain. Furthermore, calcium ion-binding activity has been demonstrated in cadherin domains, EF hands contained in calmodulin, and protein kinase C (PKC) domains. The C2 domain contained in C, the Gla domain contained in the blood coagulation protein Factor IX, the C-type lectin contained in the asialoglycoprotein receptor and the mannose-binding receptor, the A domain contained in the LDL receptor, annexin, the thrombospondin type 3 domain, and the EGF-like domain can also be used appropriately.

[0045] specific "Specific" refers to a state in which a molecule does not exhibit any significant binding to any molecules other than the one or more molecules that are its binding partners. The term also applies to cases in which an antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitopes to which an antigen-binding domain binds are contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0046] antibody As used herein, an antibody refers to a natural or partially or fully synthetically produced immunoglobulin. Antibodies can be isolated from natural sources such as plasma or serum where they occur, or from the culture supernatant of antibody-producing hybridoma cells, or can be partially or fully synthesized using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4.

[0047] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0048] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. The monoclonal antibodies of the present invention include "humanized antibodies" and "chimeric antibodies."

[0049] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0050] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R protein shown in SEQ ID NO: 15, which is used as a sensitizing antigen for antibody production, can be obtained by expressing the IL-6R gene, whose nucleotide sequence is disclosed in SEQ ID NO: 16. Specifically, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence shown in SEQ ID NO: 15, which is the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), is expressed instead of the IL-6R protein shown in SEQ ID NO: 15. Purified native IL-6R protein can also be used as a sensitizing antigen.

[0051] The purified IL-6R protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also be obtained by degrading the IL-6R protein using a protease. However, the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be any sequence selected from the amino acid sequence corresponding to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 15. The number of amino acids constituting the peptide used as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0052] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0053] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0054] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen intraperitoneally or subcutaneously to a mammal. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0055] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages: -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0056] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0057] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R is confirmed in the serum, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0058] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0059] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

[0060] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

[0061] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0062] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0063] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0064] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0065] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0066] Screening and monocloning of the desired antibody can be preferably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

[0067] To screen for hybridomas producing the monoclonal antibodies of the present invention by FACS, first, cells expressing IL-6R are prepared. Preferred cells for screening are mammalian cells overexpressing IL-6R. By using non-transformed mammalian cells as a control host cell, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, hybridomas producing IL-6R monoclonal antibodies can be obtained by selecting hybridomas producing antibodies that do not bind to host cells but bind to cells overexpressing IL-6R.

[0068] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody capable of binding to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

[0069] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

[0070] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0071] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0072] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells that produce the anti-IL-6R antibody. To do this, total RNA is usually first extracted from the hybridoma. The following methods can be used to extract mRNA from cells. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0073] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or similar. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be isolated from hybridomas. cDNA encoding antibody V regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, the SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002; Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, which will be described later, can be introduced at both ends of the cDNA.

[0074] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0075] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0076] The resulting 5'-RACE cDNA library is used as a template for PCR amplification of antibody genes. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0077] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

[0078] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibodies can be screened using the binding activity of the reconstituted immunoglobulins to IL-6R as an indicator. For example, when the goal is to obtain antibodies against IL-6R, it is more preferable that the antibodies bind to IL-6R specifically. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0079] Methods for detecting the binding of an antibody to IL-6R-expressing cells are known. Specifically, the binding of an antibody to IL-6R-expressing cells can be detected by techniques such as the above-mentioned FACS. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of an antibody.

[0080] Panning methods using phage vectors are also suitable for screening antibodies using binding activity as an index. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be enriched.

[0081] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusion in-frame of both DNAs digested with the same combination of restriction enzymes.

[0082] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that its expression is controlled by an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 113) can be used as a signal sequence, although other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells can be transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0083] For antibody gene expression, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. By co-transfecting the same host cells with vectors incorporating the H chain and L chain, antibody molecules comprising both the H chain and L chain can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (see International Publication WO 1994011523).

[0084] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. All of these expression systems can be applied to isolating the antigen-binding domains of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0085] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0086] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris -Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0087] Expression systems for antibody genes using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the antibody gene of interest is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0088] In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing a fusion gene with an antibody gene inserted therein is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the goat that received the embryo produces milk from which the desired antibody can be obtained as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0089] When the antigen-binding molecules described herein are administered to humans, the antigen-binding domain in the aggregate may be an antigen-binding domain derived from a recombinant antibody that has been artificially modified to reduce heterologous antigenicity to humans, for example. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies are produced appropriately using known methods.

[0090] The antibody variable region used to prepare the antigen-binding domain of the antigen-binding molecule described herein is typically composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are the regions that essentially determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences that make up FRs often show high identity even among antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by CDR grafting.

[0091] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which CDRs from non-human animals, such as mouse antibodies, are grafted onto human antibodies are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting mouse antibody CDRs onto human FRs. In overlap extension PCR, a nucleotide sequence encoding the mouse antibody CDR to be grafted is added to a primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting human FRs that are highly identical to the mouse FRs is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0092] The nucleotide sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform complementary strand synthesis. This reaction links the human FRs via the mouse CDR sequences.

[0093] The V region gene, in which three CDRs and four FRs are finally linked, is amplified in its entirety using primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A humanized antibody expression vector can be constructed by inserting the DNA obtained as described above and DNA encoding a human antibody C region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996 / 002576).

[0094] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select FRs of a human antibody that form a good antigen-binding site with the CDRs when linked via the CDRs. If necessary, amino acid residues in the FRs can be substituted so that the CDRs of the reshaped human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. By measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Cancer Res., (1993) 53, 851-856).

[0095] Alternatively, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0096] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) by phage display. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0097] In addition to the above, methods for obtaining antibody genes include B cell cloning techniques (such as identification and cloning of the coding sequence of each antibody, isolation thereof, and use to construct expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4)) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or WO2008 / 081008.

[0098] EU numbering According to the method used in the present invention, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Herein, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids in the variable regions are represented according to the Kabat numbering, and the amino acids in the constant regions are represented according to the EU numbering based on the Kabat amino acid positions.

[0099] Antigen uptake or promotion of uptake into cells In the present invention, "antigen uptake into cells" by an antigen-binding molecule means that the antigen is taken up into cells by endocytosis. Furthermore, in the present invention, "promoting intracellular uptake" means accelerating the rate at which an antigen-binding molecule bound to an antigen in plasma is taken up into cells and / or reducing the amount of the taken-up antigen recycled into plasma. The degree of promotion of the intracellular uptake rate in the present invention refers to the acceleration of the intracellular uptake rate compared to the antigen-binding molecule before its antigen-binding activity under low calcium concentration conditions is reduced to be lower than that under high calcium concentration conditions. Therefore, in the present invention, whether or not an antigen-binding molecule has promoted intracellular uptake of an antigen can be determined by determining whether or not the intracellular uptake rate of the antigen is increased. The intracellular uptake rate of the antigen can be calculated, for example, by adding the antigen-binding molecule and the antigen to a culture medium containing human FcRn-expressing cells and measuring the decrease in the antigen concentration in the culture medium over time, or by measuring the amount of antigen taken up into human FcRn-expressing cells over time.

[0100] By utilizing a method for accelerating the rate of intracellular antigen uptake by an antigen-binding molecule of the present invention, for example, by administering an antigen-binding molecule, the rate of antigen elimination from plasma can be accelerated. Therefore, whether intracellular antigen uptake by an antigen-binding molecule has been promoted can also be confirmed by, for example, measuring whether the rate of antigen elimination present in plasma is accelerated or whether the antigen concentration in plasma is reduced by administration of the antigen-binding molecule. In other words, it is also possible to accelerate the decrease in antigen concentration in plasma by administering an antigen-binding molecule of the present invention.

[0101] Number of antigen bindings per antigen-binding molecule In the present invention, the "number of times a single antigen-binding molecule binds to an antigen" refers to the number of times a single antigen-binding molecule can bind to an antigen before it is degraded and lost. In the present invention, "increasing the number of times a single antigen-binding molecule binds to an antigen" refers to increasing the number of cycles that a single antigen-binding molecule can go through before it is degraded and lost, where one cycle is defined as the binding of an antigen to an antigen-binding molecule in plasma, the uptake of the antigen-bound antigen-binding molecule into cells, dissociation of the antigen in an endosome, and the return of the antigen-binding molecule to plasma. In the present invention, the number of cycles may be increased compared to an antigen-binding molecule whose antigen-binding activity under low calcium concentrations is not lower than that under high calcium concentrations, or compared to an antigen-binding molecule before its antigen-binding activity under low calcium concentrations is reduced below that under high calcium concentrations. Therefore, whether the number of cycles has increased can be determined by whether "intracellular uptake has been promoted," as described above, or whether "plasma retention has been improved," as described below.

[0102] Improved plasma retention In the present invention, "improved plasma retention" can be rephrased as "improved pharmacokinetics," "improved pharmacokinetics," "excellent pharmacokinetics," "improved plasma retention," "excellent plasma retention," or "prolonged plasma retention," and these terms are used interchangeably.

[0103] In the present invention, "improving plasma retention" not only refers to extending the time from when an antigen-binding molecule is administered to an animal such as a human, mouse, rat, monkey, rabbit, or dog until it disappears from plasma (for example, until it becomes unable to return to plasma due to intracellular degradation), but also includes extending the time that an antigen-binding molecule remains in plasma in an antigen-binding state (for example, in a state where the antigen-binding molecule is not bound to an antigen) between when it is administered and when it is degraded and eliminated. In other words, it includes extending the time until an antigen-binding molecule that is not bound to an antigen (antigen-free antigen-binding molecule) is degraded and eliminated.

[0104] Even if an antigen-binding molecule is present in plasma, if an antigen is already bound to that 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 increased, the time available for binding to a new antigen is extended (increasing the opportunity for binding to a new antigen), thereby reducing the time that an antigen is not bound to an antigen-binding molecule in vivo and lengthening the time that the antigen is bound to the antigen-binding molecule. If the elimination of an antigen from plasma can be accelerated by administration of an antigen-binding molecule, the plasma concentration of the antigen-free antigen-binding molecule will increase and the time that the antigen is bound to the antigen-binding molecule will be extended. Specifically, "improving the plasma retention of an antigen-binding molecule" in the present invention includes improving any pharmacokinetic parameter of a non-binding antigen-binding molecule of the present invention (increased plasma half-life, increased mean plasma residence time, or decreased plasma clearance) compared to a non-binding antigen-binding molecule whose antigen-binding activity under low calcium concentrations is not lower than that under high calcium concentrations, or compared to a non-binding antigen-binding molecule before its antigen-binding activity under low calcium concentrations is reduced below that under high calcium concentrations, or extending the time that an antigen remains bound to the antigen-binding molecule after administration of the antigen-binding molecule, or accelerating elimination of the antigen from plasma by the antigen-binding molecule.

[0105] Whether a pharmacokinetic parameter has been improved can be determined by measuring any of the parameters, such as the plasma half-life, mean plasma residence time, or plasma clearance, of an antigen-binding molecule or an 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, and each parameter is calculated. If the plasma half-life or mean plasma residence time is prolonged, it can be said that the plasma retention of the antigen-binding molecule has been 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) according to the accompanying instructions. The plasma concentration of an antigen-free antigen-binding molecule can be measured by methods known to those skilled in the art, for example, the method described in Clin Pharmacol. 2008 Apr;48(4):406-17 can be used.

[0106] In the present invention, "improving plasma retention" also includes extending the time during which an antigen remains bound to an antigen-binding molecule after administration of the antigen-binding molecule. Whether or not the time during which an antigen remains bound to an antigen-binding molecule after administration of the antigen-binding molecule has been extended can be determined by measuring the plasma concentration of antigen unbound to the antigen-binding molecule (free antigen) or the concentration of antigen unbound to the antigen-binding molecule (free antigen concentration) relative to the total antigen concentration, and counting the time until the ratio of these concentrations increases.

[0107] In the present invention, the "antigen concentration in plasma" can be measured by measuring the plasma concentration of antigen unbound to antigen-binding molecules or the ratio of the concentration of antigen unbound to antigen-binding molecules to the total antigen concentration using methods known to those skilled in the art. For example, the method described in Pharm Res. 2006 Jan;23(1):95-103 can be used.

[0108] Furthermore, when an antigen exhibits some function in vivo, whether the antigen binds to an antigen-binding molecule (antagonist molecule) that neutralizes the antigen function can also be evaluated by determining 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 binds 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.

[0109] Measurements such as measurement of the plasma concentration of antigen-binding molecule-unbound antigen, measurement of the ratio of antigen concentration of antigen-binding molecule-unbound to total antigen concentration, and measurement of in vivo markers are not particularly limited, but are preferably performed after a certain time has elapsed since administration of the antigen-binding molecule. In the present invention, "after a certain time has elapsed since administration of the antigen-binding molecule" is not particularly limited and can be determined appropriately by a person skilled in the art depending on the properties of the administered antigen-binding molecule, etc., and examples include 1 day after administration of the antigen-binding molecule, 3 days after administration of the antigen-binding molecule, 7 days after administration of the antigen-binding molecule, 14 days after administration of the antigen-binding molecule, and 28 days after administration of the antigen-binding molecule.

[0110] In the present invention, it is preferable that plasma retention in humans is improved. When it is difficult to measure plasma retention in humans, plasma retention in humans can be predicted based on 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.).

[0111] Dissociation of antigens bound to antigen-binding molecules extracellularly from the antigen-binding molecules intracellularly The present invention can also be used as a method for promoting the intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule. In the present invention, the site where the antigen dissociates from the antigen-binding molecule may be any site within the cell, preferably within the early endosome. In the present invention, "intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule" does not necessarily mean that all antigens bound to antigen-binding molecules extracellularly and taken up into the cell dissociate from the antigen-binding molecule within the cell. It is sufficient that the proportion of antigens dissociated from antigen-binding molecules within the cell is higher than that of antigen-binding molecules whose antigen-binding activity under low calcium concentrations is not lower than that under high calcium concentrations, or compared to antigen-binding molecules before their antigen-binding activity is reduced to be lower than that under high calcium concentrations. Furthermore, a method for promoting intracellular dissociation of an antigen bound to an antigen-binding molecule from the antigen-binding molecule can also be considered a method for promoting intracellular uptake of an antigen-binding molecule bound to an antigen, thereby imparting to the antigen-binding molecule a property that makes it easier to promote intracellular dissociation of the antigen from the antigen-binding molecule.

[0112] Antigen-binding molecules that are taken up into cells in a bound state to an antigen are released outside the cell in an unbound state. The present invention can also be used as a method for promoting the extracellular release of antigen-binding molecules that have been taken up into cells in an antigen-bound state in an unantigen-bound state. In the present invention, "extracellular release of antigen-binding molecules that have been taken up into cells in an antigen-bound state in an unantigen-bound state" does not necessarily mean that all antigen-binding molecules taken up into cells in an antigen-bound state are released extracellularly in an unantigen-bound state. It is sufficient that the proportion of antigen-binding molecules released extracellularly in an unantigen-bound state is higher than that of antigen-binding molecules whose antigen-binding activity under low calcium concentration conditions is not lower than that under high calcium concentration conditions, or that the proportion of antigen-binding molecules released extracellularly is higher than that before the antigen-binding activity under low calcium concentration conditions is reduced compared to that under high calcium concentration conditions. It is preferable that the antigen-binding molecules released extracellularly maintain their antigen-binding activity. Furthermore, a method for promoting the extracellular release of an antigen-binding molecule that has been taken up into cells in a state bound to an antigen, in an unbound state, can also be said to be a method for promoting the intracellular uptake of an antigen-binding molecule bound to an antigen, and imparting to the antigen-binding molecule a property that makes it more likely that the extracellular release of the antigen-binding molecule in a state unbound to an antigen will be promoted.

[0113] Calcium concentration conditions In the present invention, "under low calcium concentration conditions" generally means an ionized calcium concentration of 0.1 μM to 30 μM, preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the ionized calcium concentration in early endosomes in vivo. Furthermore, in the present invention, "under high calcium concentration conditions" generally means an ionized calcium concentration of 100 μM to 10 mM, preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the ionized calcium concentration in plasma (blood) in vivo.

[0114] Therefore, in the present invention, the phrase "the antigen-binding activity of an antigen-binding molecule is lower under a low calcium concentration condition than under a high calcium concentration condition" means that the antigen-binding activity of the antigen-binding molecule at an ionized calcium concentration of 0.1 μM to 30 μM is weaker than that at an ionized calcium concentration of 100 μM to 10 mM. Preferably, this means that the antigen-binding activity of the antigen-binding molecule at an ionized calcium concentration of 0.5 μM to 10 μM is weaker than that at an ionized calcium concentration of 200 μM to 5 mM, and particularly preferably that the antigen-binding activity at the ionized calcium concentration in early endosomes in vivo is weaker than that at the ionized calcium concentration in plasma in vivo. Specifically, this means that the antigen-binding activity of the antigen-binding molecule at an ionized calcium concentration of 1 μM to 5 μM is weaker than that at an ionized calcium concentration of 0.5 mM to 2.5 mM.

[0115] Furthermore, in the present invention, the expression "the antigen-binding activity of an antigen-binding molecule is lower under low calcium concentrations than under high calcium concentrations" can also be expressed as "the antigen-binding activity of an antigen-binding molecule is higher under high calcium concentrations than under low calcium concentrations." Furthermore, the expression "the antigen-binding activity of an antigen-binding molecule is lower under low calcium concentrations than under high calcium concentrations" also includes cases where the antigen-binding activity of an antigen-binding molecule under low calcium concentrations is reduced compared to that under high calcium concentrations, or the antigen-binding activity of an antigen-binding molecule under high calcium concentrations is increased compared to that under low calcium concentrations, by, for example, modifying the amino acid sequence in the antigen-binding molecule. In other words, in the present invention, the ratio of the antigen-binding activity of an antigen-binding molecule under low calcium concentrations to that under high calcium concentrations may be increased. For example, as described below, an embodiment includes increasing the value of KD(Ca 3 μM) / KD(Ca 2 mM). To increase the ratio of antigen-binding activity under low calcium concentrations to that under high calcium concentrations, for example, antigen-binding molecules with low antigen-binding activity under low calcium concentrations can be selected, or the amino acid sequence in the antigen-binding molecule can be modified to lower the antigen-binding activity under low calcium concentrations; antigen-binding molecules with high antigen-binding activity under high calcium concentrations can be selected, or the amino acid sequence in the antigen-binding molecule can be modified to increase the antigen-binding activity under high calcium concentrations; or both can be performed.

[0116] In the present invention, "the antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition" may also be expressed as "the antigen-binding ability under a low calcium concentration condition is weaker than that under a high calcium concentration condition," and "the antigen-binding activity under a low calcium concentration condition is made lower than that under a high calcium concentration condition" may also be expressed as "the antigen-binding ability under a low calcium concentration condition is made weaker than that under a high calcium concentration condition."

[0117] FcRn Unlike Fcγ receptors belonging to the immunoglobulin superfamily, FcRn, particularly human FcRn, is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22–29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18 (12), 592–598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2-microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain anchors the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the Fc region of antibodies (Raghavan et al., Immunity (1994) 1, 303–315).

[0118] FcRn is expressed in the maternal placenta or yolk sac of mammals and is involved in the transfer of IgG from mother to fetus. Additionally, in the small intestine of neonatal rodents, where FcRn is expressed, FcRn is involved in the transfer of maternal IgG from ingested colostrum or milk across the brush border epithelium. FcRn is expressed in numerous other tissues across multiple species, as well as in various endothelial cell lines. It is also expressed in human adult vascular endothelium, muscle vasculature, and liver sinusoids. FcRn is thought to play a role in maintaining plasma IgG concentrations by binding IgG and recycling it to serum. The binding of FcRn to IgG molecules is usually strictly pH-dependent, with optimal binding occurring in the acidic pH range below 7.0.

[0119] Human FcRn, whose precursor is a polypeptide containing the signal sequence represented by SEQ ID NO: 17, forms a complex with human β2-microglobulin in vivo (the polypeptide containing the signal sequence is shown in SEQ ID NO: 18). Soluble human FcRn complexed with β2-microglobulin is produced using standard recombinant expression techniques. The binding activity of the FcRn-binding domain of the present invention toward such soluble human FcRn complexed with β2-microglobulin can be evaluated. Unless otherwise specified, human FcRn herein refers to a form that can bind to the FcRn-binding domain of the present invention, such as a complex between human FcRn and human β2-microglobulin.

[0120] FcRn-binding domain The antigen-binding molecules of the present invention have an antigen-binding domain and 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 at acidic and / or neutral pH, and may be a domain that has direct or indirect human FcRn-binding activity. Examples of such domains include the Fc region of an IgG immunoglobulin, albumin, albumin domain 3, an anti-human FcRn antibody, an anti-human FcRn peptide, and an anti-human FcRn scaffold molecule that have direct human FcRn-binding activity, as well as molecules that indirectly bind to IgG or albumin and have human FcRn-binding activity. In the present invention, domains that have human FcRn-binding activity in the acidic and neutral pH ranges are preferred. Such domains may be used as they are, provided that they already have human FcRn-binding activity in the acidic and neutral pH ranges. When the domain has no or weak human FcRn-binding activity in the acidic and / or neutral pH range, human FcRn-binding activity can be obtained by modifying amino acids in the antigen-binding molecule; however, it is preferable to modify amino acids in the human FcRn-binding domain to obtain human FcRn-binding activity in the acidic and / or neutral pH range. Alternatively, amino acids in a domain that already has human FcRn-binding activity in the acidic and / or neutral pH range can be modified to enhance human FcRn-binding activity. The desired amino acid modification in the human FcRn-binding domain can be identified by comparing the human FcRn-binding activity in the acidic and / or neutral pH range before and after the amino acid modification.

[0121] 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 region of an antibody. However, a region capable of binding to a polypeptide that has human FcRn-binding activity, such as albumin or IgG, can indirectly bind to human FcRn via albumin, IgG, or the like. Therefore, the human FcRn-binding region of the present invention may also be a region that binds to a polypeptide that has albumin or IgG-binding activity. In particular, a human FcRn-binding domain with high human FcRn-binding activity at neutral pH is preferred. A human FcRn-binding domain with high human FcRn-binding activity at neutral pH may be selected in advance, or amino acids in the antigen-binding molecule may be modified to confer human FcRn-binding activity at neutral pH, or human FcRn-binding activity at neutral pH may be increased.

[0122] Conditions other than pH when measuring binding activity to human FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009 / 125825, measurements can be performed in MES buffer at 37°C. Furthermore, the human FcRn-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare). The binding activity of an antigen-binding molecule to human FcRn can be assessed by injecting human FcRn or the antigen-binding molecule, respectively, as an analyte onto a chip onto which the antigen-binding molecule or human FcRn has been immobilized.

[0123] Here, the binding activity to human FcRn at an acidic pH refers to the 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, the binding activity to human FcRn at a neutral pH refers to the 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 of plasma in vivo.

[0124] When altering amino acids in an antigen-binding molecule to confer or enhance human FcRn-binding activity at neutral pH, for example, when the Fc region of an IgG immunoglobulin is used as the human FcRn-binding domain, it is possible to confer or enhance human FcRn-binding activity at neutral pH to the antigen-binding molecule by altering the amino acids in the human FcRn-binding domain. Preferred examples of IgG immunoglobulin Fc regions for alteration include the Fc region of natural human IgG (IgG1, IgG2, IgG3, IgG4). Alterations to other amino acids may be made at any amino acid position, as long as human FcRn-binding activity at neutral pH is conferred or enhanced. When an antigen-binding molecule contains the Fc region of human IgG1 as the human FcRn-binding domain, it is preferable to include alterations that result in stronger human FcRn binding at neutral pH than natural 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 such modifications include 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. More specifically, such modifications include those listed in Table 1. These modifications can be used to confer or enhance (strengthen) the human FcRn-binding activity of the Fc region of an IgG immunoglobulin at neutral pH. Table 2 also lists examples of modifications that can enhance the human FcRn-binding activity in the acidic pH range compared to native human IgG1.Among these modifications, modifications that can enhance binding to human FcRn even at neutral pH can be appropriately selected and used in the present invention.

[0125] "Amino acid modification" or "amino acid modification" of an FcRn-binding domain includes modification to an amino acid sequence different from that of the parent FcRn-binding domain. Any FcRn-binding domain can be used as the parent FcRn-binding domain as long as the modified variant of the parent FcRn-binding domain can bind to human FcRn in the neutral pH range. Furthermore, an FcRn-binding domain obtained by further modifying an already modified FcRn-binding domain can also be suitably used as the FcRn-binding domain of the present invention. The parent FcRn-binding domain may refer to the polypeptide itself, a composition comprising the parent FcRn-binding domain, or a polynucleotide sequence encoding the parent FcRn-binding domain. The parent FcRn-binding domain may include known recombinantly produced Fc regions as outlined in the antibody section. The source of the parent FcRn-binding domain may be, but is not limited to, any non-human animal or human. Preferably, the any organism is an organism selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In another embodiment, the parent FcRn-binding domain can also be obtained from cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the parent FcRn-binding domain is obtained from human IgG1, but is not limited to a particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the parent FcRn-binding domain. Similarly, as used herein, it is meant that the Fc region of any class or subclass of IgG from any of the above organisms can preferably be used as the parent FcRn-binding domain.Examples of naturally occurring IgG mutants or engineered forms are described in known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0126] Examples of modifications include one or more mutations, such as substitution of amino acid residues different from those in the parent FcRn-binding domain, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the parent FcRn-binding domain. Preferably, the amino acid sequence of the modified FcRn-binding domain comprises at least a portion of a non-naturally occurring FcRn-binding domain. Such variants necessarily have less than 100% sequence identity or similarity with the parent FcRn-binding domain. In a preferred embodiment, the variant has an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100%, and most preferably about 95% to less than 100%, to the amino acid sequence of the parent FcRn-binding domain. In one non-limiting embodiment of the present invention, there is at least one amino acid difference between a parent FcRn-binding domain and an altered FcRn-binding domain of the present invention. The amino acid difference between the parent FcRn-binding domain and an altered FcRn-binding domain can be preferably identified by the difference in a specified amino acid at an amino acid residue position specified by the EU numbering system described above.

[0127] Furthermore, examples of alterations that can enhance human FcRn binding in the acidic pH range compared to the parent human IgG are shown in Table 2. Among these alterations, alterations that can enhance human FcRn binding even in the neutral pH range can be appropriately selected and used in the present invention. Furthermore, combinations of alterations that can enhance human FcRn binding of Fv4-IgG1 under acidic conditions are shown in Tables 6-1 and 6-2. Particularly preferred modified amino acids in the Fc region of a parent human IgG include those 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).

[0128] Particularly preferred modifications include, for example, modifications of the Fc region of the parent IgG according to EU numbering an amino acid substitution of Gly at position 237 with Met; Amino acid substitution of Pro at position 238 with Ala, an amino acid substitution of Ser at position 239 with Lys; an amino acid substitution of Lys at position 248 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; an amino acid substitution of Ser at position 254 with Thr; an amino acid substitution of Arg at position 255 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; an amino acid substitution of Glu at position 258 with His; an amino acid substitution of Asp at position 265 with Ala; an amino acid substitution of Asp at position 270 with Phe; an amino acid substitution of Asn at position 286 with Ala or Glu; an amino acid substitution of Thr at position 289 with His; an amino acid substitution of Asn at position 297 with Ala; an amino acid substitution of Ser at position 298 with Gly; an amino acid substitution of Val to Ala at position 303; an amino acid substitution of Val to Ala at position 305; an amino acid substitution of 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; an 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; an amino acid substitution of Lys at position 317 with Ala; an amino acid substitution of Asn at position 325 with Gly; an amino acid substitution of Ile to Val at position 332; an amino acid substitution of Lys at position 334 with Leu; An amino acid substitution of Lys at position 360 with His, an amino acid substitution of Asp at position 376 with Ala; an amino acid substitution of Glu at position 380 with Ala; an amino acid substitution of Glu at position 382 with Ala; an amino acid substitution of Asn or Ser at position 384 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 with Glu; an amino acid substitution of Asn at position 389 with Ala or Ser; an amino acid substitution of Ser at position 424 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; an amino acid substitution of His at position 433 with Lys; an amino acid substitution of Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and an amino acid substitution of Tyr or Phe at position 436 with His; Examples include:

[0129] The number of amino acids to be altered is not particularly limited, and alterations may be made to only one amino acid position, or to two or more amino acids. Examples of combinations of alterations at two or more amino acids include those shown in Table 3. Tables 4-1 to 4-5 show combinations of alterations that can strengthen the binding to human FcRn in the acidic pH range compared to the parent human IgG. Of these alterations, combinations of alterations that can strengthen the binding to human FcRn even in the neutral pH range can be appropriately selected and used in the present invention. Tables 5-1 and 5-2 show combinations of alterations that can strengthen the binding to human FcRn for Fv4-IgG1 under neutral conditions.

[0130] 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.

[0131] [Table 1]

[0132] [Table 2]

[0133] [Table 3]

[0134] [Table 4-1]

[0135] Table 4-2 is a continuation of Table 4-1. [Table 4-2]

[0136] Table 4-3 is a continuation of Table 4-2. [Table 4-3]

[0137] Table 4-4 is a continuation of Table 4-3. [Table 4-4]

[0138] Table 4-5 is a continuation of Table 4-4. [Table 4-5]

[0139] [Table 5-1]

[0140] Table 5-2 is a continuation of Table 5-1. [Table 5-2]

[0141] [Table 6-1]

[0142] Table 6-2 is a continuation of Table 6-1. [Table 6-2]

[0143] These amino acid modifications can be appropriately carried out using known techniques, and are described, for example, 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 US A. 2006 Dec 5;103(49):18709-14, EP2154157, US20070141052, WO2000 / 042072, WO2002 / 060919, WO2006 / 020114, WO2006 / 031370, WO2010 / 033279, WO2006 / 053301, and WO2009 / 086320, modifications of the Fc region of natural human IgG1 have been carried out.

[0144] According to The Journal of Immunology, 2009, 182: 7663-7671, the human FcRn-binding activity of native human IgG1 in the acidic pH range (pH 6.0) is KD 1.7 μM, whereas native human IgG1 has almost no detectable human FcRn-binding activity in the neutral pH range. Thus, preferred embodiments of the antigen-binding molecules used in the methods of the present invention include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 20 μM or less and whose human FcRn-binding activity in the neutral pH range is equal to or greater than that of native human IgG1. More preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is KD 2.0 μM or less and whose human FcRn-binding activity in the neutral pH range is KD 40 μM or less. More preferred embodiments include antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is a KD of 0.5 μM or less and whose human FcRn-binding activity in the neutral pH range is a KD of 15 μM or less. That is, it is preferable that the antigen-binding activity of the antigen-binding molecule under acidic pH conditions is lower than that under neutral pH conditions. 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).

[0145] Although the dissociation constant (KD) can be used as a measure of human FcRn-binding activity, the human FcRn-binding activity of human native IgG1 is barely detectable in the neutral pH range (pH 7.4), making it difficult to calculate the KD. One method for determining whether human FcRn-binding activity at pH 7.4 is higher than that of human native IgG1 is to determine the magnitude of the binding response when analytes are applied at the same concentration in a 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 greater than the response when human FcRn is applied at pH 7.4 on a chip on which human native IgG1 is immobilized, then the human FcRn-binding activity of the antigen-binding molecule at pH 7.4 can be determined to be higher than that of human native IgG1.

[0146] pH 7.0 can also be used as the neutral pH range. Using pH 7.0 as the neutral pH can promote weak interactions between human FcRn and the FcRn-binding domain. Regarding the temperature used in the measurement conditions, 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 a 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 a 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 3.

[0147] In a more preferred embodiment, the antigen-binding molecule of the present invention has higher human FcRn-binding activity than native 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 native human IgG, or a KD of 3.2 μM or stronger. In a more preferred embodiment, the human FcRn-binding activity at pH 7.0 and 25° C. is 38-fold higher than native human IgG, or a KD of 2.3 μM or stronger.

[0148] Native human IgG1, IgG2, IgG3, or IgG4 is preferably used as a reference native human IgG for comparison with an antigen-binding molecule for its human FcRn-binding activity or in vivo binding activity. Preferably, a reference antigen-binding molecule can be used that contains the same antigen-binding domain as the antigen-binding molecule of interest and the native human IgG Fc region as the human FcRn-binding domain. More preferably, native human IgG1 is used as a reference native human IgG for comparison with an antigen-binding molecule for its human FcRn-binding activity or in vivo binding activity.

[0149] More specifically, antigen-binding molecules having a long-term effect on plasma antigen elimination activity described in the present invention have an FcRn-binding activity at pH 7.0 and 25°C that is 28- to 440-fold higher than that of native human IgG1, or an FcRn-binding activity with a KD ranging from 3.0 μM to 0.2 μM. To evaluate the long-term effect of antigen-binding molecules of the present invention on plasma antigen elimination activity, long-term plasma antigen concentrations are determined by measuring the total or free antigen concentration and the antigen / antigen-binding molecule molar ratio in plasma 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule. Whether a reduction in plasma antigen concentration or antigen / antigen-binding molecule molar ratio is achieved by an antigen-binding molecule described in the present invention can be determined by evaluating the reduction at any one or more of the time points described above.

[0150] More specifically, antigen-binding molecules having a short-term effect on plasma antigen elimination activity described in the present invention have a human FcRn-binding activity that is 440-fold higher than that of native human IgG at pH 7.0 and 25° C., or have an FcRn-binding activity with a KD of 0.2 μM or stronger. To evaluate the short-term effect of antigen-binding molecules of the present invention on plasma antigen elimination activity, short-term plasma antigen concentrations are determined by measuring the total 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.

[0151] The methods of the present invention are applicable to any antigen-binding molecule regardless of the type of target antigen.

[0152] For example, when an antigen-binding molecule is an antibody that binds to a membrane antigen, the antibody binds to the antigen after administration and is then internalized into intracellular endosomes along with the antigen. The antibody then migrates to lysosomes while still bound to the antigen, where it is degraded along with the antigen. This elimination from plasma via internalization is called antigen-dependent elimination and has been reported for many antibody molecules (Drug Discov Today. 2006 Jan;11(1-2):81-8). When a single IgG antibody molecule binds to an antigen in a bivalent manner, the antibody is internalized while bound to two antigen molecules and is degraded in the lysosome. Therefore, in the case of a conventional antibody, a single IgG antibody molecule cannot bind to three or more antigen molecules. For example, a single IgG antibody molecule with neutralizing activity cannot neutralize three or more antigen molecules.

[0153] 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 the acidic conditions of the endosome. IgG molecules that cannot bind to human FcRn proceed to lysosomes, where they are degraded. However, IgG molecules that have bound to human FcRn migrate to the cell surface and dissociate from human FcRn under the neutral conditions of plasma, returning to the plasma.

[0154] Furthermore, when the antigen-binding molecule is an antibody that binds to a soluble antigen, the antibody administered into the body binds to the antigen and is 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 because they are released outside the cells while still bound to the antigen, they cannot re-bind to the antigen. 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.

[0155] Calcium concentration-dependent antigen-binding antibodies, which bind strongly to antigens under high calcium concentrations in plasma and dissociate from antigens under low calcium concentrations in endosomes, can dissociate from antigens in endosomes. After dissociating from an antigen, calcium concentration-dependent antigen-binding antibodies can re-bind to antigens when recycled into plasma by FcRn, enabling a single antibody to repeatedly bind to multiple antigens. Furthermore, because antigens bound to antigen-binding molecules are dissociated in endosomes and are not recycled into plasma, this promotes the uptake of antigens into cells by antigen-binding molecules, and administration of the antigen-binding molecules promotes antigen elimination, enabling a decrease in antigen concentration in plasma.

[0156] antigen binding molecule The present invention provides antigen-binding molecules that have an antigen-binding domain and a human FcRn-binding domain, and that have different antigen-binding activities under two different calcium concentration conditions, with the antigen-binding activity of the antigen-binding molecule being lower under a low calcium concentration condition than under a high calcium concentration condition.

[0157] 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. Preferred examples of antigen-binding domains include domains that have an antibody antigen-binding region. Examples of antibody antigen-binding regions include CDRs and variable regions. When the antibody antigen-binding region is a CDR, it may contain all six CDRs contained in a full-length antibody, or it may contain one or more CDRs. When the antibody binding region contains a CDR, the CDR may contain amino acid deletions, substitutions, additions, and / or insertions, or may be a portion of the CDR.

[0158] Furthermore, antigen-binding molecules targeted by the methods of the present invention include antigen-binding molecules with antagonistic activity (antagonist antigen-binding molecules), antigen-binding molecules with agonist activity (agonist antigen-binding molecules), and molecules with cytotoxic activity. Preferred embodiments include antagonistic antigen-binding molecules, particularly antagonistic antigen-binding molecules that recognize antigens such as receptors and cytokines.

[0159] 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 antigen-binding activity (antigen-binding domain) and a human FcRn-binding domain. In particular, antigen-binding molecules comprising a human FcRn-binding domain are preferred in the present invention.

[0160] Antigen-binding molecules having an antigen-binding domain and a human FcRn-binding domain include antibodies. Preferred examples of the antibodies of the present invention include IgG antibodies. 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. Furthermore, the antigen-binding molecules of the present invention may contain an antibody constant region, and amino acid mutations may be introduced into the constant region. Examples of amino acid mutations to be introduced include, but are not limited to, those that increase or decrease binding to Fcγ receptors (Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10). Furthermore, pH-dependent binding can be altered by selecting an appropriate constant region, such as IgG2.

[0161] 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, human antibody, rat antibody, rabbit antibody, goat antibody, or camel antibody. Furthermore, the antibody may be a modified antibody in which the amino acid sequence has been substituted, such as a chimeric antibody, particularly a humanized antibody. Furthermore, the antibody may be a bispecific antibody, a modified antibody bound to various molecules, or a polypeptide containing an antibody fragment.

[0162] 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.

[0163] A "humanized antibody," also known as a reshaped human antibody, is an antibody derived from a non-human mammal, such as a mouse, in which the complementarity-determining regions (CDRs) of the antibody have been grafted onto the CDRs 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 genetic recombination techniques are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0164] 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.

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

[0166] Furthermore, antigen-binding molecules to which the present invention can be applied may be antibody-like molecules. Antibody-like molecules (scaffold molecules, peptide molecules) are molecules that exert their functions by binding to target molecules (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). These antibody-like molecules also bind to target molecules in a calcium concentration-dependent manner, promote the intracellular uptake of antigens by the antigen-binding molecules, promote the decrease in plasma antigen concentration by administration of the antigen-binding molecules, improve the plasma retention of antigen-binding molecules, and can increase the number of times a single antigen-binding molecule binds to an antigen.

[0167] Alternatively, the antigen-binding molecule may be a protein in which a human FcRn-binding domain is fused to a target-binding receptor protein, such as a TNFR-Fc fusion protein, an IL1R-Fc fusion protein, a VEGFR-Fc fusion protein, or a CTLA4-Fc fusion protein (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60). These receptor-human FcRn-binding domain fusion proteins also bind to target molecules in a calcium concentration-dependent manner, promote the intracellular uptake of antigens by the antigen-binding molecule, promote the decrease in plasma antigen concentration upon administration of the antigen-binding molecule, improve the plasma retention of the antigen-binding molecule, and increase the number of times a single antigen-binding molecule binds to an antigen.

[0168] The antigen-binding molecule may also be a fusion protein of a human FcRn-binding domain and an artificial ligand protein that binds to a target and has a neutralizing effect, such as a mutant IL-6 (EMBO J. 1994 Dec 15;13(24):5863-70.). These artificial ligand fusion proteins also bind to target molecules in a calcium concentration-dependent manner, promote the uptake of antigens into cells by the antigen-binding molecule, promote a decrease in the antigen concentration in plasma by administration of the antigen-binding molecule, improve the plasma retention of the antigen-binding molecule, and increase the number of times a single antigen-binding molecule binds to an antigen.

[0169] Furthermore, the antibodies of the present invention may have modified sugar chains. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (WO99 / 54342, etc.), antibodies lacking fucose attached 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 the ionized calcium concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurement can be performed under conditions of HEPES buffer and 37°C. Measurement can be performed using, for example, Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane-type antigen, the binding activity to the membrane-type antigen can be evaluated by passing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0171] In the antigen-binding molecules of the present invention, the ratio of antigen-binding activity under low calcium concentrations to that under high calcium concentrations is not particularly limited, as long as the antigen-binding activity under low calcium concentrations is weaker than that under high calcium concentrations, but preferably the ratio of the KD (Dissociation constant) for the antigen under low calcium concentrations to the KD under high calcium concentrations, KD(Ca 3 μM) / KD(Ca 2 mM), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of KD(Ca 3 μM) / KD(Ca 2 mM) is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be produced by those skilled in the art.

[0172] When the antigen is a soluble antigen, the KD (dissociation constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, or a flow cytometer.

[0173] Furthermore, in the case of an antigen-binding molecule of the present invention, other indicators that indicate the ratio of antigen-binding activity under low calcium concentration conditions to antigen-binding activity under high calcium concentration conditions include, 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 binding activity ratio. d When using the dissociation rate constant, k d (dissociation rate constant) and k under high calcium concentration conditions d (dissociation rate constant) ratio kd (under low calcium concentration conditions) / k d The value of k (under high calcium concentration conditions) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. d (under low calcium concentration conditions) / k d The upper limit of the value (under high calcium concentration conditions) is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be produced within the technical common sense of a person skilled in the art.

[0174] The value of antigen binding activity is k when the antigen is a soluble antigen. d (dissociation rate constant) can be used, and when the antigen is a membrane antigen, the apparent k d (Apparent dissociation rate constant) can be used. d (dissociation rate constant), and apparent k d The (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.

[0175] In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different calcium concentrations, it is preferable that all conditions other than the calcium concentration be the same.

[0176] To obtain antigen-binding molecules whose antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations, methods for reducing (weakening) the antigen-binding activity of an antigen-binding molecule under low calcium concentrations compared to that under high calcium concentrations (methods for conferring calcium concentration-dependent antigen-binding activity) are not particularly limited. Antigen-binding molecules whose antigen-binding activity under low calcium concentrations is reduced (weaker) than that under high calcium concentrations (antigen-binding molecules that exhibit calcium concentration-dependent binding) can be obtained directly, for example, by screening an in vitro-presented antibody library using calcium concentration-dependent binding to the antigen as an index.

[0177] Other methods include methods for directly obtaining antigen-binding molecules with such properties. For example, antibodies with the desired properties can be directly obtained by immunizing animals (mouse, rat, hamster, rabbit, human immunoglobulin transgenic mouse, human immunoglobulin transgenic rat, human immunoglobulin transgenic rabbit, llama, camel, etc.) with an antigen and screening the resulting antibodies using calcium concentration-dependent binding to the antigen as an index. Alternatively, random mutations can be introduced into the amino acid sequence of the antigen-binding molecule, and the antigen-binding activity of the antigen-binding molecule can be measured under different calcium concentrations according to the method described above. Antigen-binding molecules with lower antigen-binding activity under low calcium concentrations than under high calcium concentrations can be selected, compared with the unmodified antigen-binding molecule.

[0178] When the antigen-binding activity of an antigen-binding molecule under low calcium concentration conditions is reduced (weakened) compared to that under high calcium concentration conditions (the KD(under low calcium concentration) / KD(under high calcium concentration) value is increased) by the above-mentioned methods or the like, it is preferred that the KD(under low calcium concentration) / KD(under high calcium concentration) value is generally at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold that of the original antibody, although this is not particularly limited.

[0179] Furthermore, when the method of the present invention for conferring calcium concentration-dependent antigen-binding activity is used for an antigen-binding molecule that has human FcRn-binding activity at neutral pH, or when combined with a method for conferring or enhancing human FcRn-binding activity at neutral pH, it is possible to enhance the functions of promoting antigen uptake into cells, increasing the number of antigen bindings per antigen-binding molecule, promoting the decrease in plasma antigen concentration following administration of the antigen-binding molecule, and improving the plasma retention of the antigen-binding molecule. Examples of methods for conferring or enhancing human FcRn-binding activity at neutral pH include the above-mentioned method of modifying amino acids in the human FcRn-binding domain. Here, "human FcRn-binding activity at neutral pH" refers to binding activity to human FcRn at pH 6.7 to pH 10.0. Preferred human FcRn-binding activity includes human FcRn-binding activity between pH 7.0 and pH 8.0, and more preferred human FcRn-binding activity includes human FcRn-binding activity at pH 7.4.

[0180] Furthermore, when the method of the present invention for conferring calcium concentration-dependent antigen-binding activity is used with an antigen-binding molecule that has pH-dependent antigen-binding activity, or when combined with a method for conferring pH-dependent antigen-binding activity, it is possible to enhance the functions of promoting antigen uptake into cells, increasing the number of antigen bindings per antigen-binding molecule, promoting the decrease in plasma antigen concentration following administration of the antigen-binding molecule, or improving the plasma retention of the antigen-binding molecule. Examples of methods for conferring pH-dependent antigen-binding activity include the methods described in WO2009 / 125825.

[0181] Specifically, for example, calcium concentration-dependent antigen-binding molecules of the present invention can be used in combination with a method for reducing (weakening) the antigen-binding activity of an antigen-binding molecule at acidic pH compared to that at neutral pH. Here, "reducing (weakening) the antigen-binding activity at acidic pH compared to that at neutral pH" means weakening the antigen-binding activity of an antigen-binding molecule at pH 4.0 to pH 6.5 compared to that at pH 6.7 to pH 10.0. Preferably, it means weakening the antigen-binding activity of an antigen-binding molecule at pH 5.5 to pH 6.5 compared to that at pH 7.0 to pH 8.0, and particularly preferably weakening the antigen-binding activity of an antigen-binding molecule at pH 5.8 compared to that at pH 7.4. In the present invention, acidic pH generally refers to pH 4.0 to pH 6.5, preferably pH 5.5 to pH 6.5, and particularly preferably pH 5.8. In the present invention, the neutral pH is generally pH 6.7 to pH 10.0, preferably pH 7.0 to pH 8.0, and particularly preferably pH 7.4.

[0182] Furthermore, "reducing the antigen-binding activity of an antigen-binding molecule at acidic pH compared to that at neutral pH" can also be expressed as "increasing the antigen-binding activity of the antigen-binding molecule at neutral pH compared to that at acidic pH." That is, in the present invention, the difference between the antigen-binding activity of an antigen-binding molecule at acidic pH and that at neutral pH may be increased (for example, the value of KD(pH 5.8) / KD(pH 7.4) may be increased, as described below). To increase the difference between the antigen-binding activity of an antigen-binding molecule at acidic pH and that at neutral pH, for example, the antigen-binding activity at acidic pH may be decreased, the antigen-binding activity at neutral pH may be increased, or both may be performed.

[0183] In the present invention, as long as the antigen-binding activity at acidic pH is weaker than that at neutral pH, the difference between the antigen-binding activity at acidic pH and that at neutral pH is not particularly limited, but preferably the ratio of the KD at pH 5.8 to the KD (Dissociation constant) at pH 7.4 (KD(pH5.8) / KD(pH7.4)) is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. There is no particular upper limit to the KD(pH5.8) / KD(pH7.4) value, and it may be any value, such as 400, 1,000, or 10,000, as long as it can be produced by those skilled in the art.

[0184] In addition, other indicators that show the difference in antigen-binding activity between at acidic pH and at neutral pH include, for example, the dissociation rate constant k d It is also possible to use the k (dissociation rate constant) instead of KD (dissociation constant) as an index of the difference in binding activity. d When using the dissociation rate constant, the k d (dissociation rate constant) and k at pH 7.4 d (dissociation rate constant) ratio k d (pH5.8) / k d The value of k (pH 7.4) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. d (pH5.8) / k d The upper limit of the pH (pH 7.4) is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be prepared within the technical common sense of a person skilled in the art.

[0185] The method for conferring pH-dependent antigen-binding activity is not particularly limited, and examples include substituting at least one amino acid in an antigen-binding molecule with histidine or inserting at least one histidine into the antigen-binding molecule to weaken the antigen-binding activity at pH 5.8 compared to that at pH 7.4. It is already known that substituting an amino acid in an antibody with histidine can confer pH-dependent antigen-binding activity to an antibody (FEBS Letter, 309(1), 85-88, (1992)). In the present invention, the position at which the histidine mutation (substitution) or insertion is introduced (is performed) into the antigen-binding molecule is not particularly limited, and may be any site as long as the antigen-binding activity at pH 5.8 is weaker than that at pH 7.4 (the KD(pH5.8) / KD(pH7.4) value is higher) compared to before the mutation or insertion. When the antigen-binding molecule is an antibody, examples include the variable region of the antibody. The number of histidine mutations or insertions to be introduced (carried out) can be appropriately determined by those skilled in the art. 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. Mutations other than histidine mutations (mutations to amino acids other than histidine) may also be introduced simultaneously. Furthermore, histidine mutations and histidine insertions may be performed simultaneously. Histidine substitutions or histidine insertions may be performed randomly by a method known to those skilled in the art, such as histidine scanning, in which alanine is replaced with histidine. Antigen-binding molecules with a higher KD(pH5.8) / KD(pH7.4) value than before mutation may be selected from a library of antigen-binding molecules into which histidine mutations or insertions have been randomly introduced.

[0186] When at least one amino acid in an antigen-binding molecule is substituted with histidine or at least one histidine is inserted into an amino acid in the antigen-binding molecule, it is preferred, but not limited to, that the antigen-binding activity of the antigen-binding molecule after the histidine substitution or insertion at pH 7.4 be equivalent to that of the antigen-binding molecule before the histidine substitution or insertion. Here, "the antigen-binding activity of the antigen-binding molecule after the histidine substitution or insertion at pH 7.4 being equivalent to that of the antigen-binding molecule before the histidine substitution or insertion" means that the antigen-binding molecule after the histidine substitution or insertion maintains at least 10%, preferably at least 50%, more preferably at least 80%, and more preferably at least 90% of the antigen-binding activity of the antigen-binding molecule before the histidine substitution or insertion. If 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 is made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after such histidine substitution or insertion.

[0187] Other methods for weakening the antigen-binding activity of an antigen-binding molecule at pH 5.8 compared to that at pH 7.4 include substituting an amino acid in the antigen-binding molecule with a non-natural amino acid or inserting a non-natural amino acid into an amino acid in the antigen-binding molecule. It is known that the pKa of non-natural amino acids can be artificially controlled (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, non-natural amino acids can be used instead of the above-mentioned histidine. Furthermore, the above-mentioned histidine substitution and / or insertion and the non-natural amino acid substitution and / or insertion may be performed simultaneously. The non-natural amino acids used in the present invention may be any non-natural amino acids, and non-natural amino acids known to those skilled in the art can be used.

[0188] Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, another method for weakening the antigen-binding activity of the antigen-binding molecule at pH 5.8 compared to that at pH 7.4 includes a method of modifying the antibody constant region contained in the antigen-binding molecule. Specific examples of such antibody constant region modifications include the constant region substitution methods described in WO2009 / 125825.

[0189] Another method for modifying an antibody constant region is to examine multiple constant region isotypes (IgG1, IgG2, IgG3, IgG4) and select an isotype with reduced antigen-binding activity at pH 5.8 (faster dissociation rate at pH 5.8). Another method involves introducing amino acid substitutions into the amino acid sequence of a wild-type isotype (wild-type IgG1, IgG2, IgG3, IgG4 amino acid sequence) to reduce antigen-binding activity at pH 5.8 (faster dissociation rate at pH 5.8). The sequences of the hinge regions of antibody constant regions vary significantly depending on the isotype (IgG1, IgG2, IgG3, IgG4), and differences in the amino acid sequence of the hinge region significantly affect antigen-binding activity. Therefore, by selecting an appropriate isotype depending on the type of antigen or epitope, it is possible to select an isotype with reduced antigen-binding activity at pH 5.8 (faster dissociation rate at pH 5.8). Furthermore, because differences in the amino acid sequence of the hinge region significantly affect antigen-binding activity, the hinge region is considered to be the preferred site for amino acid substitutions in the amino acid sequence of the wild-type isotype.

[0190] 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(pH5.8) / KD(pH7.4) value is increased) by the methods described above or the like, it is preferred that the KD(pH5.8) / KD(pH7.4) value is generally at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold that of the original antibody, although this is not particularly limited.

[0191] antigen binding molecule Furthermore, the present invention provides antigen-binding molecules that have different antigen-binding activities under two different calcium concentration conditions, with the antigen-binding activity being lower under low calcium concentration conditions than under high calcium concentration conditions. Preferably, the present invention provides antigen-binding molecules whose antigen-binding activity is lower under low calcium concentration conditions (ionized calcium concentration of 0.1 μM to 30 μM) than under high calcium concentration conditions (ionized calcium concentration of 100 μM to 10 mM). More specifically, examples of such antigen-binding molecules include those whose antigen-binding activity is lower at ionized calcium concentrations in early endosomes in vivo (low calcium concentrations, e.g., 1 μM to 5 μM) than at ionized calcium concentrations in plasma in vivo (high calcium concentrations, e.g., 0.5 mM to 2.5 mM).

[0192] With regard to the antigen-binding molecule of the present invention whose antigen-binding activity is lower under a low calcium concentration condition than under a high calcium concentration condition, the difference in binding activity is not limited as long as the antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition, and it is sufficient that the antigen-binding activity under a low calcium concentration condition is slightly lower.

[0193] In a preferred embodiment of the antigen-binding molecule of the present invention whose antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations, the ratio of KD under low calcium concentrations to KD under high calcium concentrations, KD(low Ca) / KD(high Ca), is 2 or more, more preferably 10 or more, and even more preferably 40 or more. There are no particular upper limits to the KD(low Ca) / KD(high Ca) value, and it may be any value, such as 400, 1,000, or 10,000, as long as it can be prepared by those skilled in the art.

[0194] Furthermore, in another preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations, and the kd and k at pH 7.4 d The ratio of k d (low Ca) / k d (high Ca) value is 2 or more, and more preferably k d (low Ca) / k d (high Ca) value is 5 or more, and more preferably k d (low Ca) / k d (high Ca) value is 10 or more, and more preferably k d (low Ca) / k d (High Ca) value is 30 or more. d (low Ca) / k d The upper limit of the (high Ca) value is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be produced using the techniques of a person skilled in the art.

[0195] The antigen-binding molecules of the present invention may further have human FcRn-binding activity under neutral pH conditions as described above. Combining the human FcRn-binding activity under neutral pH conditions with calcium concentration-dependent antigen-binding activity can enhance the functions of promoting antigen uptake into cells, increasing the number of antigen bindings per antigen-binding molecule, promoting the decrease in plasma antigen concentration following administration of the antigen-binding molecule, or improving the plasma retention of the antigen-binding molecule.

[0196] Furthermore, the antigen-binding molecules of the present invention may further have the above-mentioned pH-dependent antigen-binding activity, i.e., the antigen-binding activity is lower under acidic pH conditions than under neutral pH conditions. Combining this pH-dependent antigen-binding activity with calcium concentration-dependent antigen-binding activity can enhance the functions of promoting antigen uptake into cells, increasing the number of antigen bindings per antigen-binding molecule, promoting the decrease in plasma antigen concentration following administration of the antigen-binding molecule, or improving the plasma retention of the antigen-binding molecule.

[0197] Furthermore, the antigen-binding molecules of the present invention may have any other properties, such as agonist or antagonist antigen-binding molecules, as long as their antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations. Preferred examples of antigen-binding molecules of the present invention include antagonist antigen-binding molecules. Antagonist antigen-binding molecules typically inhibit the binding of a ligand (agonist) to a receptor and inhibit receptor-mediated intracellular signal transduction.

[0198] Furthermore, an antigen-binding molecule to which pH-dependent antigen-binding activity has been conferred may have at least one amino acid substituted with histidine or at least one histidine inserted therein.

[0199] Furthermore, the antigen to which the antigen-binding molecule of the present invention binds is not particularly limited, and any antigen may be bound. Examples include membrane antigens such as receptor proteins (membrane-bound receptors and soluble receptors) and cell surface markers, and soluble antigens such as cytokines. Specific examples of other antigens are as described above.

[0200] Screening Methods The present invention provides a method for screening for antigen-binding molecules whose antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations. (i) the function of promoting the uptake of antigens into cells; (ii) the ability to bind to the antigen more than once; (iii) promoting the reduction of antigen concentrations in plasma; and (iv) Excellent plasma retention function; The present invention provides a method for screening for antigen-binding molecules having at least one function selected from the above.

[0201] Specifically, the present invention provides a method for screening an antigen-binding molecule, comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding molecule under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding molecule under a high calcium concentration condition; (c) selecting antigen-binding molecules whose antigen-binding activity under low calcium concentration conditions is lower than that under high calcium concentration conditions.

[0202] The present invention further provides a method for screening for an antigen-binding molecule, comprising the following steps (a) to (c): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) placing the antigen-binding molecule bound to the antigen in step (a) under a low calcium concentration condition; (c) recovering the antigen-binding molecules dissociated in step (b).

[0203] The present invention further provides a method for screening for an antigen-binding molecule, comprising the following steps (a) to (d): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under low calcium concentration conditions; (b) selecting antigen-binding molecules that do not bind to the antigen in step (a); (c) allowing the antigen-binding molecule selected in step (b) to bind to an antigen under high calcium concentration conditions; (d) a step of obtaining the antigen-binding molecule bound to the antigen in step (c).

[0204] The present invention further provides a method for screening for an antigen-binding molecule, comprising the following steps (a) to (c): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen-immobilized column under high calcium concentration conditions; (b) eluting the antigen-binding molecules bound to the column in step (a) from the column under low calcium concentration conditions; (c) collecting the antigen-binding molecules eluted in step (b).

[0205] The present invention further provides a method for screening for an antigen-binding molecule, comprising the following steps (a) to (d): (a) passing an antigen-binding molecule or an antigen-binding molecule library through an antigen-immobilized column under low calcium concentration conditions; (b) recovering the antigen-binding molecules that did not bind to the column and were eluted in step (a); (c) allowing the antigen-binding molecules recovered in step (b) to bind to antigens under high calcium concentration conditions; (d) a step of obtaining the antigen-binding molecule bound to the antigen in step (c).

[0206] The present invention further provides a method for screening for an antigen-binding molecule, comprising the following steps (a) to (d): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) obtaining the antigen-binding molecule bound to the antigen in step (a); (c) placing the antigen-binding molecule obtained in step (b) under a low calcium concentration condition; (d) a step of obtaining in step (c) antigen-binding molecules whose antigen-binding activity is weaker than the criterion selected in step (b).

[0207] These steps may be repeated two or more times. Therefore, the present invention provides the above-mentioned screening method, further comprising the step of repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is usually within 10 times.

[0208] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding molecule under low calcium concentration conditions is not particularly limited as long as it is an antigen-binding activity at an ionized calcium concentration of 0.1 μM to 30 μM, with a preferred ionized calcium concentration being 0.5 μM to 10 μM. A more preferred ionized calcium concentration is the ionized calcium concentration in early endosomes in vivo, specifically 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding molecule under high calcium concentration conditions is not particularly limited as long as it is an antigen-binding activity at an ionized calcium concentration of 100 μM to 10 mM, with a preferred ionized calcium concentration being 200 μM to 5 mM. A more preferred ionized calcium concentration is the ionized calcium concentration in plasma in vivo, specifically 0.5 mM to 2.5 mM.

[0209] The antigen-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding molecule can be measured using the KD (Dissociation constant), apparent KD (Apparent dissociation constant), and dissociation rate k d (Dissociation rate constant), or apparent k d These can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, FACS, etc.

[0210] In the present invention, the step of selecting antigen-binding molecules whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is the same as the step of selecting antigen-binding molecules whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0211] As long as the antigen-binding activity under a high calcium concentration is higher than that under a low calcium concentration, the difference between the antigen-binding activity under a high calcium concentration and that under a low calcium concentration is not particularly limited; however, the antigen-binding activity under a high calcium concentration is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times that under a low calcium concentration.

[0212] The antigen-binding molecules to be screened by the screening methods of the present invention may be any antigen-binding molecules, and for example, the antigen-binding molecules described above can be screened. For example, antigen-binding molecules having native sequences may be screened, or antigen-binding molecules with substituted amino acid sequences may be screened.

[0213] Antigen-binding molecules to be screened by the screening methods of the present invention may be prepared in any manner, and examples thereof include 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, and antibodies or libraries obtained by introducing amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations into these antibodies or libraries (libraries with an increased content of amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acids, libraries in which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced at specific sites).

[0214] By the screening method of the present invention, when administered to animals such as humans, mice, and monkeys, (i) the function of promoting the uptake of antigens into cells, (ii) the ability to bind to the antigen more than once; (iii) the ability to promote the reduction of antigen concentrations in plasma; and (iv) Excellent plasma retention function; It is possible to obtain antigen-binding molecules having at least one function selected from the following: Therefore, the screening method of the present invention can be used as a screening method for obtaining antigen-binding molecules having at least one of these functions.

[0215] Furthermore, these antigen-binding molecules obtained by the screening method of the present invention 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 in pharmaceutical compositions.

[0216] Method for producing antigen-binding molecules The present invention provides a method for producing an antigen-binding molecule whose antigen-binding activity under low calcium concentrations is lower than that under high calcium concentrations. The present invention also provides a method for producing an antigen-binding molecule that exhibits (i) the function of promoting antigen uptake into cells, (ii) the ability to bind to the antigen more than once; (iii) promoting the reduction of antigen concentrations in plasma; and (iv) Excellent plasma retention function; The present invention provides a method for producing an antigen-binding molecule having at least one function selected from the above.

[0217] Specifically, the present invention provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (e): (a) determining the antigen-binding activity of an antigen-binding molecule under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding molecule under a high calcium concentration condition; (c) selecting antigen-binding molecules whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition; (d) obtaining a gene encoding the antigen-binding molecule selected in step (c); (e) producing an antigen-binding molecule using the gene obtained in step (d).

[0218] The present invention further provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (e): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) placing the antigen-binding molecule bound to the antigen in step (a) under a low calcium concentration condition; (c) obtaining the antigen-binding molecules dissociated in step (b); (d) obtaining a gene encoding the antigen-binding molecule obtained in step (c); (e) producing an antigen-binding molecule using the gene obtained in step (d). Note that steps (a) to (d) may be repeated two or more times. Therefore, the present invention provides the above-mentioned method further comprising the step of repeating steps (a) to (d) two or more times. The number of times steps (a) to (d) are repeated is not particularly limited, but is usually within 10 times.

[0219] The present invention further provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under low calcium concentration conditions; (b) selecting antigen-binding molecules that do not bind to the antigen in step (a); (c) contacting the antigen-binding molecule selected in step (b) with an antigen under high calcium concentration conditions; (d) obtaining the antigen-binding molecule bound to the antigen in step (c); (e) obtaining a gene encoding the antigen-binding molecule obtained in step (d); (f) producing an antigen-binding molecule using the gene obtained in step (e). Note that steps (a) to (e) may be repeated two or more times. Therefore, the present invention provides the above-mentioned method further comprising the step of repeating steps (a) to (e) two or more times. The number of times steps (a) to (e) are repeated is not particularly limited, but is usually within 10 times.

[0220] Furthermore, the present invention provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (e): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen-immobilized column under high calcium concentration conditions; (b) eluting the antigen-binding molecules bound to the column in step (a) from the column under low calcium concentration conditions; (c) obtaining the antigen-binding molecules eluted in step (b); (d) obtaining a gene encoding the antigen-binding molecule obtained in step (c); (e) producing an antigen-binding molecule using the gene obtained in step (d). Note that steps (a) to (d) may be repeated two or more times. Therefore, the present invention provides the above-mentioned method further comprising the step of repeating steps (a) to (d) two or more times. The number of times steps (a) to (d) are repeated is not particularly limited, but is usually within 10 times.

[0221] The present invention further provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f): (a) passing an antigen-binding molecule or an antigen-binding molecule library through an antigen-immobilized column under low calcium concentration conditions; (b) recovering the antigen-binding molecules that did not bind to the column and were eluted in step (a); (c) allowing the antigen-binding molecules recovered in step (b) to bind to antigens under high calcium concentration conditions; (d) obtaining the antigen-binding molecule bound to the antigen in step (c); (e) obtaining a gene encoding the antigen-binding molecule obtained in step (d); (f) producing an antigen-binding molecule using the gene obtained in step (e). Note that steps (a) to (e) may be repeated two or more times. Therefore, the present invention provides the above-mentioned method further comprising the step of repeating steps (a) to (e) two or more times. The number of times steps (a) to (e) are repeated is not particularly limited, but is usually within 10 times.

[0222] The present invention further provides a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f): (a) contacting an antigen-binding molecule or an antigen-binding molecule library with an antigen under high calcium concentration conditions; (b) obtaining the antigen-binding molecule bound to the antigen in step (a); (c) placing the antigen-binding molecule obtained in step (b) under a low calcium concentration condition; (d) obtaining in step (c) antigen-binding molecules whose antigen-binding activity is weaker than the criterion selected in step (b); (e) obtaining a gene encoding the antigen-binding molecule obtained in step (d); (f) producing an antigen-binding molecule using the gene obtained in step (e). Note that steps (a) to (e) may be repeated two or more times. Therefore, the present invention provides the above-mentioned method further comprising the step of repeating steps (a) to (e) two or more times. The number of times steps (a) to (e) are repeated is not particularly limited, but is usually within 10 times.

[0223] Antigen-binding molecules used in the production methods of the present invention may be prepared in any manner, and examples thereof include 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, and libraries into which calcium-chelating amino acids (e.g., aspartic acid and glutamic acid) or unnatural amino acid mutations have been introduced (libraries with an increased content of calcium-chelating amino acids (e.g., aspartic acid and glutamic acid) or unnatural amino acids, libraries into which calcium-chelating amino acids (e.g., aspartic acid and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites).

[0224] In the above-described production method, the antigen-binding activity of an antigen-binding molecule under low calcium concentration conditions is not particularly limited as long as the ionized calcium concentration is between 0.1 μM and 30 μM, with preferred examples including antigen-binding activity at an ionized calcium concentration between 0.5 μM and 10 μM. A more preferred ionized calcium concentration is the ionized calcium concentration in early endosomes in vivo, specifically 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding molecule under high calcium concentration conditions is not particularly limited as long as the ionized calcium concentration is between 100 μM and 10 mM, with preferred examples including antigen-binding activity at an ionized calcium concentration between 200 μM and 5 mM. A more preferred ionized calcium concentration is the ionized calcium concentration in plasma in vivo, specifically 0.5 mM to 2.5 mM.

[0225] The antigen-binding activity of an antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art.

[0226] The step of selecting antigen-binding molecules whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is the same as the step of selecting antigen-binding molecules whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0227] As long as the antigen-binding activity under a high calcium concentration is higher than that under a low calcium concentration, the difference between the antigen-binding activity under a high calcium concentration and that under a low calcium concentration is not particularly limited; however, the antigen-binding activity under a high calcium concentration is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times that under a low calcium concentration.

[0228] In the above-mentioned production methods, binding of an antigen to an antigen-binding molecule may be carried out under any conditions, and is not particularly limited. For example, the antigen may be bound to an antigen-binding molecule by contacting the antigen with an immobilized antigen-binding molecule, or the antigen may be bound to an antigen-binding molecule by contacting the antigen with an immobilized antigen. Alternatively, the antigen-binding molecule may be bound to the antigen by contacting the antigen in solution.

[0229] Furthermore, the production methods of the present invention can be used for antigen-binding molecules that have human FcRn-binding activity at neutral pH, as described above, and can be combined with methods for conferring or enhancing human FcRn-binding activity at neutral pH. When the production methods of the present invention are combined with methods for conferring or enhancing human FcRn-binding activity at neutral pH, the method may further comprise the step of modifying amino acids in the antigen-binding molecule to confer or enhance human FcRn-binding activity at neutral pH. Furthermore, preferred examples of the human FcRn-binding domain of an antigen-binding molecule that has human FcRn-binding activity at neutral pH include the above-described human FcRn-binding domain that has human FcRn-binding activity at neutral pH. Therefore, the production methods of the present invention may further comprise the step of previously selecting an antigen-binding molecule that has a human FcRn-binding domain with high human FcRn-binding activity at neutral pH and / or modifying amino acids in the antigen-binding molecule to confer or enhance human FcRn-binding activity at neutral pH.

[0230] Furthermore, the production methods of the present invention can be used for antigen-binding molecules with pH-dependent antigen-binding activity, or can be combined with a method for conferring pH-dependent antigen-binding activity (WO 2009 / 125825). When combining the production methods of the present invention with a method for conferring pH-dependent antigen-binding activity, the method may further comprise the step of selecting an antigen-binding molecule whose antigen-binding activity under acidic pH conditions is lower than that under neutral pH conditions and / or modifying amino acids in the antigen-binding molecule to reduce its antigen-binding activity under acidic pH conditions to be lower than that under neutral pH conditions.

[0231] Furthermore, preferred examples of antigen-binding molecules with pH-dependent antigen-binding activity include antigen-binding molecules in which at least one amino acid has been substituted with histidine or in which at least one histidine has been inserted. Therefore, the production methods of the present invention may use antigen-binding molecules in which at least one amino acid has been substituted with histidine or in which at least one histidine has been inserted, or may further comprise the step of substituting at least one amino acid of the antigen-binding molecule with histidine or inserting at least one histidine.

[0232] In the production method of the present invention, an unnatural amino acid may be used in place of histidine, and therefore the present invention can be understood by replacing the above-mentioned histidine with an unnatural amino acid.

[0233] When administered to animals such as humans, mice, and monkeys using the production method of the present invention, (i) the function of promoting the uptake of antigens into cells; (ii) the ability to bind to the antigen more than once; (iii) the ability to promote the reduction of antigen concentrations in plasma; and (iv) Excellent plasma retention function; It is possible to produce antigen-binding molecules having at least one function selected from the following: Therefore, the production methods of the present invention can be used to obtain antigen-binding molecules having at least one of these functions.

[0234] Furthermore, these antigen-binding molecules are considered to be particularly excellent as pharmaceuticals because they allow for a reduction in the dosage and frequency of administration to patients, thereby enabling a reduction in the total dosage. Therefore, the production method of the present invention can be used as a method for producing antigen-binding molecules for use as pharmaceutical compositions.

[0235] The gene obtained in the production method of the present invention is usually carried (inserted) into 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 Escherichia coli is used as the host, a cloning vector such as the pBluescript vector (Stratagene) is preferred, although various commercially available vectors can also 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 Escherichia coli, in cultured cells, or in an individual organism. For example, preferred expression vectors include the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms. The DNA of the present invention can be inserted into a vector by conventional methods, for example, by 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).

[0236] The host cells are not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing antigen-binding molecules 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. Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, edited by Ausubel et al. (1987) Published by John Wiley & Sons, Sections 9.1-9.9), lipofection, and microinjection.

[0237] Host cells can be cultured according to known methods. For example, when animal cells are used as hosts, culture media such as DMEM, MEM, RPMI1640, and IMDM can be used. In this case, serum supplements such as FBS and fetal calf serum (FCS) can be used in combination, or the cells can be cultured in a serum-free culture. The pH during culture is preferably about 6 to 8. Culture is usually carried out at about 30 to 40°C for about 15 to 200 hours, with medium replacement, aeration, and stirring as necessary.

[0238] To secrete an antigen-binding molecule expressed in a host cell into the lumen of the endoplasmic reticulum, the periplasmic space, or 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.

[0239] 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 recovered. In the present invention, the "host" includes these animals and plants.

[0240] 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.

[0241] 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 specifically produced in milk, such as caprine β-casein. A polynucleotide fragment containing this fusion gene is then injected into a goat embryo, and the embryo is then implanted into a female goat. The antigen-binding molecule of interest can be obtained from the milk produced by the transgenic goat born to the goat that received the embryo, or from the milk produced by its offspring. Appropriate hormones may be administered to the transgenic goat to increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat (Ebert et al., Bio / Technology (1994) 12: 699-702).

[0242] Furthermore, silkworms, for example, can be used as insects for producing the antigen-binding molecules of the present invention. When using silkworms, the antigen-binding molecules of interest can be obtained from the body fluids of the silkworms by infecting them with a baculovirus into which a polynucleotide encoding the antigen-binding molecule of interest has been inserted.

[0243] Furthermore, when using plants to produce the antigen-binding molecules of the present invention, tobacco, for example, can be used. When using tobacco, a polynucleotide encoding the antigen-binding molecule of interest is inserted into a plant expression vector, such as pMON 530, and this vector is then introduced into bacteria such as Agrobacterium tumefaciens. This bacterium is then used to infect tobacco, such as Nicotiana tabacum, and the desired antigen-binding molecule can be isolated from the tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-8). Alternatively, the same bacteria can be used to infect duckweed (Lemna minor), and after cloning, the desired antigen-binding molecule can be isolated from the duckweed cells (Cox KM et al. Nat. Biotechnol. 2006 Dec;24(12):1591-1597).

[0244] The antigen-binding molecules obtained in this manner can be isolated from inside or outside the host cells (culture medium, milk, etc.) and purified as substantially pure and homogeneous antigen-binding molecules. The separation and purification of antigen-binding molecules can be performed using any separation and purification method commonly used for purifying polypeptides, and is not limited in any way. For example, the antigen-binding molecules can be separated and purified by appropriately selecting and combining methods such as chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0245] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These 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).

[0246] If necessary, before or after purification of the antigen-binding molecule, an appropriate protein-modifying enzyme can be used to optionally modify or partially remove peptides. Examples of protein-modifying enzymes that can be used include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

[0247] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising the antigen-binding molecules of the present invention, antigen-binding molecules isolated by the screening methods of the present invention, or antigen-binding molecules produced by the production methods of the present invention. The antigen-binding molecules of the present invention, antigen-binding molecules isolated by the screening methods of the present invention, or antigen-binding molecules produced by the production methods of the present invention are (i) the function of promoting the uptake of antigens into cells; (ii) the ability to bind to the antigen more than once; (iii) the ability to promote the reduction of antigen concentrations in plasma; and (iv) Excellent plasma retention function; and is useful as a pharmaceutical composition since it is expected to reduce the administration frequency of the antigen-binding molecule. The pharmaceutical composition of the present invention can also contain a pharmaceutically acceptable carrier.

[0248] In the present invention, the pharmaceutical composition generally refers to a drug for treating or preventing a disease, or for testing or diagnosing a disease.

[0249] The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is set so that an appropriate volume within the specified range is obtained. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.

[0250] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50), may be used in combination.

[0251] Oily liquids include sesame oil and soybean oil, and may contain benzyl benzoate and / or benzyl alcohol as solubilizers. Buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection solution is usually filled into suitable ampoules.

[0252] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, it can be in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

[0253] The administration method can be selected appropriately depending on the patient's age and symptoms. The dose of a pharmaceutical composition containing an antigen-binding molecule can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dose can be set, for example, at 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these numerical values. The dose and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dose and administration method taking these conditions into consideration.

[0254] The pharmaceutical composition of the present invention may also be a pharmaceutical composition used to promote the uptake of an antigen into cells or to promote the reduction of antigen concentration in plasma.

[0255] The present invention also relates to methods for promoting intracellular uptake of antigens by antigen-binding molecules or methods for promoting a decrease in plasma antigen concentration by administering the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention. Administration may be carried out in vivo or in vitro. Subjects to be administered include, for example, non-human animals (e.g., mice, monkeys), or humans.

[0256] The present invention also relates to methods for increasing the number of antigen bindings per antigen-binding molecule or methods for improving the plasma retention of an antigen-binding molecule, by using the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention.

[0257] It should be noted that 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), and even when such amino acids are modified after translation, they are naturally included in the amino acid sequences described in the present invention.

[0258] The present invention also provides kits for use in the methods of the present invention, which contain at least the antigen-binding molecules of the present invention. The kits may also contain other packaged components such as pharmaceutically acceptable carriers, vehicles, and instructions describing the method of use.

[0259] The present invention also relates to agents that promote intracellular antigen uptake by antigen-binding molecules, promote the reduction of plasma antigen concentrations, increase the number of antigen bindings by a single antigen-binding molecule, or improve the plasma retention of antigen-binding molecules, each of which contains as an active ingredient an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention.

[0260] The present invention also relates to use of the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention in the production of an agent that promotes antigen uptake into cells by the antigen-binding molecule, an agent that promotes a decrease in plasma antigen concentration, an agent that increases the number of antigen bindings by a single antigen-binding molecule, or an agent that improves the plasma retention of an antigen-binding molecule.

[0261] The present invention also relates to the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention, for use in methods for promoting intracellular antigen uptake by antigen-binding molecules, methods for promoting a decrease in plasma antigen concentration, methods for increasing the number of antigen bindings per antigen-binding molecule, or methods for improving the plasma retention of antigen-binding molecules.

[0262] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0263] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Concept of the antigen elimination acceleration effect of calcium-dependent antigen-binding antibodies (1-1) pH-dependent binding of antibodies accelerates antigen elimination by antigen-binding antibodies H54 / L28-IgG1, described in WO 2009 / 125825, is a humanized anti-IL-6 receptor antibody, and Fv4-IgG1 is a humanized anti-IL-6 receptor antibody that has been conferred with the property of pH-dependent binding to soluble human IL-6 receptor (binding under neutral conditions and dissociating under acidic conditions). In vivo testing in mice described in WO 2009 / 125825 showed that elimination 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.

[0264] Soluble human IL-6 receptor bound to antibodies that bind to conventional soluble human IL-6 receptor is recycled into plasma along with the antibody by FcRn, whereas antibodies that bind to soluble human IL-6 receptor in a pH-dependent manner dissociate the antibody-bound soluble human IL-6 receptor under the acidic conditions of the endosome. The dissociated soluble human IL-6 receptor is degraded in lysosomes, significantly accelerating the elimination of soluble human IL-6 receptor. Furthermore, antibodies that bind to soluble human IL-6 receptor in a pH-dependent manner are recycled into plasma by FcRn, and the recycled antibody can again bind to soluble human IL-6 receptor. This process is repeated, allowing a single antibody molecule to bind to soluble human IL-6 receptor multiple times (Figure 1).

[0265] Furthermore, as described in WO 2009 / 125825, after binding to membrane-type human IL-6 receptor, a typical humanized anti-IL-6 receptor antibody is internalized as a complex between the humanized anti-IL-6 receptor antibody and membrane-type human IL-6 receptor, and then degraded in the lysosome. In contrast, a humanized anti-IL-6 receptor antibody that binds in a pH-dependent manner binds to membrane-type human IL-6 receptor and is internalized, then dissociates from the membrane-type human IL-6 receptor under acidic conditions in the endosome and is recycled into plasma. The recycled antibody can again bind to membrane-type human IL-6 receptor, and this process can be repeated, allowing a single antibody molecule to bind to membrane-type human IL-6 receptor multiple times (Figure 2).

[0266] (1-2) pH and calcium concentration in plasma and endosomes In the action of the pH-dependent binding antibody shown in Figures 1 and 2, it is important to take advantage of the difference in the environment between plasma and endosomes, i.e., the difference in pH (plasma: pH 7.4, endosome: pH 6.0), to allow the antibody to bind strongly to the antigen in plasma and dissociate from the antigen in endosomes. To create such a difference in the antigen-binding ability of the pH-dependent binding antibody between plasma and endosomes, the magnitude of the difference in the environment between plasma and endosomes is important. The difference in pH is, in other words, a difference in hydrogen ion concentration. In other words, the hydrogen ion concentration in plasma at pH 7.4 is approximately 40 nM, while the hydrogen ion concentration in endosomes at pH 6.0 is approximately 1000 nM, resulting in an approximately 25-fold difference in the concentration of the factor (hydrogen ion) between plasma and endosomes.

[0267] To more easily achieve or enhance the effects shown in Figures 1 and 2, we considered using antibodies that depend on factors whose concentrations differ more significantly than the difference in hydrogen ion concentrations between plasma and endosomes. A search for factors whose concentrations differ significantly between plasma and endosomes revealed calcium. The ionized calcium concentration in plasma is approximately 1.1–1.3 mM, while the ionized calcium concentration in endosomes is approximately 3 μM. This indicates a roughly 400-fold difference in the concentration of the factor (calcium) between plasma and endosomes, a difference greater than the hydrogen ion concentration difference (25-fold). Therefore, we considered that the effects shown in Figures 1 and 2 could be more easily achieved or enhanced by using an ionized calcium concentration-dependent binding antibody that binds to the antigen under high calcium concentrations (1.1–1.3 mM) and dissociates from the antigen under low calcium concentrations (3 μM).

[0268] Furthermore, WO 2009 / 125825 describes the preparation of a pH-dependent binding antibody whose properties change between pH 7.4 and pH 6.0 by introducing histidine. Histidine is neutrally charged under the neutral conditions of plasma, but positively charged under the acidic conditions of endosomes. This change in histidine charge can be utilized to impart pH dependence to the antibody-antigen interaction. On the other hand, as shown in Figure 3, when histidine is used, in order for the antibody to bind to an antigen in plasma and dissociate from the antigen in endosomes, the histidine residue of the antibody must interact with a positively charged amino acid or an amino acid that can act as a hydrogen bond donor on the antigen. Therefore, in order for the pH-dependent binding antibody to exert its intended effect, the epitope on the antigen to which it binds must contain a positively charged amino acid or an amino acid that can act as a hydrogen bond donor.

[0269] On the other hand, as shown in Figure 4, calcium-dependent binding antibodies are thought to bind to antigens via calcium ions, and therefore the epitopes on the antigen are negatively charged amino acids that can chelate calcium ions or amino acids that can act as hydrogen bond acceptors. This makes it possible to target epitopes that cannot be targeted by pH-dependent binders prepared by introducing histidine. Furthermore, as shown in Figure 5, the use of antibodies that are both calcium-dependent and pH-dependent may enable targeting of epitopes with a wide range of properties.

[0270] [Example 2] Obtaining Ca-dependent binding antibodies from a human antibody library using phage display technology (2-1) Construction of a naive human antibody phage display library Using polyA RNA prepared from human PBMCs and commercially available human polyA RNA as templates, we constructed multiple human antibody phage display libraries displaying Fab domains consisting of human antibody sequences, following the method described in Methods Mol Biol. 2002;178:87-100.

[0271] (2-2) Isolation of Ca-dependent binding antibody fragments from a library by bead panning The first selection from the constructed human antibody phage display library was to enrich only antibody fragments that bind to the antigen, or to enrich for antibody fragments that exhibit Ca-dependent binding. To enrich for antibody fragments with Ca-dependent binding, phages were allowed to bind to the antigen in the presence of Ca ions, and then eluted by chelating the Ca ions with EDTA. Biotin-labeled human IL-6 receptor was used as the antigen.

[0272] Phages were produced from E. coli harboring the constructed phagemids for phage display. The resulting culture medium was precipitated with 2.5 M NaCl / 10% PEG and diluted with TBS to prepare a phage library solution. BSA and CaCl2 were added to the phage library solution to a final concentration of 4% BSA and 1.2 mM ionized calcium. Panning was performed using a conventional method using antigens immobilized on magnetic beads (J Immunol Methods. 2008 Mar 20;332(1-2):2-9., J Immunol Methods. 2001 Jan 1;247(1-2):191-203., Biotechnol Prog. 2002 Mar-Apr;18(2):212-20., Mol Cell Proteomics. 2003 Feb;2(2):61-9.). The magnetic beads used were NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin).

[0273] Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution and allowed to contact with the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added and allowed to bind for 15 minutes at room temperature. The beads were washed once with 1 mL of 1.2 mM CaCl2 / TBS (TBS containing 1.2 mM CaCl2). Subsequently, phage were recovered by standard elution methods to concentrate antibody fragments with binding activity. To concentrate antibody fragments with Ca-dependent binding activity, the beads were suspended in 2 mM EDTA / TBS (TBS containing 2% EDTA). 10 mL of E. coli strain TG1 in logarithmic growth phase (OD600 0.4-0.5) was added to the recovered phage solution and infected by gently stirring at 37°C for 1 hour. The infected E. coli were plated onto a 225 mm x 225 mm plate. A new culture was started from this E. coli and phage culture was performed.

[0274] In subsequent panning rounds, enrichment was performed based on Ca-dependent binding. Specifically, 40 pmol of biotin-labeled antigen was added to the prepared phage library solution and allowed to react with the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added and allowed to bind for 15 minutes at room temperature. The beads were washed once with 1 mL of 1.2 mM CaCl2 / TBST (TBS containing 1.2 mM CaCl2 and 0.1% Tween-20) and once with 1.2 mM CaCl2 / TBS. The beads were then suspended in 0.1 mL of 2 mM EDTA / TBS (TBS containing 2% EDTA) at room temperature. The beads were immediately separated using a magnet stand, and the phage suspension was recovered. The recovered phage suspension was added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.5) and infected by gentle agitation at 37°C for 1 hour. The infected E. coli was plated onto a 225 mm x 225 mm plate. Culture was started again from this E. coli, and phage culture was performed in the same manner as above, and panning was repeated twice.

[0275] (2-3) Evaluation by phage ELISA Phage-containing culture supernatant was collected from the E. coli single colony obtained by the above method, following the method described in (Methods Mol Biol. 2002;178:133-145.). Phage-containing culture supernatants were supplemented with BSA and CaCl2 to a final concentration of 4% BSA and 1.2 mM calcium ion concentration, and then subjected to ELISA. StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of PBS containing biotin-labeled antigen. After washing with PBST (PBS containing 0.1% Tween 20) to remove the antigen, the plates were blocked with 250 μL of 4% BSA-TBS for at least 1 hour. The 4% BSA-TBS was removed, and the prepared culture supernatant was added and incubated at 37°C for 1 hour to allow binding of the phage-displayed antibody. After washing with 1.2 mM CaCl2 / TBST (TBS containing 1.2 mM CaCl2 and 0.1% Tween 20), the plates were incubated with 1.2 mM CaCl2 / TBS or 1 mM EDTA / TBS for 30 minutes at 37°C. After washing with 1.2 mM CaCl2 / TBST, the cells were incubated for 1 hour with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS containing 4% BSA and 1.2 mM ionized calcium. After washing with 1.2 mM CaCl2 / TBST, the cells were detected with TMB single solution (ZYMED). The reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was measured. Antibody fragments determined to have Ca-dependent binding activity were sequenced using specific primers.

[0276] (2-4) Antibody expression and purification Clones determined to have Ca-dependent binding ability by phage ELISA were introduced into an animal cell expression plasmid. Antibody expression was carried out using the following method. Human fetal kidney cell-derived FreeStyle 293-F strain (Invitrogen) was suspended in FreeStyle 293 Expression Medium (Invitrogen) and 1.33 × 10 6The cells were seeded at a cell density of 1000 cells / mL in each well of a 6-well plate (3 mL per well). The prepared plasmid was introduced into the cells by lipofection. The cells were cultured for 4 days in a CO2 incubator (37°C, 8% CO2, 90 rpm). rProtein A Sepharose was extracted from the culture supernatant. TM Antibodies were purified using Fast Flow (Amersham Biosciences) by methods known to those skilled in the art. The concentration of purified antibodies was determined by measuring absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

[0277] [Example 3] Evaluation of the Ca-dependent binding ability of the obtained antibodies to the human IL-6 receptor The antibodies 6RL#9-IgG1 (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 2), 6RK#12-IgG1 (heavy chain SEQ ID NO: 66, light chain SEQ ID NO: 67), and FH4-IgG1 (heavy chain SEQ ID NO: 3, light chain SEQ ID NO: 4) obtained in Example 2 were assayed for human interleukin-6 receptor (hIL6R) binding activity at pH 7.4 using a Biacore T100 (GE Healthcare). Measurements were performed using a running buffer containing 0.05% Surfactant P20, 10 mmol / L ACES, and 150 mmol / L NaCl (pH 7.4 or pH 6.0) containing 3 μM or 2 mM CaCl.

[0278] An appropriate amount of recombinant protein A (Thermo Scientific) was immobilized on a sensor chip CM4 (GE Healthcare) by the amino coupling method, and then the antibody was bound to it. An appropriate concentration of hIL-6R was injected as an analyte to allow it to interact with the antibody on the sensor chip. 10 mmol / L glycine-HCl (pH 1.5) was then injected to regenerate the sensor chip. Measurements were performed at 37°C. The resulting sensorgrams are shown in Figure 6. These results indicate that the 6RL#9-IgG1, 6RK#12-IgG1, and FH4-IgG1 antibodies all bind to Ca. 2+ At a concentration of 3 μM, Ca 2+ It was clearly demonstrated that the binding to hIL6R was weaker than when the concentration was 2 mM.

[0279] Among these antibodies, 6RL#9-IgG1 (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 2) and FH4-IgG1 (heavy chain SEQ ID NO: 3, light chain SEQ ID NO: 4) were selected as Ca-dependent antibodies and further kinetic analysis was performed. H54 / L28-IgG1 (heavy chain SEQ ID NO: 5, light chain SEQ ID NO: 6), described in WO 2009 / 125825, was used as an antibody without Ca-dependent activity. A high calcium ion concentration of 2 mM was used, and a low calcium ion concentration of 3 μM was used. Human IL-6 receptor (IL-6R) was used as the antigen. An appropriate amount of protein A (Invitrogen) was immobilized on a Sensor Chip CM4 (GE Healthcare) by the amine coupling method, and the target antibody was captured onto the immobilized antibody. The running buffer used was either 10 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, 2 mmol / L CaCl2, pH 7.4 or 10 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, 3 μmol / L CaCl2, pH 7.4. All measurements were performed at 37°C, and the IL-6R was diluted in the respective buffers.

[0280] For H54L28-IgG1, IL-6R dilution and blank running buffer were injected at a flow rate of 20 μL / min for 3 minutes to allow IL-6R to interact with the antibody captured on the sensor chip. The running buffer was then injected at a flow rate of 20 μL / min for 10 minutes to observe the dissociation of IL-6R, after which 10 mmol / L Glycine-HCl, pH 1.5, was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the sensor chip. The kinetic parameters, the binding rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s), were calculated from the sensorgrams obtained in the measurements, and the dissociation constant K of each antibody for the human IL-6 receptor was calculated based on these values. D (M) was calculated. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare).

[0281] For FH4-IgG1 and 6RL#9-IgG1, the IL-6R dilution solution and blank running buffer were injected at a flow rate of 5 μL / min for 15 minutes to allow IL-6R to interact with the antibody captured on the sensor chip. The sensor chip was then regenerated by injecting 10 mmol / L Glycine-HCl, pH 1.5, at a flow rate of 30 μL / min for 30 seconds. The dissociation constant K was calculated using a steady-state affinity model for the sensorgram obtained by the measurement. D (M) was calculated using Biacore T100 Evaluation Software (GE Healthcare).

[0282] The dissociation constant K between each antibody and IL-6R in the presence of 2 mM CaCl2 was determined by this method. D The results are shown in Table 7. For H54 / L28-IgG1, no difference in IL-6R binding due to differences in Ca concentration was observed, but for FH4-IgG1 and 6RL#9-IgG1, a significant decrease in binding was observed under low Ca concentrations (Figures 7, 8, and 9).

[0283] [Table 7]

[0284] For H54 / L28-IgG1, the K under the condition of 3 μM Ca was measured in the same manner as in the presence of 2 mM Ca. D For FH4-IgG1 and 6RL#9-IgG1, almost no binding to IL-6R was observed at a Ca concentration of 3 μM. D Although it is difficult to calculate K D It is possible to predict the following (Biacore T100 Software Handbook, BR-1006-48, AE 01 / 2007).

[0285] [Formula 1] TIFF0007817318000014.tif5128

[0286] The meaning of each item in the above formula 1 is as follows: R eq (RU): Steady state binding levels R max (RU): Analyte binding capacity of the surface RI (RU): Bulk refractive index contribution in the sample C (M): Analyte concentration K D (M): Equilibrium dissociation constant

[0287] Using this formula 1, the predicted dissociation constant K between each antibody and IL-6R at a Ca concentration of 3 μmol / L was calculated. D The estimated results are shown in Table 8.

[0288] [Table 8]

[0289] In Table 8 above, R eq , R max , RI, and C are assumed values ​​based on measurement results.

[0290] These results suggest that FH4-IgG1 and 6RL#9-IgG1 have increased K activity against IL-6R by increasing the CaCl2 concentration from 2 mM to 3 μM. D It was predicted that the affinity would increase by approximately 60-fold and approximately 120-fold, respectively (a 60-fold and more than 120-fold decrease in affinity). Table 9 shows the K D value, and K D The Ca dependence of the values ​​was summarized.

[0291] [Table 9]

[0292] [Example 4] Evaluation of calcium ion binding to the obtained antibodies Next, to evaluate calcium ion binding to the antibody, the thermal denaturation midpoint temperature (Tm) was measured by differential scanning calorimetry (DSC) (MicroCal VP-Capillary DSC, MicroCal). The thermal denaturation midpoint temperature (Tm) is an indicator of stability. When calcium ions bind and stabilize the protein, the Tm becomes higher than when calcium ions are not bound (J Bio Chem. 2008 Sep 12; Vol. 283; No. 37: pp. 25140-25149). The purified antibody was dialyzed (EasySEP, TOMY) against a solution of 20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, pH 7.4, or 20 mM Tris-HCl, 150 mM NaCl, 3 μM CaCl2, pH 7.4. The protein solution was adjusted to 0.1 mg / mL with the solution used for dialysis, and DSC measurement was performed at a heating rate of 240°C / hr from 20°C to 115°C. The thermal denaturation midpoint temperature (Tm value) of the Fab domain of each antibody was calculated based on the obtained DSC denaturation curves and is shown in Table 10.

[0293] [Table 10]

[0294] The results in Table 10 show that the Fab Tm values ​​of FH4 and 6RL#9, which exhibit calcium-dependent binding ability, change with calcium ion concentration, whereas the Tm value of H54 / L28, which does not exhibit calcium-dependent binding ability, does not change. The change in the Fab Tm values ​​shown for FH4 and 6RL#9 indicates that calcium ions bind to these antibodies, stabilizing the Fab portion. This indicates that calcium ions bind to FH4 ​​and 6RL#9, whereas calcium ions do not bind to H54 / L28.

[0295] [Example 5] Evaluation of the effect of Ca-dependent binding antibodies on antigen plasma retention using normal mice (5-1) In vivo test using normal mice Normal mice (C57BL / 6J mice, Charles River Japan) were administered either hsIL-6R (soluble human IL-6 receptor: prepared in Reference Example 1) alone or hsIL-6R and anti-human IL-6 receptor antibody simultaneously, and the pharmacokinetics of hsIL-6R and anti-human IL-6 receptor antibody were evaluated. A single dose of hsIL-6R solution (5 μg / mL) or a mixed solution of hsIL-6R and anti-human IL-6 receptor antibody was administered via the tail vein at 10 mL / kg. The anti-human IL-6 receptor antibodies used were the aforementioned H54 / L28-IgG1, 6RL#9-IgG1, and FH4-IgG1.

[0296] The hsIL-6R concentration in the mixed solution was 5 μg / mL, while the anti-human IL-6 receptor antibody concentration varied depending on the antibody: H54 / L28-IgG1 was 0.1 mg / mL, and 6RL#9-IgG1 and FH4-IgG1 were 10 mg / mL. Since the anti-human IL-6 receptor antibody was present in sufficient excess relative to the hsIL-6R, it is believed that most of the hsIL-6R was bound to the antibody. Blood samples were collected 15 minutes, 7 hours, 1 day, 2 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 12,000 rpm at 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20°C or below until assayed.

[0297] (5-2) Measurement of anti-human IL-6 receptor antibody concentrations in normal mouse plasma 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 Fragment of Antibody (SIGMA) was dispensed into a Nunc-Immuno Plate, MaxiSoup (Nalge nunc International), and the plate was left to stand overnight at 4°C to create an anti-human IgG-coated plate. Plasma calibration curve samples with plasma concentrations of 0.64, 0.32, 0.16, 0.08, 0.04, 0.02, and 0.01 μg / mL and 100-fold diluted mouse plasma samples were prepared. The plate was then dispensed into the anti-human IgG-coated plate and incubated at 25°C for 1 hour. The plate was then incubated with biotinylated anti-human IL-6R antibody (R&D) for 1 hour at 25°C, followed by Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 0.5 hours at 25°C. A color reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. The reaction was stopped with 1N-Sulfuric acid (Showa Chemical), and the absorbance at 450 nm was measured using a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). Figure 10 shows the time course of plasma antibody concentrations of H54 / L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 in normal mice after intravenous administration, as measured using this method.

[0298] (5-3) Measurement of plasma hsIL-6R concentration by electrochemiluminescence Mouse plasma hsIL-6R concentrations were measured using electrochemiluminescence. Standard hsIL-6R samples were prepared at 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg / mL. Mouse plasma samples were diluted 50-fold or more. These 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 tocilizumab (heavy chain SEQ ID NO: 13, light chain SEQ ID NO: 14) solutions and incubated overnight at 4°C. The assay buffer contained 10 mM EDTA to reduce the free Ca concentration in the sample, allowing almost all of the hsIL-6R in the sample to dissociate from 6RL#9-IgG1 or FH4-IgG1 and remain bound to the added tocilizumab. The samples were then dispensed onto an MA400 PR Streptavidin Plate (Meso Scale Discovery). After further incubation at 25°C for 1 hour and washing, Read Buffer T (x4) (Meso Scale Discovery) was dispensed and immediately measured using a SECTOR PR 400 reader (Meso Scale Discovery). hsIL-6R concentrations were calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma hsIL-6R concentrations in normal mice after intravenous administration measured using this method is shown in Figure 11.

[0299] The results showed that hsIL-6R alone showed a very rapid elimination, whereas coadministration of H54 / L28-IgG1, a conventional antibody that does not bind to hsIL-6R via calcium-dependent binding, significantly delayed the elimination of hsIL-6R. In contrast, coadministration of 6RL#9-IgG1 or FH4-IgG1, which have over 100-fold higher calcium-dependent binding to hsIL-6R, significantly accelerated the elimination of hsIL-6R. Compared with coadministration of H54 / L28-IgG1, coadministration of 6RL#9-IgG1 and FH4-IgG1 reduced plasma hsIL-6R concentrations on Day 1 by 39-fold and 2-fold, respectively. These results confirmed that calcium-dependent binding antibodies can accelerate the elimination of antigens from plasma.

[0300] [Example 6] Examination of the improvement of the antigen elimination acceleration effect of Ca-dependent antigen-binding antibodies (antibody production) (6-1) Binding of IgG antibodies to FcRn IgG antibodies have a long plasma retention time due to their binding to FcRn. Binding of IgG to FcRn is only observed under acidic conditions (pH 6.0), with almost no binding 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 the endosome, and dissociate from FcRn under neutral conditions in plasma. Introducing a mutation into the Fc region of IgG to abolish binding to FcRn under acidic conditions results in the antibody no longer being recycled from endosomes to plasma, significantly impairing its plasma retention time.

[0301] One method reported to improve the plasma retention of IgG antibodies is to enhance their binding to FcRn under acidic conditions. Introducing amino acid substitutions into the Fc region of IgG antibodies to improve FcRn binding under acidic conditions increases the recycling efficiency from endosomes to plasma, resulting in improved plasma retention. When introducing amino acid substitutions, it is important not to enhance FcRn binding under neutral conditions. If IgG antibodies bind to FcRn under neutral conditions, even if they return to the cell surface by binding to FcRn under the acidic conditions of the endosome, they will not be recycled into plasma unless they dissociate from FcRn under neutral plasma conditions, thereby impairing plasma retention.

[0302] For example, as described in J Immunol. 2002;169(9):5171-80, it has been reported that when an antibody that binds to mouse FcRn under neutral conditions (pH 7.4) due to amino acid substitutions introduced 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 introducing amino acid substitutions into IgG1 improves human FcRn binding 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 in plasma retention was observed. Therefore, antibody engineering techniques for improving antibody function have focused solely on improving the plasma retention of antibodies by increasing human FcRn binding under acidic conditions without increasing human FcRn binding under neutral conditions (pH 7.4), and no reports have been published to date of the benefits of introducing amino acid substitutions into the Fc region of IgG antibodies to increase human FcRn binding under neutral conditions (pH 7.4).

[0303] Antibodies that bind to antigens in a Ca-dependent manner are extremely useful because they accelerate the elimination of soluble antigens and allow a single antibody molecule to bind to soluble antigens multiple times. To further improve this effect, we investigated a method to enhance FcRn binding under neutral conditions (pH 7.4).

[0304] (6-2) Preparation of Ca-dependent human IL-6 receptor-binding antibodies that bind to FcRn under neutral conditions Amino acid mutations that increase FcRn binding under neutral conditions (pH 7.4) were introduced into FH4-IgG1 and 6RL#9-IgG1, which have calcium-dependent antigen-binding ability, and into H54 / L28-IgG1, a control that does not have calcium-dependent antigen-binding ability. The amino acid mutations were introduced using PCR, a method known to those skilled in the art. Specifically, FH4-N434W (heavy chain SEQ ID NO: 7, light chain SEQ ID NO: 8), 6RL#9-N434W (heavy chain SEQ ID NO: 9, light chain SEQ ID NO: 10), and H54 / L28-N434W (heavy chain SEQ ID NO: 11, light chain SEQ ID NO: 12) were prepared by substituting Asn at position 434 (EU numbering) in the IgG1 heavy chain constant region with Trp. The amino acid substitutions were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described in the accompanying instructions to generate mutants, and the resulting plasmid fragments were inserted into an animal cell expression vector to generate the desired expression vector. Antibody expression, purification, and concentration measurement were performed as described in Example 2.

[0305] [Example 7] Evaluation of the effect of accelerating the disappearance of Ca-dependent binding antibodies using normal mice (7-1) In vivo test using normal mice Normal mice (C57BL / 6J mice, Charles River Japan) were administered either hsIL-6R (soluble human IL-6 receptor, prepared as described in Reference Example 1) alone or hsIL-6R and anti-human IL-6 receptor antibody simultaneously, and the pharmacokinetics of hsIL-6R and anti-human IL-6 receptor antibody were evaluated. A single dose of hsIL-6R solution (5 μg / mL) or a mixture of hsIL-6R and anti-human IL-6 receptor antibody was administered via the tail vein at 10 mL / kg. The anti-human IL-6 receptor antibodies used were the aforementioned H54 / L28-N434W, 6RL#9-N434W, and FH4-N434W.

[0306] The hsIL-6R concentration in the mixed solution was 5 μg / mL, while the anti-human IL-6 receptor antibody concentration varied for each antibody: 0.042 mg / mL for H54 / L28-N434W, 0.55 mg / mL for 6RL#9-N434W, and 1 mg / mL for FH4-N434W. Because the anti-human IL-6 receptor antibody was present in sufficient excess relative to the hsIL-6R, it is believed that most of the hsIL-6R was bound to the antibody. Blood samples were collected 15 minutes, 7 hours, 1 day, 2 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 12,000 rpm at 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at ≤−20°C until assayed.

[0307] (7-2) Measurement of anti-human IL-6 receptor antibody concentrations in normal mouse plasma by ELISA The anti-human IL-6 receptor antibody concentrations in mouse plasma were measured by the same ELISA method as in Example 6. The time courses of plasma antibody concentrations of H54 / L28-N434W, 6RL#9-N434W, and FH4-N434W in normal mice after intravenous administration, as measured by this method, are shown in Figure 12.

[0308] (7-3) Measurement of plasma hsIL-6R concentration by electrochemiluminescence Mouse plasma hsIL-6R concentrations were measured by electrochemiluminescence. Standard hsIL-6R samples were prepared at 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg / mL. Mouse plasma samples were diluted 50-fold or more and 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). The assay buffer contained 10 mM EDTA to reduce the free Ca concentration in the samples, ensuring that almost all hsIL-6R dissociated from 6RL#9-N434W or FH4-N434W and remained free. The mixture was then dispensed into an MA400 PR Streptavidin Plate (Meso Scale Discovery). After further incubation at 25°C for 1 hour and washing, Read Buffer T (x4) (Meso Scale Discovery) was dispensed and immediately measured using a SECTOR PR 400 reader (Meso Scale Discovery). hsIL-6R concentrations were calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma hsIL-6R concentrations in normal mice after intravenous administration, measured using this method, is shown in Figure 13.

[0309] Coadministration of H54 / L28-N434W, a conventional antibody that enhances FcRn binding at pH 7.4 but lacks Ca-dependent binding to hsIL-6R, significantly slowed the elimination of hsIL-6R compared with hsIL-6R alone. In contrast, coadministration of 6RL#9-N434W or FH4-N434W, which have 100-fold more Ca-dependent binding to hsIL-6R and enhanced FcRn binding at pH 7.4, accelerated the elimination of hsIL-6R compared with hsIL-6R alone. Coadministration of 6RL#9-N434W and FH4-N434W reduced plasma hsIL-6R concentrations on Day 1 by 3-fold and 8-fold, respectively, compared with hsIL-6R alone. This confirmed that antigen elimination from plasma can be further accelerated by enhancing the FcRn-binding ability of calcium-dependent binding antibodies at pH 7.4.

[0310] Compared with H54 / L28-IgG1, a conventional antibody that does not bind to hsIL-6R in a Ca-dependent manner, 6RL#9-IgG1 and FH4-IgG1, which have 100-fold higher Ca-dependent binding to hsIL-6R, were confirmed to enhance hsIL-6R elimination. 6RL#9-N434W and FH4-N434W, which have 100-fold higher Ca-dependent binding to hsIL-6R and enhanced FcRn binding at pH 7.4, were confirmed to accelerate hsIL-6R elimination compared to hsIL-6R alone. These data suggest that, similar to the pH-dependent binding of antibodies shown in Figure 1, Ca-dependent binding antibodies dissociate antigens in endosomes. As described in Example 1, the epitopes that can be targeted by antibodies that bind to antigens in a pH-dependent manner are limited (Figure 3). However, by using the antibodies that bind to antigens in a Ca-dependent manner discovered in this study (Figures 4 and 5), it is thought that the epitopes that can be targeted by antibodies that can dissociate antigens in an endosome-dependent manner can be broadened.

[0311] [Example 8] Identification of the calcium ion binding site of the 6RL#9 antibody by X-ray crystal structure analysis (8-1)X-ray crystal structure analysis As shown in Example 4, measurements of the thermal denaturation temperature (Tm) suggested that the 6RL#9 antibody binds to calcium ions. However, it was not possible to predict which site of the 6RL#9 antibody binds to calcium ions. Therefore, X-ray crystal structure analysis was used to identify the residues in the 6RL#9 antibody sequence that interact with calcium ions.

[0312] (8-2) Expression and purification of 6RL#9 antibody The expressed 6RL#9 antibody was purified for use in X-ray crystal structure analysis. Specifically, animal expression plasmids prepared to express the heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) of the 6RL#9 antibody were transiently transfected into animal cells. The final cell density was 1 x 10 6 The prepared plasmid was introduced by lipofection into 800 mL of human fetal kidney cell-derived FreeStyle 293-F (Invitrogen) cells suspended in FreeStyle 293 Expression Medium (Invitrogen) at 100 cells / mL. The plasmid-introduced cells were cultured in a CO2 incubator (37°C, 8% CO2, 90 rpm) for 5 days. rProtein A Sepharose TM Antibodies were purified from the culture supernatants obtained as described above using Fast Flow (Amersham Biosciences) according to methods known to those skilled in the art. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0313] (8-3) Purification of Fab fragment from 6RL#9 antibody Antibody 6RL#9 was concentrated to 21 mg / mL using a 10,000 MWCO ultrafiltration membrane. A 2.5 mL sample of the antibody was diluted to 5 mg / mL with 4 mM L-cysteine, 5 mM EDTA, and 20 mM sodium phosphate buffer (pH 6.5). 0.125 mg of papain (Roche Applied Science) was added, the mixture was stirred, and the mixture was incubated at 35°C for 2 hours. After incubation, 10 mL of 25 mM MES buffer (pH 6) containing one tablet of protease inhibitor cocktail mini, EDTA-free (Roche Applied Science) was added to the mixture, and the mixture was then incubated on ice to stop the protease reaction caused by papain. The sample was then loaded onto a 1 mL cation exchange column, HiTrap SP HP (GE Healthcare), equilibrated with 25 mM MES buffer, pH 6, connected in tandem with a 1 mL Protein A carrier column, HiTrap MabSelect Sure (GE Healthcare). A purified fraction of the 6RL#9 antibody Fab fragment was obtained by elution with a linear increase in NaCl concentration in the same buffer up to 300 mM. The purified fraction was then concentrated to approximately 0.8 mL using a 5000 MWCO ultrafiltration membrane. The concentrate was loaded onto a gel filtration column, Superdex 200 10 / 300 GL (GE Healthcare), equilibrated with 100 mM HEPES buffer (pH 8) containing 50 mM NaCl. The purified 6RL#9 antibody Fab fragment for crystallization was eluted from the column using the same buffer. All of the above column procedures were performed at a low temperature of 6 to 7.5°C.

[0314] (8-4) Crystallization of the Fab fragment of 6RL#9 antibody in the presence of Ca Seed crystals of the 6RL#9 Fab fragment were obtained using standard conditions. The purified 6RL#9 Fab fragment, to which CaCl2 had been added to make a 5 mM concentration, was then concentrated to 12 mg / mL using a 5000 MWCO ultrafiltration membrane. The concentrated sample was then crystallized by the hanging drop vapor diffusion method. A 100 mM HEPES buffer solution (pH 7.5) containing 20-29% PEG4000 was used as the reservoir solution. Crystallization drops were prepared by adding 0.2 μl of a 100-10,000-fold dilution series of the seed crystals, crushed in 100 mM HEPES buffer (pH 7.5) containing 29% PEG4000 and 5 mM CaCl2, to a mixture of 0.8 μl of the reservoir solution and 0.8 μl of the concentrated sample on a cover glass. The crystallized drops were allowed to stand at 20° C. for 2 to 3 days, and the X-ray diffraction data of the resulting thin plate-like crystals was measured.

[0315] (8-5) Crystallization of the Fab fragment of 6RL#9 antibody in the absence of Ca Purified 6RL#9 antibody Fab fragments were concentrated to 15 mg / ml using a 5000 MWCO ultrafiltration membrane. The concentrated sample was then crystallized by the hanging drop vapor diffusion method. A 100 mM HEPES buffer solution (pH 7.5) containing 18-25% PEG4000 was used as the reservoir solution. Crystallization drops were prepared by adding 0.2 μl of a 100-10,000-fold dilution series of 6RL#9 antibody Fab fragment crystals obtained in the presence of Ca in 100 mM HEPES buffer solution (pH 7.5) containing 25% PEG4000 to a mixture of 0.8 μl of the reservoir solution and 0.8 μl of the concentrated sample on a cover glass. The crystallization drops were left at 20°C for 2 to 3 days, and X-ray diffraction data of the resulting thin, plate-like crystals were measured.

[0316] Measurement of X-ray diffraction data of crystals of the Fab fragment of (8-6)6RL#9 antibody in the presence of Ca A single crystal of the 6RL#9 antibody Fab fragment, grown in the presence of Ca, was immersed in a solution of 35% PEG4000 and 5 mM CaCl2 in 100 mM HEPES buffer (pH 7.5) and frozen in liquid nitrogen by scooping it up along with the external solution using a pin with a small nylon loop. X-ray diffraction data were measured on the frozen crystal at beamline BL-17A of the Photon Factory, a synchrotron radiation facility of the High Energy Accelerator Research Organization (KEK). The frozen state was maintained by placing the crystal in a nitrogen stream at -178°C throughout the measurement. A total of 180 diffraction images were collected by rotating the crystal 1° at a time using a CCD detector Quantum315r (ADSC) installed on the beamline. Lattice constant determination, diffraction spot indexing, and diffraction data processing were performed using the programs Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensity data was obtained up to a resolution of 2.2 Å. The crystal belongs to the space group P212121 with lattice constants a = 45.47 Å, b = 79.86 Å, c = 116.25 Å, α = 90°, β = 90°, γ = 90°.

[0317] (8-7) Measurement of X-ray diffraction data of crystals of the Fab fragment of 6RL#9 antibody in the absence of Ca A single crystal of the 6RL#9 antibody Fab fragment, grown in the absence of Ca, was immersed in 100 mM HEPES buffer (pH 7.5) containing 35% PEG4000 and frozen in liquid nitrogen by scooping it up along with the external solution using a pin with a small nylon loop. X-ray diffraction data were measured on the frozen crystal at beamline BL-5A of the Photon Factory, a synchrotron radiation facility of the High Energy Accelerator Research Organization (KEK). The frozen state was maintained by placing the frozen crystal in a nitrogen stream at -178°C throughout the measurements. A total of 180 diffraction images were collected by rotating the crystal 1° at a time using a CCD detector, Quantum210r (ADSC), installed on the beamline. Lattice constant determination, diffraction spot indexing, and diffraction data processing were performed using the programs Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensity data were obtained at a resolution of 2.3 Å. The crystals belonged to the space group P212121 with lattice constants a = 45.40 Å, b = 79.63 Å, c = 116.07 Å, α = 90°, β = 90°, and γ = 90°, and were isomorphic to the crystals in the presence of Ca.

[0318] Crystal structure analysis of the Fab fragment of (8-8)6RL#9 antibody in the presence of Ca The crystal structure of the 6RL#9 antibody Fab fragment in the presence of Ca was determined using molecular replacement with the program Phaser (CCP4 Software Suite). Based on the size of the resulting crystal lattice and the molecular weight of the 6RL#9 antibody Fab fragment, the number of molecules in the asymmetric unit was predicted to be one. Based on primary sequence homology, amino acid residues 112-220 of the A chain and 116-218 of the B chain were extracted from the structure coordinates of PDB code: 1ZA6 and used as model molecules for the CL and CH1 regions. Next, amino acid residues 1-115 of the B chain were extracted from the structure coordinates of PDB code: 1ZA6 and used as model molecules for the VH region. Finally, amino acid residues 3-147 of the light chain were extracted from the structure coordinates of PDB code: 2A9M and used as model molecules for the VL region. Following this procedure, the orientation and position of each model molecule within the crystal lattice were determined using rotation and translation functions, resulting in an initial structural model of the 6RL#9 antibody Fab fragment. Rigid-body refinement of the initial structure model, involving the movement of the VH, VL, CH1, and CL domains, resulted in a crystallographic confidence factor R value of 46.9% and a free R value of 48.6% for the reflection data between 25 and 3.0 Å. Further refinement was performed using the program Refmac5 (CCP4 Software Suite) and repeated model modification with the program Coot (Paul Emsley) by referring to the electron density map calculated using the experimentally determined structure factor Fo and the structure factor Fc and phase calculated from the model, with coefficients 2Fo-Fc and Fo-Fc. Finally, calcium ions and water molecules were incorporated into the model based on the electron density map with coefficients 2Fo-Fc and Fo-Fc, and refinement was performed using the program Refmac5 (CCP4 Software Suite). By using 21,020 reflection data at 25-2.2 Å resolution, the final crystallographic reliability factor R value for the 3,440-atom model was 20.0% and the free R value was 27.9%.

[0319] X-ray diffraction data of the crystals of the Fab fragment of (8-9)6RL#9 antibody in the absence of Ca The crystal structure of the 6RL#9 antibody Fab fragment in the absence of calcium was determined using the structure of the isomorphic crystal in the presence of calcium. Water and calcium ions were removed from the structural coordinates of the 6RL#9 antibody Fab fragment in the presence of calcium, and rigid-body refinement was performed by moving the VH, VL, CH1, and CL domains. The crystallographic reliability factor R value for the reflection data from 25-3.0 Å was 30.3%, and the free R value was 31.7%. Further refinement of the model was performed using the program Refmac5 (CCP4 Software Suite). Furthermore, the model was refined by repeated model corrections in the program Coot (Paul Emsley) with reference to the electron density map calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated from the model, and the phases, with coefficients 2Fo-Fc and Fo-Fc. Finally, the model was refined using the program Refmac5 (CCP4 Software Suite) by incorporating water molecules into the model based on the electron density map with coefficients 2Fo-Fc and Fo-Fc. Using 18,357 reflection data at 25-2.3 Å resolution, the final crystallographic reliability factor R value for the 3,351-atom model was 20.9% and the free R value was 27.7%.

[0320] Comparison of X-ray diffraction data from crystals of the Fab fragment of (8-10)6RL#9 antibody in the presence and absence of Ca Comparing the crystal structures of the 6RL#9 antibody Fab fragment in the presence and absence of Ca revealed significant changes in the heavy chain CDR3. The structure of the heavy chain CDR3 of the 6RL#9 antibody Fab fragment determined by X-ray crystal structure analysis is shown in Figure 14. Specifically, in the 6RL#9 antibody Fab fragment crystal in the presence of Ca, a calcium ion was present in the center of the heavy chain CDR3 loop. The calcium ion appeared to interact with positions 95, 96, and 100a (Kabat numbering) of the heavy chain CDR3. In the presence of Ca, the heavy chain CDR3 loop, which is important for antigen binding, is stabilized by calcium binding, presumably achieving an optimal structure for antigen binding. Calcium binding to the heavy chain CDR3 of an antibody has not been reported previously, and the structure of calcium-bound antibody heavy chain CDR3 is novel. Heavy chain CDR3 is known to be the most important region for antigen binding, and the motif discovered in this example, which requires calcium ions to maintain the structure of heavy chain CDR3, is thought to suggest that calcium ions play an important role in antigen binding. In other words, it is highly likely that antibodies having this motif will bind to antigens in a calcium ion-dependent manner. For example, if a synthetic library containing this motif could be constructed, it would be possible to efficiently obtain calcium-dependent binding antibodies from such a library.

[0321] [Example 9] Isolation of antibodies that bind to IL-6 in a Ca-dependent manner from a human antibody library using phage display technology (9-1) Construction of a naive human antibody phage display library A human antibody phage display library consisting of multiple phages displaying Fab domains of different human antibody sequences was constructed according to methods known to those skilled in the art using poly(A) RNA prepared from human PBMCs or commercially available human poly(A) RNA as a template.

[0322] (9-2) Isolation of Ca-dependent antigen-binding antibody fragments from a library by bead panning The initial selection from the constructed naive human antibody phage display library was carried out by enriching only antibody fragments capable of binding to the antigen (IL-6), using biotin-labeled IL-6 as the antigen.

[0323] Phages were produced from E. coli harboring the constructed phage display phagemid. The culture medium of the E. coli cells in which phage production was performed was supplemented with 2.5 M NaCl / 10% PEG to precipitate the phage population. The resulting phage library solution was then diluted with TBS. Next, BSA and CaCl2 were added to the phage library solution to a final concentration of 4% BSA and 1.2 mM calcium ion. Panning was performed using a commonly used method using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; Mol. Cell Proteomics (2003) 2 (2), 61-9). NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

[0324] Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was allowed to contact the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added, and the antigen-phage complexes were allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with 1.2 mM CaCl2 / TBST (TBST containing 1.2 mM CaCl2) and then washed twice more with 1 mL of 1.2 mM CaCl2 / TBS (TBS containing 1.2 mM CaCl2). 0.5 mL of 1 mg / mL trypsin was then added, and the beads were suspended at room temperature for 15 minutes. The beads were then immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.5). The E. coli cells were cultured at 37°C for 1 hour with gentle agitation to infect the phages. The infected E. coli was inoculated onto a 225 mm x 225 mm plate, and then phages were collected from the culture medium of the inoculated E. coli to prepare a phage library solution.

[0325] In subsequent panning rounds, phage enrichment was performed based on Ca-dependent binding. Specifically, 40 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library was exposed to the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added, and the antigen-phage complexes were allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed with 1 mL of 1.2 mM CaCl2 / TBST and 1.2 mM CaCl2 / TBS. 0.1 mL of 2 mM EDTA / TBS was then added, and the beads were suspended at room temperature. The beads were then immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was then added with 5 μL of 100 mg / mL trypsin to cleave the pIII protein (pIII protein derived from the helper phage) of the non-Fab-displaying phages, thereby eliminating their ability to infect E. coli. The phages recovered from the trypsinized phage solution were added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.7). The E. coli were cultured at 37°C for 1 hour with gentle agitation to infect the phages. The infected E. coli were plated onto a 225 mm x 225 mm plate. Next, the phages were recovered from the plated E. coli culture, yielding a phage library solution. Panning using Ca-dependent binding activity as an indicator was repeated three times.

[0326] (9-3) Evaluation by phage ELISA Phage-containing culture supernatant was recovered from single E. coli colonies obtained by the above method according to a standard method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant, to which BSA and CaCl2 were added to achieve a final concentration of 4% BSA and 1.2 mM calcium ion concentration, was subjected to ELISA as follows: StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of PBS containing biotin-labeled antigen. After washing each well of the plate with PBST to remove the antigen, the wells were blocked with 250 μL of 4% BSA-TBS for at least 1 hour. After removing the 4% BSA-TBS, the prepared culture supernatant was added to each well, and the plate was left to stand at 37°C for 1 hour, allowing the phage-displaying antibodies to bind to the antigen present in each well. After washing with 1.2 mM CaCl2 / TBST, 1.2 mM CaCl2 / TBS or 1 mM EDTA / TBS was added to each well, and the plate was incubated at 37°C for 30 minutes. After washing with 1.2 mM CaCl2 / TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS to a final concentration of 4% BSA and 1.2 mM ionized calcium was added to each well, and the plate was incubated for 1 hour. After washing with 1.2 mM CaCl2 / TBST, TMB single solution (ZYMED) was added to each well, and the color reaction was stopped by adding sulfuric acid, and the color development was measured by absorbance at 450 nm.

[0327] Phage ELISA was performed using the 96 isolated clones to identify the 6KC4-1#85, 6LC4-1#15, and 6LC4-2#16 antibodies, which possess Ca-dependent binding to IL-6. Based on the results of the phage ELISA, antibody fragments determined to have Ca-dependent antigen binding were used as templates to amplify the genes using specific primers, and the resulting amplified genes were sequenced. The sequences of the heavy chain variable region and light chain variable region of the 6KC4-1#85 antibody are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively. A polynucleotide encoding the heavy chain variable region of the 6KC4-1#85 antibody (SEQ ID NO: 25) was linked by PCR to a polynucleotide encoding an IgG1-derived sequence (SEQ ID NO: 65), resulting in a DNA fragment. This DNA fragment was then inserted into an animal cell expression vector, and a vector expressing the heavy chain represented by SEQ ID NO: 27 was constructed. A polynucleotide encoding the light chain variable region of the 6KC4-1#85 antibody (SEQ ID NO: 26) was linked by PCR to a polynucleotide encoding the natural Kappa chain constant region (SEQ ID NO: 28), resulting in a DNA fragment encoding the sequence shown in SEQ ID NO: 29. This fragment was then incorporated into an animal cell expression vector. Using a similar method, the 6LC4-1#15 antibody (heavy chain SEQ ID NO: 68, light chain SEQ ID NO: 69) and the 6LC4-2#16 antibody (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) were incorporated into cell expression vectors. The sequences of the constructed variants were confirmed using methods known to those skilled in the art.

[0328] (9-4) Antibody expression and purification Clones determined to have Ca-dependent antigen binding ability by phage ELISA were introduced into an animal cell expression plasmid. Antibody expression was carried out using the following method. Human fetal kidney cell-derived FreeStyle 293-F strain (Invitrogen) was suspended in FreeStyle 293 Expression Medium (Invitrogen) and incubated at 1.33 x 10 6The cells were seeded at a density of 3000 cells / mL in each well of a 6-well plate. The prepared plasmid was introduced into the cells by lipofection. The cells were cultured for 4 days in a CO2 incubator (37°C, 8% CO2, 90 rpm). rProtein A Sepharose TM Antibodies were purified from the culture supernatants obtained above using Fast Flow (Amersham Biosciences) by methods known to those skilled in the art. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the measured values ​​using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0329] (9-5) Evaluation of calcium-dependent binding of anti-IL6 antibodies The obtained antibodies were analyzed for human interleukin-6 (hIL6) binding activity (dissociation constant K D Measurements were performed using a running buffer containing 0.05% Tween 20, 10 mmol / L ACES, and 150 mmol / L NaCl (pH 7.4) containing 3 μM or 1.2 mM CaCl.

[0330] An appropriate amount of recombinant Protein A / G (Thermo Scientific) was immobilized on a sensor chip CM5 (GE Healthcare) by the amino coupling method, and then the antibody was bound to it. An appropriate concentration of hIL6 (human interleukin 6, Kamakura Techno Science) was injected as an analyte to allow it to interact with the antibody on the sensor chip. 10 mmol / L Glycine-HCl (pH 1.5) was then injected to regenerate the sensor chip. Measurements were performed at 37°C. The resulting sensorgram is shown in Figure 15. These results indicate that the 6LC4-1#15-IgG1, 6LC4-2#16-IgG1, and 6KC4-1#85-IgG1 antibodies are Ca 2+ At a concentration of 3 μM, Ca2+ The binding to hIL-6 was clearly weaker than that at a concentration of 1.2 mM. The above results show that calcium-dependent antigen binding is observed not only for IL-6R as shown in Example 3 but also for IL-6, indicating that the present invention may be applicable to other antigens.

[0331] [Example 10] Evaluation of calcium ion binding of 6KC4-1#85 antibody (10-1) Evaluation of calcium ion binding of 6KC4-1#85 antibody The calcium-dependent antigen-binding antibody 6KC4-1#85 isolated from a human antibody library was evaluated for calcium binding. The Tm values ​​measured under different ionized calcium concentrations were evaluated using the method described in Example 4.

[0332] The Tm value of the Fab domain of the 6KC4-1#85 antibody is shown in Table 11. As shown in Table 11, the Tm value of the Fab domain of the 6KC4-1#85 antibody varied depending on the calcium ion concentration, demonstrating that the 6KC4-1#85 antibody binds to calcium.

[0333] [Table 11]

[0334] (10-2) Identification of the calcium ion binding site of 6KC4-1#85 antibody Although the 6KC4-1#85 antibody was shown to bind to calcium ions in Example 10 (10-1), 6KC4-1#85 does not have a calcium-binding motif like the hVk5-2 sequence described below. Therefore, to identify which residues in the 6KC4-1#85 antibody bind to calcium ions, modified heavy chains (6_H1-11 (SEQ ID NO: 30), 6_H1-12 (SEQ ID NO: 31), 6_H1-13 (SEQ ID NO: 32), 6_H1-14 (SEQ ID NO: 33), and 6_H1-15 (SEQ ID NO: 34)) and modified light chains (6_L1-5 (SEQ ID NO: 35) and 6_L1-6 (SEQ ID NO: 36)) were constructed in which the Asp (D) residues in the CDRs of the 6KC4-1#85 antibody were replaced with Ala (A) residues, which are not involved in calcium ion binding or chelating. The modified antibodies were purified from the culture medium of animal cells into which expression vectors containing the modified antibody genes had been introduced, according to the method described in Example 2. The calcium binding of the purified modified antibodies was measured according to the method described in Example 4. The measurement results are s...

Claims

1. A method for producing an antibody having at least one function selected from (i) the function of promoting intracellular uptake of an antigen, (ii) the function of binding to an antigen two or more times, (iii) the function of promoting a decrease in the antigen concentration in plasma, and (iv) the function of excellent plasma retention, the method comprising the following steps (a) and (b): (a) an antibody comprising an antigen-binding domain and a human FcRn-binding domain, which has different antigen-binding activities under two different calcium concentration conditions, and whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition; preparing a gene encoding an antibody comprising a light chain variable region in which the antigen-binding domain of the light chain contains a calcium-binding motif, at least two of the amino acids at positions 30, 31, 32, and 50, as determined by the Kabat numbering system, are metal-chelating amino acids selected from serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, and tyrosine, and the amino acid at position 92 is serine; (b) A step of introducing the gene prepared in step (a) into a host to express the antibody.

2. 2. The method of claim 1, wherein the low calcium concentration is an ionized calcium concentration of 0.1 μM to 30 μM.

3. 2. The method of claim 1, wherein the high calcium concentration is an ionized calcium concentration of 100 μM to 10 mM.

4. 2. The method of claim 1, wherein the low calcium concentration is an ionized calcium concentration within an endosome.

5. 2. The method of claim 1, wherein the elevated calcium concentration is ionized calcium concentration in plasma.

6. The method according to any one of claims 1 to 5, wherein the antigen to which the antibody binds is a soluble receptor or a soluble antigen.

7. The method of claim 1, comprising an FcRn-binding domain having binding activity to FcRn under a neutral pH condition.

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