Antigen-binding molecule that promotes clearance of antigens containing carbohydrate receptor-binding domains from plasma

Abstract

To provide an antigen-binding molecule in which antigen uptake into cells is promoted; to provide an antigen-binding molecule capable of promoting a decrease in antigen concentration in plasma; to provide an antigen-binding molecule capable of binding an antigen multiple times; to provide an antigen-binding molecule with improved pharmacokinetics; to provide a pharmaceutical composition comprising the antigen-binding molecule; and to provide methods for producing the same.SOLUTION: Disclosed is an antigen-binding molecule containing a sugar chain receptor binding domain and having a weaker antigen-binding activity at pH in early endosomes compared to the antigen-binding activity at pH in plasma; as well as a pharmaceutical composition comprising the antigen-binding molecule; and methods for producing the same. Using the antigen-binding molecule of the present invention can promote antigen uptake into cells while increasing the number of antigens that can be bound by one antibody molecule, and administering the antibody can prolong the pharmacokinetics of the antibody while promoting the reduction of the antibody in plasma.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority from Japanese Patent Application No. 2011-221400 (filed October 5, 2011). No. 60 / 699,999, filed on Dec. 1, 2003, the contents of which are incorporated herein by reference.

[0002] Technical Field The present invention relates to antigen-binding molecules that promote intracellular uptake of antigens, antigen-binding molecules that can promote a decrease in plasma antigen concentration, antigen-binding molecules that can bind to antigens multiple times, antigen-binding molecules with improved pharmacokinetics, pharmaceutical compositions containing the antigen-binding molecules, and methods for producing them. [Background technology]

[0003] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among them, many IgG-type antibody drugs have been commercialized, and many antibody drugs are currently being developed. (Non-Patent Documents 1 and 2). Meanwhile, various technologies applicable to second-generation antibody drugs have been developed, and techniques for improving effector function, antigen-binding ability, pharmacokinetics, and stability, or reducing the risk of immunogenicity, have been reported (Non-Patent Document 3). Antibody drugs generally require very high doses, which can be considered to pose challenges such as the difficulty of preparing subcutaneous formulations and the high manufacturing costs. Possible methods for reducing the dose of antibody drugs include methods that improve the pharmacokinetics of antibodies and methods that improve the affinity between antibodies and antigens.

[0004] Artificial amino acid substitution in the constant region has been reported as a method for improving the pharmacokinetics of antibodies (Non-Patent Documents 4 and 5). Affinity maturation technology (Non-Patent Document 6) has been reported as a technique for enhancing antigen binding ability and antigen neutralization ability, and the CDR region of the variable region has been reported as a method for enhancing the antigen binding ability and antigen neutralization ability. It is possible to enhance the binding activity to antigens by introducing mutations into amino acids such as . Enhancement of antigen binding ability can improve in vitro biological activity or reduce the dosage. It is possible to improve the efficacy of the compound in vivo (Non-Patent Document 7).

[0005] 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 molecule of antigen (two antigens in the case of a bivalent antibody). However, previous methods were limited to a stoichiometric neutralization reaction of one molecule of antigen (two antigens in the case of a bivalent antibody) with one antibody molecule, making it impossible to completely neutralize an antigen with an antibody amount less than the amount of antigen. 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 described above. Therefore, in order to maintain the neutralizing effect of an antigen for the desired period with an antibody amount less than the antigen amount, one antibody must produce multiple It is necessary to neutralize the antigen. Recently, an antibody that binds to the antigen in a pH-dependent manner has been reported as a new method for achieving this (Patent Document 1). A pH-dependent antigen-binding antibody that binds strongly to the antigen under the neutral conditions in plasma and dissociates from the antigen under the acidic conditions in the endosome is capable of dissociating from the antigen in the endosome. pH-dependent antigen-binding antibody After dissociating from the antigen, the antibody is recycled into the plasma by FcRn and can bind to the antigen again, so a single antibody can repeatedly bind to multiple antigens. do.

[0006] Furthermore, the plasma retention time of antigens is extremely short compared to antibodies that bind to FcRn and are recycled. When such an antibody with long plasma retention time binds to the antigen, the plasma retention time of the antibody-antigen complex becomes as long as that of the antibody. Therefore, by binding to the antibody, the antigen actually retains its plasma retention time longer, 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).

[0007] 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, no antibody engineering techniques have been reported to date that further improve the effects of pH-dependent antigen-binding antibodies, such as the ability to repeatedly bind to antigens and the ability to accelerate antigen elimination from plasma.

[0008] On the other hand, glycoproteins present in plasma bind to their glycan-specific receptors and are eliminated from the plasma (Non-Patent Document 10). At this time, the glycoprotein binds to a glycan receptor present on the cell surface and is taken up into the cell. After dissociating from the glycan receptor within the cell, it is degraded by lysosomes, but the glycan receptor is recycled back to the cell surface. Specifically, this glycoprotein and the glycan receptor have a pH-dependent binding ability, where they bind strongly under the neutral conditions of plasma and dissociate under the acidic conditions of endosomes (Non-Patent Documents 11 and 12). Such pH-dependent binding between glycoproteins and glycan receptors is related to the glycans attached to the glycoprotein. An example of such a glycan and glycan receptor is an N-linked glycan with a terminal galacrose and an asialoglycoprotein. Examples include a sugar chain having mannose at its terminal and a mannose receptor (Non-Patent Document 11), and a sugar chain having mannose at its terminal and a mannose receptor (Non-Patent Document 12).

[0009] Due to the properties of such N-linked glycans, the addition of functionally unnecessary N-linked glycans to biopharmaceuticals, including antibodies, is considered undesirable from the viewpoint of their efficacy or maintaining plasma retention (Non-Patent Documents 13 and 14), and there have been no examples of their use with the aim of further improving the effect of promoting antigen elimination from plasma.

[0010] The references cited herein are as follows: The contents of these references are incorporated herein by reference in their entirety. No admission is made that any of these references is prior art to this specification. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2009 / 125825 [Non-patent literature]

[0012] [Non-Patent Document 1] Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology (2005) 23, 1073 - 1078 [Non-patent document 2] Pavlou AK, Belsey MJ., Eur J Pharm Biopharm. (2005) 59 (3), 389-96 [Non-patent document 3] Kim SJ, Park Y, Hong HJ., Mol Cells. (2005) 20 (1), 17-29 [Non-patent document 4] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., J Immunol. (2006) 176 (1), 346-56 [Non-patent document 5] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Nat Biotechnol. (1997) 15 (7), 637-40

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patent document 9

Non-patent document 10

Non-patent document 11

Non-patent document 12

Non-patent document 13

[0013] The present inventors have conducted extensive research into methods for promoting the intracellular uptake of antigens by antigen-binding molecules with antigen-binding activity, methods for antigens binding multiple times, methods for promoting a decrease in plasma antigen concentration by administering antigen-binding molecules, and methods for improving the plasma retention of antigen-binding molecules. As a result, the present inventors have found that an antigen-binding molecule that has an antigen-binding domain, an FcRn-binding domain (particularly human FcRn) and a glycosyl receptor-binding domain, and that exhibits weaker binding ability to the glycosyl receptor under endosomal ion concentration conditions than under plasma ion concentration conditions, can promote the intracellular uptake of antigens; and that an antigen-binding molecule having an antigen-binding domain whose antigen-binding activity is weaker under early endosome ion concentration conditions than under plasma ion concentration conditions can further promote the intracellular uptake of antigens by the antigen-binding molecule, can increase the number of antigens that can be bound by a single antigen-binding molecule, can promote a decrease in plasma antigen concentration by administering an antigen-binding molecule, and can improve the pharmacokinetics of the antigen-binding molecule.

[0014] Specifically, the present invention relates to methods for promoting intracellular antigen uptake by antigen-binding molecules, methods for increasing the number of antigens that can be bound by a single antigen-binding molecule, methods for promoting a decrease in plasma antigen concentration by administering the same, methods for improving the pharmacokinetics of antigen-binding molecules, antigen-binding molecules with promoted intracellular antigen uptake, antigen-binding molecules with an increased number of antigens that can be bound, antigen-binding molecules whose administration can promote a decrease in plasma antigen concentration, antigen-binding molecules with improved pharmacokinetics, pharmaceutical compositions containing the antigen-binding molecules, and methods for producing them. More specifically, the present invention relates to: [1] A method for producing an antigen-binding molecule, comprising the steps of: (a) The polypeptide sequence of an antigen-binding molecule containing an antigen-binding domain and an FcRn-binding domain is provided. Supply process (b) A candidate motif for the carbohydrate receptor-binding domain in the polypeptide sequence Identifying the amino acid sequence (c) designing a motif for a carbohydrate receptor-binding domain comprising an amino acid sequence that differs from the amino acid sequence identified in (b) by at least one amino acid. (d) preparing a gene encoding a polypeptide of an antigen-binding molecule containing the motif of the sugar chain receptor-binding domain designed in (c); (e) recovering the antigen-binding molecule from the culture medium of the host cells transformed with the gene obtained in (d). [2] A method comprising further treating the antigen-binding molecules obtained in (e) with an enzyme. a method for producing [3] The production method according to [1] or [2], wherein the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions. [4] The production method according to [3], wherein the ion concentration conditions are pH conditions. [5] The production method according to [4], wherein the antigen-binding domain has higher antigen-binding activity under a neutral pH range than under an acidic pH range. [6] The method of [5], comprising substituting at least one amino acid in the antigen-binding domain with an amino acid having a side chain pKa of 4.0-8.0, or inserting at least one amino acid having a side chain pKa of 4.0-8.0 into the antigen-binding domain, thereby providing an antigen-binding domain whose antigen-binding activity in a neutral pH range is higher than that in an acidic pH range. [7] The manufacturing method according to [3], wherein the ion concentration condition is a calcium ion concentration condition. [8] The production method according to [7], wherein the antigen-binding domain has a higher antigen-binding activity under a high calcium ion concentration than under a low calcium ion concentration. [9] The method of [8], comprising substituting at least one amino acid in the antigen-binding domain with a calcium-binding motif or inserting a calcium-binding motif into the antigen-binding domain, thereby providing an antigen-binding domain whose antigen-binding activity is higher under a high calcium ion concentration than under a low calcium ion concentration.

[10] The method according to any one of [1] to [9], wherein the antigen-binding domain comprises an antibody variable region.

[11] The method according to any one of [1] to

[10] , wherein the FcRn-binding domain comprises an antibody Fc region.

[12] The method according to

[11] , wherein the antibody is an IgG antibody.

[13] The method according to

[12] , wherein the IgG antibody is any one of IgG1, IgG2, IgG3, and IgG4. method,

[14] The method according to any one of [1] to

[12] , wherein the binding activity of the sugar chain receptor-binding domain to a sugar chain receptor changes depending on ion concentration conditions.

[15] The method according to

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

[16] The method according to any one of [1] to

[15] , wherein the binding activity of the glycan receptor-binding domain to the glycan receptor under a neutral pH range is higher than that under an acidic pH range.

[17] The method according to

[14] , wherein the ion concentration condition is a calcium ion concentration condition.

[18] The method according to

[17] , wherein the binding activity of the glycan receptor-binding domain to the glycan receptor under a high calcium ion concentration condition is higher than that under a low calcium ion concentration condition.

[19] The method according to any one of [1] to

[18] , wherein the sugar chain receptor binding domain is a sugar chain.

[20] The method according to

[19] , wherein the sugar chain is an O-linked sugar chain.

[21] The method according to

[19] , wherein the sugar chain is an N-linked sugar chain.

[22] Designing the motif of the sugar chain receptor binding domain allows N-linked sugar chains to be attached. The method according to

[21] , comprising designing a motif that

[23] The N-linked sugar chain according to

[21] or

[22] , wherein the terminal of the N-linked sugar chain contains galactose. method,

[24] The method according to

[23] , wherein the N-linked sugar chain has three or more galactose ends. ,

[25] The method according to any one of

[21] to

[24] , wherein the sugar chain receptor is an asialoglycoprotein receptor.

[26] The method according to

[21] or

[22] , wherein the terminal of the N-linked sugar chain contains mannose. law,

[27] The method according to

[26] , wherein the sugar chain receptor is a mannose receptor.

[28] An antigen-binding molecule produced by the method according to any one of [1] to

[27] .

[29] An antigen-binding molecule comprising an FcRn-binding domain, an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, and one or more carbohydrate receptor-binding domains whose carbohydrate receptor-binding activity changes depending on ion concentration conditions.

[30] The antigen-binding molecule of

[29] , wherein the antigen-binding activity of the antigen-binding domain changes depending on pH conditions.

[31] The antigen-binding molecule of

[30] , wherein the antigen-binding activity of the antigen-binding domain is higher under a neutral pH condition than under an acidic pH condition.

[32] The antigen-binding molecule of

[31] , wherein at least one amino acid in the antigen-binding domain contains at least one amino acid with a side chain pKa of 4.0 to 8.0.

[33] The antigen-binding molecule of

[29] , wherein the antigen-binding activity of the antigen-binding domain changes depending on calcium ion concentration.

[34] The antigen-binding molecule of

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

[35] The antigen-binding molecule of

[34] , wherein at least one amino acid in the antigen-binding domain comprises a calcium-binding motif.

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

[29] to

[35] , wherein the antigen-binding domain comprises an antibody variable region.

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

[29] to

[36] , wherein the FcRn-binding domain comprises an antibody Fc region.

[38] The antigen-binding molecule of

[37] , wherein the antibody is an IgG antibody.

[39] The method according to

[38] , wherein the IgG antibody is any one of IgG1, IgG2, IgG3, and IgG4. antigen-binding molecules,

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

[29] to

[39] , wherein the binding activity of the sugar chain receptor-binding domain to a sugar chain receptor changes depending on ion concentration conditions.

[41] The antigen-binding molecule of

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

[42] The antigen-binding molecule of

[41] , wherein the sugar chain receptor-binding domain has a higher binding activity to the sugar chain receptor under a neutral pH range than under an acidic pH range.

[43] The antigen-binding molecule of

[40] , wherein the ion concentration condition is a calcium ion concentration condition.

[44] The antigen-binding molecule of

[43] , wherein the sugar chain receptor-binding domain has a higher binding activity to the sugar chain receptor under a high calcium ion concentration condition than under a low calcium ion concentration condition.

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

[29] to

[44] , wherein the sugar chain receptor-binding domain is a sugar chain.

[46] The antigen-binding molecule of

[45] , wherein the sugar chain is an O-linked sugar chain or an N-linked sugar chain.

[47] The sugar chain receptor-binding domain contains a motif for N-linked sugar chain binding. 6. The antigen-binding molecule according to

[48] ​​The method according to

[46] or

[47] , wherein the terminal of the N-linked sugar chain contains galactose. antigen-binding molecules,

[49] The antigen-binding molecule of

[48] , wherein the N-linked sugar chain has three or more terminal galactoses.

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

[47] to

[49] , wherein the sugar chain receptor is an asialoglycoprotein receptor.

[51] The method according to

[46] or

[47] , wherein the terminal of the N-linked sugar chain contains mannose. antigen-binding molecules,

[52] The antigen-binding molecule of

[46] or

[47] , wherein the sugar chain receptor is a mannose receptor.

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

[28] to

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

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

[28] to

[53] , wherein the sugar chain receptor-binding domain is contained in the antigen-binding domain.

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

[28] to

[53] , wherein the sugar chain receptor-binding domain is contained in an FcRn-binding domain.

[56] A pharmaceutical composition comprising the antigen-binding molecule of any one of

[28] to

[55] .

[57] A method for intracellular uptake of an antigen-binding molecule of a cell expressing a sugar chain receptor, comprising contacting the antigen-binding molecule of any one of

[28] to

[55] with the cell in vivo or ex vivo.

[58] A method for intracellular uptake of an antigen bound to an antigen-binding molecule, comprising contacting the antigen-binding molecule of any one of

[28] to

[55] with a cell expressing a sugar chain receptor in vivo or ex vivo.

[59] A method for increasing the number of antigens bound by a single antigen-binding molecule, comprising contacting the antigen-binding molecule of any one of

[28] to

[55] with a cell expressing a sugar chain receptor in vivo or ex vivo.

[60] A method for reducing extracellular antigens, comprising contacting the antigen-binding molecule of any one of

[28] to

[55] with a cell expressing a sugar chain receptor in vivo or ex vivo.

[61] The method according to

[58] , wherein the extracellular space is plasma.

[62] A method for improving the pharmacokinetics of an antigen-binding molecule, comprising contacting the antigen-binding molecule of any of

[28] to

[55] with a cell expressing a sugar chain receptor in vivo.

[63] A method for promoting dissociation of an antigen bound to the antigen-binding molecule outside of a cell from the antigen-binding molecule, the method comprising contacting the antigen-binding molecule of any one of

[28] to

[55] with a cell expressing a sugar chain receptor in vivo or in vitro.

[64] A method selected from any of the following, comprising increasing the number of binding domains for a carbohydrate receptor in an antigen-binding molecule that contains an antigen-binding domain, an FcRn-binding domain, and one or more binding domains for the carbohydrate receptor: (i) Intracellular antigen binding of cells expressing the sugar chain receptor in vivo or ex vivo Methods for promoting uptake of molecules (ii) A method for promoting the incorporation of an antigen bound to an antigen-binding molecule into cells expressing the sugar chain receptor in vivo or ex vivo. (iii) Increasing the number of antigens bound by a single antigen-binding molecule in vivo or in vitro How to add (iv) a method for increasing the in vivo or in vitro antigen elimination ability of an antigen-binding molecule; (v) a method for improving the pharmacokinetics of an antigen-binding molecule; or (vi) A method for promoting the dissociation of an extracellularly bound antigen from an antigen-binding molecule.

[65] The method according to

[64] , wherein the antigen-binding activity of the antigen-binding molecule varies depending on ion concentration conditions.

[66] The method according to

[64] , wherein the antigen-binding activity of the antigen-binding domain changes depending on pH conditions.

[67] The method according to

[66] , wherein the antigen-binding activity of the antigen-binding domain is higher under a neutral pH condition than under an acidic pH condition.

[68] The method according to

[67] , wherein at least one amino acid in the antigen-binding domain contains at least one amino acid with a side chain pKa of 4.0 to 8.0.

[69] The method according to

[64] , wherein the antigen-binding activity of the antigen-binding domain changes depending on calcium ion concentration.

[70] The method according to

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

[71] The method of

[70] , wherein at least one amino acid in the antigen-binding domain comprises a calcium-binding motif.

[72] The method of any one of

[64] to

[71] , wherein the antigen-binding domain comprises an antibody variable region.

[73] The method according to any one of

[64] to

[72] , wherein the FcRn-binding domain comprises an Fc region of an antibody.

[74] The method according to

[73] , wherein the antibody is an IgG antibody.

[75] The method according to

[74] , wherein the IgG antibody is any one of IgG1, IgG2, IgG3, and IgG4. method,

[76] The method according to any one of

[64] to

[75] , wherein the binding activity of the sugar chain receptor-binding domain to a sugar chain receptor changes depending on ion concentration conditions.

[77] The method according to

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

[78] The method according to

[76] , wherein the binding activity of the glycan receptor-binding domain to the glycan receptor under a neutral pH range is higher than that under an acidic pH range.

[79] The method according to

[76] , wherein the ion concentration conditions are calcium ion concentration conditions.

[80] The method according to

[79] , wherein the binding activity of the sugar chain receptor-binding domain to the sugar chain receptor under a high calcium ion concentration condition is higher than that under a low calcium ion concentration condition.

[81] The method according to any one of

[64] to

[80] , wherein the sugar chain receptor binding domain is a sugar chain.

[82] The method according to

[81] , wherein the sugar chain is an O-linked sugar chain.

[83] The method according to

[81] , wherein the sugar chain is an N-linked sugar chain.

[84] The sugar chain receptor-binding domain contains a motif for N-linked sugar chain binding. 3).

[85] The method according to

[83] or

[84] , wherein the terminal of the N-linked sugar chain contains galactose. method,

[86] The method according to

[85] , wherein the N-linked sugar chain has three or more terminal galactoses.

[87] The method according to any one of

[84] to

[86] , wherein the sugar chain receptor is an asialoglycoprotein receptor.

[88] The method according to

[83] or

[84] , wherein the terminal of the N-linked sugar chain contains mannose. method,

[89] The method according to

[83] or

[84] , wherein the sugar chain receptor is a mannose receptor.

[90] The method according to any one of

[64] to

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

[91] The method according to any one of

[64] to

[90] , wherein the sugar chain receptor-binding domain is contained in an antigen-binding domain.

[92] The method according to any one of

[64] to

[90] , wherein the sugar chain receptor-binding domain is contained in an FcRn-binding domain in the Fc region. to provide. [Brief explanation of the drawings]

[0015] [Figure 1] This is a schematic diagram showing that IgG antibody molecules dissociate from soluble antigens in endosomes, accelerating the elimination of the antigens and allowing them to rebind to new antigens. [Figure 2] This is a schematic diagram showing that IgG antibody molecules bind to carbohydrate receptors in blood vessels, are taken up into cells, and dissociate from soluble antigens together with carbohydrate receptors in endosomes, accelerating antigen elimination and allowing them to bind to new antigens again. [Figure 3] FIG. 1 shows heavy and light chains detected by reducing SDS-PAGE. [Figure 4] FIG. 1 shows chromatograms of anion exchange chromatography before and after neuraminidase treatment. [Figure 5] FIG. 1 shows a mass chromatogram of N-linked glycans attached to light chains obtained by RP-LC / ESI-MS analysis of reduced GL-M111. [Figure 6] FIG. 1 shows the results of observing neuraminidase activity by anion exchange chromatography. [Figure 7] This figure shows the results of detecting the heavy and light chains by reducing SDS-PAGE of a sample in which the constant region had been restored to IgG1. [Figure 8] FIG. 1 shows time courses of antibody plasma concentrations in normal mice. [Figure 9]FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in normal mice. [Figure 10] FIG. 1 shows time courses of antibody plasma concentrations in normal mice. [Figure 11] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in normal mice. [Figure 12] FIG. 1 shows heavy and light chains detected by reducing SDS-PAGE. [Figure 13] FIG. 10 shows a mass chromatogram of N-linked glycans attached to light chains obtained by RP-LC / ESI-MS analysis of reduced GL5-G1_kif+. [Figure 14] FIG. 1 shows time courses of antibody plasma concentrations in normal mice. [Figure 15] FIG. 1 shows the time course of plasma concentration of soluble human IL-6 receptor in normal mice. [Figure 16] FIG. 10 shows Biacore sensorgrams showing the interaction of H54 / L28-IgG1 with soluble human IL-6 receptor at Ca 2+ 2 mM and Ca 2+ 3 μM. [Figure 17] FIG. 10 shows Biacore sensorgrams showing the interaction of FH4-IgG1 with soluble human IL-6 receptor at Ca 2+ 2 mM and Ca 2+ 3 μM. [Figure 18] FIG. 10 shows Biacore sensorgrams showing the interaction of 6RL#9-IgG1 with soluble human IL-6 receptor at Ca 2+ 2 mM and Ca 2+ 3 μM. [Figure 19] 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 20] 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 21]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 22] 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: 63, and light chain: hVk5-2, a fusion molecule of SEQ ID NOs: 6 and 42), and the dashed line represents the chromatogram of the antibody having the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 63), light chain: hVk5-2_L65 (SEQ ID NO: 62)). DETAILED DESCRIPTION OF THE INVENTION

[0016] The following definitions and detailed description are provided to facilitate understanding of the invention described herein.

[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] and / or As used herein, the term "and / or" means that "and" and "or" are used interchangeably. Specifically, for example, "position 33, position 55, and / or or 96 amino acid substitution" includes the following amino acid modification variations: (a) 33rd place, (b) 55th place, (c) 96th place, (d) 33rd and 55th place, (e) 33rd and 96th place, (f) 55th and 96th place, (g) 33rd, 55th and 96th place.

[0019] 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. Suitable examples of antigen-binding molecules that improve pharmacokinetics using the methods of the present invention include antigen-binding molecules that recognize membrane antigens such as receptor proteins (membrane-bound receptors, soluble receptors) and cell surface markers, and antigen-binding molecules that recognize soluble antigens such as cytokines. Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, and adenosine receptor. Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 Antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM , BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin , bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen 35 (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin Cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2 , CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL 22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR 1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, C D7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25 , CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40 , CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Welch bacillus toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 , CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement control factor (Decay accelerating factor), des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2 , EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin receptor, Enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Follicle-activating protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1) , GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMVUL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, Human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23 , interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin Integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14 , kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP(TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer , METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR , PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP TfR, TGF, TGF-protein, TGF-protein, TGF-protein Pan Specific TGF-link RI(ALK-5), TGF-link RII, TGF-link RIIb, TGF-link RII I, TGF-factor 1, TGF-factor 2, TGF-factor 3, TGF-factor 4, TGF-factor 5, transcription factor Ck-1 Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF- TNF-protease 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2、DR4)、TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAILR3 DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK RFN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFRp75NTR) TNFRSF17(BCMA) TNFRSF18(GITR AITR). )、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF RI CD120a、p55-60)、TNFRSF1B(TNF RII CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL). R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK Gand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B(BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa , TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR-in Tegrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A , WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA , Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa , factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminog en, plasmin, PAI-1, PAI-2、GPC3、Syndecan-1、Syndecan-2、Syndecan-3、Syndecan-4、LPA、S1P、Acetylcholine receptor、AdipoR1、AdipoR2、ADP ribosyl cyclase-1、alpha-4 / beta-7 integrin、alpha-5 / beta-1 integrin、alpha-v / beta-6 integrin、alphavbeta1 integrin、Angiopoietin ligand-2、Angptl2、Anthrax、Cadherin、Carbonic anhydrase-IX、CD105、CD155、CD158a、CD37、CD49b、CD51、CD70、CD72、Claudin 18、Clostridium difficile toxin、CS1、Delta-like protein ligand 4、DHICA oxidase、Dickkopf-1 ligand、Dipeptidyl peptidase IV、EPOR、F protein of RSV、Factor Ia、FasL、Folate receptor alpha、Glucagon receptor、Glucagon-like peptide 1 receptor、Glutamate carboxypeptidase II、GMCSFR、Hepatitis C virus E2 glycoprotein、Hepcidin、IL-17 receptor、IL-22 receptor、IL-23 receptor、IL-3 receptor、Kit tyrosine kinase、Leucine Rich Alpha-2-Glycoprotein 1 (LRG1), Lysosphingolipid receptor, Membrane glycoprotein OX2, Mesothelin, MET, MICA, MUC-16, Myelin associated glycoprotein, Neuropilin-1, Neuropilin-2, Nogo receptor, PLXNA1, PLXNA2, PLXNA3, PLXNA4A, PLXNA4B , PLXNB1, PLXNB2, PLXNB3 , PLXNC1 , PLXND1 , Programmed cell death ligand 1, Proprotein convertase PC9, P-selectin glycoprotein ligand-1, RAGE, Reticulon 4, RF, RON-8 , SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, SEMA7A, Shiga like toxin II, Sphingosine-1-phosphate receptor-1, ST2, Staphylococcal lipoteichoic acid, Tenascin, TG2, Thymic stromal lymphoprotein receptor , TNF superfamily receptor 12A, Transmembrane glycoprotein NMB, TREM-1, TREM-2, Trophoblast glycoprotein, TSH receptor, TTR, Tubulin, ULBP2 and receptors for hormones and growth factors can be mentioned as examples.

[0020] 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.

[0021] 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.

[0022] A conformational epitope, in contrast to a linear epitope, is an epitope 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). A conformational epitope may encompass an increased number of amino acids relative to a linear epitope. In recognizing a conformational epitope, the antibody recognizes 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 moieties form a conformational epitope. The chains are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the structure of a molecule include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific scanning electron microscopy. These include, but are not limited to, pin labeling and electromagnetic paramagnetic resonance spectroscopy. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0023] Binding activity The epitope bound by a test antigen-binding molecule containing an antigen-binding domain for IL-6R is shown below. Examples of methods for confirming binding include those for test compounds containing antigen-binding domains for antigens other than IL-6R. The binding of an antigen-binding molecule to an epitope can also be confirmed by an appropriate method according to the following examples.

[0024] For example, whether a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For the above purpose, a linear epitope consisting of the amino acid sequence constituting the extracellular domain of IL-6R is prepared. The peptide can be synthesized chemically. Alternatively, the cDNA of IL-6R can be synthesized. The antigen-binding molecule is obtained by genetic engineering using a region encoding the amino acid sequence corresponding to the extracellular domain of the IL-6R. Next, the binding activity of a linear peptide consisting of the amino acid sequence constituting the extracellular domain to a test antigen-binding molecule containing an antigen-binding domain for IL-6R is evaluated. For example, the peptide can be detected by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity of the antigen-binding molecule to IL-6R-expressing cells can be evaluated. The binding activity of the antigen-binding molecule to a linear peptide can be determined based on the level of inhibition by the linear peptide of the binding of the antigen-binding molecule to the linear peptide.

[0025] In addition, a test antigen-binding molecule containing an antigen-binding domain for IL-6R is For the above purpose, recognition of IL-6R can be confirmed as follows. A test antigen-binding molecule containing an antigen-binding domain for IL-6R binds strongly to IL-6R-expressing cells upon contact with the cells, while the antigen-binding molecule binds substantially no binding to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R. Here, the term "not substantially binding" refers to the case where the human IL-6R expressing cells The term "binding activity" refers to a binding activity that is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the target antigen.

[0026] Methods for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain for IL-6R to IL-6R-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual. (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420) are examples of such methods. These include ELISA and FACS (fluorescence activated chromatographic assay) using IL-6R-expressing cells as antigens. This can be evaluated by the principle of cell sorting.

[0027] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain to IL-6R-expressing cells was assessed by comparing the signal levels generated by the enzymatic reaction. The binding activity of the test antigen-binding molecule to IL-6R-expressing cells can be quantitatively assessed by adding the test antigen-binding molecule to an ELISA plate on which IL-6R-expressing cells have been immobilized, and detecting the cell-bound test antigen-binding molecule using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody-binding titer to IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule to IL-6R-expressing cells to be compared.

[0028] 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) )

[0029] For example, binding of a test antigen-binding molecule containing an antigen-binding domain to an IL-6R to an antigen A suitable example of a method for measuring the activity is as follows. First, cells expressing IL-6R are The cells are then 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 antigen-binding molecule at a desired concentration, for example, between 10 μg / ml and 10 ng / ml. Then, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody binding to the cells was analyzed using CELL QUEST Software (BD). The fluorescence intensity obtained by the method (i.e., the geometric mean value) is reflected in the fluorescence intensity obtained by the method (i.e., the geometric mean value). In other words, by obtaining the geometric mean value, the binding activity of the test antigen-binding molecule, which is represented by the amount of binding of the test antigen-binding molecule, can be measured.

[0030] A test antigen-binding molecule containing an antigen-binding domain for IL-6R is identified as having an epitope with a certain antigen-binding molecule. Sharing of a common epitope can be confirmed by competition between the two antigen-binding molecules 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.

[0031] Specifically, in the cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is incubated in the presence or absence of a candidate competing antigen-binding molecule. After pre-incubation in the presence of IL-6R, a test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to the IL-6R protein in the well is determined based on the amount of test antigen-binding molecule that competes for binding to the same epitope. This indirectly correlates with the binding activity of a candidate competing antigen-binding molecule that binds to the same epitope. That is, the greater the affinity of a 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.

[0032] The amount of test antigen-binding molecule bound to the wells via the IL-6R protein was determined by measuring the amount of antigen-binding molecule in advance. By labeling the molecule, it can be easily measured. For example, biotin-labeled antigen-binding molecules can be measured by using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly called competitive ELISA assays. Antigen-binding molecules can be easily detected or measured by other methods. Specifically, radiolabeling, fluorescent labeling, and the like are well known.

[0033] The binding activity of a candidate competitor antigen-binding molecule is compared with that obtained in a control test performed in the absence of the candidate competitor antigen-binding molecule. If the test antigen-binding molecule can block the binding of the original binding molecule by at least 20%, preferably at least 20-50%, and more preferably at least 50%, then the test antigen-binding molecule binds to substantially the same epitope as the competing antigen-binding molecule, or competes for binding to the same epitope.

[0034] Structure of the epitope to which a test antigen-binding molecule containing an antigen-binding domain for IL-6R binds When an epitope has been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activities of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0035] As a method for measuring such binding activity, for example, the above-mentioned ELISA format The binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which mutations have been introduced can be measured by comparing the binding activity of the test antigen-binding molecule and the control antigen-binding molecule to a linear peptide into which mutations have been introduced. The binding activity of the mutant peptide can also be measured by passing a test antigen-binding molecule and a control antigen-binding molecule down the column and then quantifying the antigen-binding molecules eluted in the eluate. A method for adsorbing the mutant peptide to a column, for example, as a fusion peptide with GST, is as follows: It is publicly known.

[0036] Furthermore, if the identified epitope is a conformational epitope, whether the test antigen-binding molecule and the control antigen-binding molecule share the same epitope can be assessed by the following method. First, IL-6R is bound to the control antigen-binding molecule. 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 solution such as PBS, and the cells are reacted with the test antigen-binding molecule. Then, FITC-labeled antibodies that can recognize the test and control antigen-binding molecules are added to the cell suspension, which is then washed with an appropriate buffer solution. The fluorescence intensity and cell count of the cells stained with the labeled antibodies are measured using a FACSCalibur (BD). The binding activity of the test and control antigen-binding molecules is measured by diluting them appropriately with a suitable buffer solution to the desired concentration. 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 CELLQUEST 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 labeled antibody bound, can be measured.

[0037] In this method, for example, "not substantially binding to mutant IL-6R-expressing cells" can be expressed by the following: First, the binding of IL-6R to cells expressing mutant IL-6R was examined. Test and control antigen-binding molecules are stained with labeled antibodies. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used as a flow cytometer for fluorescence detection, the obtained fluorescence intensity is The fluorescence intensity can be analyzed using CELL QUEST Software. The comparison value (ΔGeo-Mean) is calculated from the Geometric Mean value in the presence and absence of the substance using the following formula: The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculation based on the above formula. ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules)

[0038] The amount of binding of the test antigen-binding molecule to mutant IL-6R-expressing cells obtained by the analysis is reflected. The resulting Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value) is compared with a ΔGeo-Mean comparison value that reflects the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. It is particularly preferred that the concentrations of the test antigen-binding molecules used to determine the ΔGeo-Mean comparison value for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. It is used as a reference antigen-binding molecule.

[0039] The ΔGeo-Mean comparison value of the test antigen-binding molecule for the mutant IL-6R-expressing cells is At least 80%, preferably 50%, and more preferably 50% of the ΔGeo-Mean comparison value for the IL-6R-expressing cells of the offspring. If the ratio is preferably less than 30%, and particularly preferably less than 15%, it is considered that the mutant IL-6R-expressing cells are "substantially The formula for calculating the Geo-Mean value is CELL Q The comparison values ​​are described in the UEST Software User's Guide (BD Biosciences). When the comparison reveals that the epitopes of the test and control antigen-binding molecules are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be evaluated as being identical.

[0040] antigen-binding domain As used herein, the term "antigen-binding domain" refers to any domain with any structure that 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 the A domain, which is a 35-amino acid module contained in Avimer, a cell membrane protein present in living organisms (WO2004 / 044011, WO2005 / 040229), and the cell membrane Adnectin (WO2002 / 032925) contains the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed in the mouse. Affibody (WO1995 / 001937) uses an IgG-binding domain composed of a 58-amino acid three-helix bundle of Protein A as a scaffold. Affibody (WO1995 / 001937) contains a subunit consisting of a turn containing 33 amino acid residues, two antiparallel helices, and a loop. Analysis of ankyrin repeats (ARs), which have a structure consisting of repeated stacks of ARs. DARPins (Designed Ankyrin Repeat proteins) (WO2002 / 020565), which are regions exposed on the surface of the molecule; Anticalin (WO2003 / 029462), which is a four-loop region supporting one side of a barrel structure in which eight antiparallel strands highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) twist toward the center; and Immunoglobulins (IM) as an adaptive immune system in jawless fish such as lampreys and hagfish. The leucine-rich-repeat (LRR) module of the variable lymphocyte receptor (VLR) lacks the globulin structure A preferred example of the antigen-binding domain of the present invention is a concave region of a parallel sheet structure within a horseshoe-shaped structure in which multiple sheets are repeatedly stacked (WO2008 / 016854). Examples of such antigen-binding domains include those comprising the variable regions of heavy and light chains. Preferred examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "diabody," "Fab," "F(ab')2," domain antibody (dAb) (WO2004 / 058821, WO2003 / 002609), scFv-sc (WO2005 / 037989), and Fc fusion proteins. Molecules comprising an Fc region can use the Fc region as a binding domain to FcRn, particularly human FcRn. Furthermore, these molecules Molecules in which a human FcRn-binding domain is fused to the antibody can also be used.

[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 in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (IL-6R_PP; NP_000556.1), for example. It is present in a protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in No. 1. Alternatively, the antigen-binding domains in the antigen-binding molecule of the present invention can bind to different epitopes. Here, the different epitopes can be present, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. 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: 1. The antigen-binding domains contained in bispecific antibodies can be appropriately utilized for such purposes. A bispecific antibody is an antibody that has variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody can be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

[0042] Furthermore, the antigen-binding domain of the present invention can preferably be a domain involved in binding to a target (antigen) in a receptor protein that binds to that target (antigen). That is, the antigen-binding molecule may be a protein in which an FcRn-binding domain, particularly an FcRn-binding domain, and a carbohydrate receptor-binding domain are fused to a receptor protein that binds to the target (antigen). Examples of such antigen-binding molecules include TNFR-Fc fusion proteins. , IL1R-Fc fusion protein, VEGFR-Fc fusion protein, CTLA4-Fc fusion protein, etc. (Nat Med. (2003) 9 (1), 47-52, BioDrugs. (2006) 20 (3), 151-160). Even when the antigen-binding molecules of the present invention are fusion proteins of these receptor proteins and FcRn-binding domains, particularly human FcRn, their target molecule-binding activity changes depending on ion concentration conditions. Antigen-binding molecules that have binding activity for carbohydrate receptors and FcRn, particularly human FcRn, can promote antigen uptake into cells and accelerate the decrease in plasma antigen concentration by administration of the antigen-binding molecules. Furthermore, the pharmacokinetics of such antigen-binding molecules are improved, making it possible to increase the number of antigens that can be bound by a single antigen-binding molecule.

[0043] Furthermore, the antigen-binding domain of the present invention can preferably be a domain involved in binding to a target in a natural or artificial ligand that binds to the target. That is, the antigen-binding molecule can also be a molecule in which an FcRn-binding domain, particularly an FcRn-binding domain, and a carbohydrate receptor-binding domain are fused to a natural or artificial protein ligand that binds to the target and has antagonistic or neutralizing activity. Examples of such artificial ligands include mutant IL-6 (EMBO J. (1994) 13(24), 5863-70). Even when the antigen-binding molecule of the present invention is an artificial ligand fusion molecule, its binding to the target molecule changes depending on ion concentration conditions. Antigen-binding molecules that have binding activity to carbohydrate receptors and FcRn, particularly human FcRn, can promote antigen uptake into cells and reduce plasma antigen concentrations by administration of the antigen-binding molecule. Furthermore, the pharmacokinetics of such antigen-binding molecules are improved, making it possible to increase the number of antigens that can be bound by a single antigen-binding molecule.

[0044] specific "Specific" refers to a state in which one of the specifically binding molecules does not exhibit any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies when an antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. Furthermore, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing that epitope.

[0045] 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.

[0046] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Examples of methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are given below. The antibody to be combined can also be prepared appropriately according to the examples below.

[0047] The anti-IL-6R antibody can be prepared as a polyclonal or monoclonal antibody by known methods. As the anti-IL-6R antibody, a monoclonal antibody derived from a mammal is preferably produced. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed by genetic engineering techniques with an expression vector containing an antibody gene. The monoclonal antibodies of the present invention are also referred to as "human monoclonal antibodies." These include "antibody" and "chimeric antibody."

[0048] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized 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 are screened by a conventional screening method to identify hybrids that produce anti-IL-6R antibodies. A ridoma may be selected.

[0049] Specifically, monoclonal antibodies are produced, for example, as follows. First, the IL-6R protein shown in SEQ ID NO: 1, which is used as a sensitizing antigen for antibody production, can be obtained by expressing the IL-6R gene, the nucleotide sequence of which is disclosed in SEQ ID NO: 2 (IL-6R_PN; NM_000565.3). That is, a suitable host cell is transformed by inserting the gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or culture supernatant by a known method. To obtain a soluble form of IL-6R from, for example, the soluble form of IL-6R described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968), a soluble form of IL-6R, such as that shown in SEQ ID NO: A protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by 1. The protein is expressed in place of the IL-6R protein represented by SEQ ID NO: 1. Natural IL-6R protein can also be used as a sensitizing antigen.

[0050] 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. It can also be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it, but the region and size of the IL-6R peptide used as the partial peptide are not particularly limited. The desired 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: 1. 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, preferably 10 to 30, residues can be used as a sensitizing antigen.

[0051] In addition, a desired partial polypeptide or peptide of the IL-6R protein can be synthesized by combining it with a different polypeptide. The fused fusion protein can be used as a sensitizing antigen. For example, an Fc fragment of an antibody or a peptide tag can be suitably used to produce a fusion protein to be used as a sensitizing antigen. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. The method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R used in the present invention and immunization methods using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0052] 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.

[0053] The above-mentioned animals are immunized with a sensitizing antigen according to a known method. For example, a common method is to administer the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection to carry out immunization. Specifically, immunization is carried out using PBS (Phosphate-Buffered Saline), physiological saline, etc. The sensitizing antigen diluted at an appropriate dilution ratio is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then the sensitizing antigen is administered to a mammal for 4 to 21 days. The sensitizing antigen is administered several times per day. An appropriate carrier can be used for 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.

[0054] Alternatively, hybridomas producing the desired antibodies can be isolated using DNA immunization as follows: DNA immunization is the process of expressing a gene encoding an antigen protein in an immunized animal. In the immunized animal to which the vector DNA constructed in such a manner as to be able to be administered, the sensitizing antigen This is an immunization method in which immune stimulation is achieved by the expression of the gene in the living body of the immunized animal. Compared to the general immunization method in which a protein antigen is administered to the immunized animal, DNA immunization has the following advantages: Such advantages are expected. -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0055] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be suitably used. A commonly used method can be used to administer the vector to a living body. For example, gold particles to which the expression vector is adsorbed can be injected into the cells of an immunized animal using a gene gun. DNA immunization is achieved by introducing the antibody into the host. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO2003 / 104453.

[0056] In this way, mammals are immunized, and an increase in the antibody titer that binds to IL-6R in the serum is confirmed. After the recognition, immune cells are collected from the mammal and subjected to cell fusion. As preferred immune cells, splenocytes can be used in particular.

[0057] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably have an appropriate selection marker for screening. The selection marker indicates a trait that allows (or prevents) survival under specific culture conditions. The selection marker may be hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). It is known that cells lacking HGPRT or TK are hypoxanthine-aminopeptide-dependent. HAT-sensitive cells are HAT-selected. In selective medium, they cannot synthesize DNA and die, but when they fuse with normal cells, they They can continue to synthesize DNA using the salvage cycle, allowing them to grow even in HAT selection medium.

[0058] 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. Cells lacking these enzymes and unable to incorporate the log 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.

[0059] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and I mmunology (1978) 81, 1-7), NS-1 (C. Eur. 269-270), FO(J. Immunol. Methods(1980)35 (1-2), 1-21), 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.

[0060] 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 normal nutrient culture medium in the presence of a cell fusion promoter. Examples of the fusion promoter include polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and if desired, dimethicone may be used to further increase the fusion efficiency. When used, an auxiliary agent such as phenyl sulfoxide is added.

[0061] The ratio of immune cells to myeloma cells used can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. Examples of suitable medium for growing the myeloma cell line include RPMI1640 culture medium, MEM culture medium, and the like. In addition, the usual culture medium used for this type of cell culture is used, and fetal calf serum (FCS) ) or other serum replacement fluids may be suitably added.

[0062] 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.

[0063] The hybridomas thus obtained are cultured in a conventional selective culture medium, such as HAT medium (hypothelial growth factor AT). The hybridomas can be selected by culturing them in a culture medium containing xanthine, aminopterin, and thymidine. The culture can be continued in the above HAT culture medium for a sufficient time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibodies are then screened and single-cloned by the conventional limiting dilution method.

[0064] The hybridomas thus obtained can be selected by using a selective medium that corresponds 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). In other words, HAT-sensitive myeloma cells When cells are used for cell fusion, cells that have successfully fused with normal cells are selected in the HAT culture medium. The culture is continued in the HAT medium for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the period is generally from several days to several weeks. The desired hybridomas can be selected by culturing for a period of time. Hybridomas that produce the desired antibody can then be screened and single-cloned by the conventional limiting dilution method.

[0065] Screening and single cloning of the desired antibody can be suitably carried out by a screening method based on known antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can be Such monoclonal antibodies can bind to IL-6R expressed on the cell surface. Nucleotide antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS analyzes cells contacted with fluorescent antibodies using laser light, and measures the fluorescence emitted by individual cells, making it possible to measure the binding of antibodies to the cell surface. It is a system that

[0066] To screen for hybridomas producing the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. The cells used as host cells were mammalian cells in which IL-6R was forcibly expressed. By using untreated mammalian cells as a control, the expression of IL-6R on the cell surface was The binding activity of the antibody can be selectively detected, i.e., it does not bind to host cells and induces IL-6R expression. By selecting hybridomas that produce antibodies that bind to expressing cells, IL-6R monoclonal antibodies were identified. Hybridomas producing monoclonal antibodies can be obtained.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Recombinant Antibody 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.

[0071] 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)

[0072] The extracted mRNA was purified using the mRNA Purification Kit (GE Healthcare Biosciences). 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 obtained from hybridomas. cDNA encoding antibody V regions can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. For cDNA synthesis and amplification, SMART RACE cDNA amplification kit (Clontech) and PCR were used in the 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), Furthermore, in the process of synthesizing such cDNA, both ends of the cDNA can be Appropriate restriction enzyme sites, as described below, can be introduced into the ends.

[0073] 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.

[0074] 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.

[0075] The antibody gene was amplified by PCR using the obtained 5'-RACE cDNA library as a template. 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).

[0076] Specifically, when the objective is to obtain a gene encoding mouse IgG, It is possible to amplify genes encoding γ1, γ2a, γ2b, and γ3 as the main chains, and κ and λ as the light chains. To amplify the IgG variable region gene, a suitable primer can be used. The primer used for the 5' end anneals to the region corresponding to the constant region close to the variable region. On the other hand, the primer for the 5' end anneals to the region corresponding to the constant region close to the variable region. A primer that matches the sequence is used.

[0077] The amplified PCR products are used to generate immunoglobulins consisting of a combination of heavy and light chains. The binding activity of the reconstituted immunoglobulin to IL-6R is shown. For example, to obtain an antibody against IL-6R, For this purpose, it is more preferable that the antibody binds specifically to IL-6R. 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.

[0078] 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.

[0079] A panning method using a phage vector is also preferably used as a method 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, a screening method using a phage vector is advantageous.

[0080] The genes encoding the heavy and light chain variable regions are linked with an appropriate linker sequence. It can form single-chain Fv (scFv) (Nat. Biotechnol. (2005) 23(9), 1126-1136). By inserting the gene encoding the scFv into a phage vector, By this procedure, phages expressing scFv on their surface can be obtained. After contacting the phages with the desired antigen, the phages bound to the antigen can be recovered, thereby recovering DNA encoding scFv with the desired binding activity. By repeating this procedure as necessary, the desired scFv can be recovered. scFvs having a binding activity of 100% or more 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. A preferred restriction enzyme recognizes and digests a nucleotide sequence that appears 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 a sticky end. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, if a gene encoding the antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody 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. Inserting the V region gene into an expression vector already containing a constant region can produce a chimeric antibody. A vector for expressing a human antibody can be constructed. Specifically, for example, a vector encoding a desired antibody constant region (C The V region gene is inserted into the 5' end of an expression vector carrying DNA encoding the V region Restriction enzyme recognition sequences can be appropriately positioned. The two fragments are digested with the same combination of restriction enzymes and fused in frame to construct a chimeric antibody expression vector.

[0082] To produce anti-IL-6R monoclonal antibodies, the antibody gene is regulated by the expression control region. The antibody is incorporated into an expression vector so that it is expressed under the conditions specified in the above. Expression control regions for expressing the antibody 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. In the examples described later, the signal sequence has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3). Although a peptide has been used, a suitable signal sequence can also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above 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 that express DNA encoding the anti-IL-6R antibody.

[0083] For the expression of antibody genes, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is Each is incorporated into a separate expression vector. By co-transfecting the same host cell with vectors incorporating the H and L chains, antibody molecules with both H and L chains can be expressed. Alternatively, DNA encoding the H and L chains can be incorporated into a single expression vector. The host cell can be transformed by incorporating the vector into the host cell (see International Publication WO 1994 / 011523). (See reference).

[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, HEK (human embryonic kidney) 293, etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

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

[0086] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces cerevisiae and other saccharomyces Saccharomyces genus, Pichia genus such as Pichia pastoria -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 E. coli and Bacillus subtilis can be used appropriately. An expression vector containing the desired antibody gene is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody gene is extracted from the culture of the transformed cells. The desired antibody can be obtained.

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

[0089] Humanized antibodies, human antibodies When the antigen-binding molecules described herein are administered to humans, the antigen-binding domain of the antigen-binding molecule may be an antigen-binding domain derived from a recombinant antibody that has been artificially modified for the purpose of reducing heterologous antigenicity to humans, etc. In addition to the chimeric antibodies described above, recombinant antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced using known methods.

[0090] The variable region of an antibody used to prepare the antigen-binding domain of the antigen-binding molecule described herein is generally composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are essentially the regions that 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, in general, CDR grafting can improve the quality of a given antibody. It is believed that the binding specificity of the antibody can be transferred to other antibodies.

[0091] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which the CDRs of non-human animals, such as mouse antibodies, are transplanted into human antibodies are well known. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for grafting the CDR of a mouse antibody onto a human FR. In overlap extension PCR, primers for synthesizing the FR of a human antibody are used. The primers are: It is prepared for each of the four FRs. Generally, in grafting mouse CDRs to human FRs, Therefore, it is advantageous to select human FRs that are highly identical to mouse FRs in order to maintain the function of the CDRs. That is, in general, the amino acids of the FR adjacent to the mouse CDR to be transplanted are It is preferable to use human FRs consisting of amino acid sequences highly identical to the amino acid sequences of the human FRs.

[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, mouse CDRs are inserted into each FR. The resulting product has DNA encoding the mouse CDRs added to it. The base sequences encoding the mouse CDRs of each product are designed to overlap with each other. The overlapping CDR portions of the products synthesized using the human antibody gene as a template are then annealed to each other to form complementary strands. This reaction links the human FRs to the mouse CDRs via their sequences.

[0093] The V region gene, which finally contains three CDRs and four FRs, is amplified in its entirety using primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences. The DNA obtained as described above is fused in frame with DNA encoding the C region of a human antibody. A humanized antibody expression vector can be prepared by inserting the vector into an expression vector as described above. After the vector is introduced into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, thereby producing the humanized antibody. The cultured cells produce the α-glucan derivatives (see European Patent Publication EP239400 and International Patent Publication WO1996 / 002576). (see).

[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 when linked via the CDRs. If necessary, it is possible to select suitable CDRs of a reshaped human antibody. It is also possible to substitute amino acid residues in the FR to form a suitable antigen-binding site. For example, the PCR method used to graft mouse CDRs onto human FRs can be applied to introduce amino acid sequence mutations into the FR. Specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FR. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. Mutant FR sequences with desired properties can be selected by measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above-mentioned method (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, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-chain fragment (scFv) on a phage display. The scFv is expressed on the surface of the phage by the method (a). Phages expressing scFv that bind to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, An expression vector can be constructed by fusing the V region sequence in frame with the sequence of the C region of a desired human antibody and then inserting the resulting fusing sequence into an appropriate expression vector. The human antibody can be obtained by introducing the expression vector into a suitable expression cell such as those listed above and expressing the gene encoding the human antibody. These methods are already known (see International Publication Nos. WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

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

[0098] EU numbering and Kabat numbering According to the method used in the present invention, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National 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 region are represented according to the Kabat numbering system, and the amino acids in the constant region are represented according to the EU numbering system based on the Kabat amino acid positions.

[0099] FcRn Unlike Fcγ receptors belonging to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides. It shares 22 to 29% sequence identity with MHC molecules of type I (Ghetie et al., Immunol. Today (1997) 18 (12), 592-598). FcRn is complexed with soluble β or light chain (β2 microglobulin). It is expressed as a heterodimer consisting of an integrated transmembrane α or heavy chain. The α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain that 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)).

[0100] 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 transport of maternal IgG from ingested colostrum or milk across the brush border epithelium. FcRn is expressed in numerous other tissues, as well as in various endothelial cell lines. It is also expressed in human adult vascular endothelium, muscle vasculature, and liver sinusoids. FcRn binds IgG and By recycling it back into the serum, it plays a role in maintaining the plasma concentration of IgG. The binding of FcRn to IgG molecules is usually strictly pH-dependent, and the optimal binding This is observed in the acidic pH range below 7.0.

[0101] Human FcRn, whose precursor is a polypeptide containing the signal sequence shown in SEQ ID NO: 4 (FcRn; NP_004098.1), is expressed in vivo (SEQ ID NO: 5 (beta2-microglobulin; NP_004039.1)). ) and forms a complex with human β2-microglobulin. As shown later in the Reference Examples, soluble human FcRn complexed with β2-microglobulin can be produced using standard recombinant expression techniques. The binding activity of the Fc region of the present invention to such soluble human FcRn complexed with β2-microglobulin can be evaluated. Unless otherwise specified, in the present invention, human FcRn refers to a form that is capable of binding to the Fc region of the present invention, and examples include a complex between human FcRn and human β2-microglobulin.

[0102] FcRn-binding domain The antigen-binding molecules of the present invention have an FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antigen-binding molecule has FcRn-binding activity in the acidic pH range, and may be a domain that has FcRn-binding activity directly or indirectly. Examples of such domains include IgG immunoglobulins that have direct FcRn-binding activity. Fc region, albumin, albumin domain 3, anti-FcRn antibody, anti-FcRn peptide, anti-FcRn peptide Scaffold molecules, or IgG or albumin that indirectly have binding activity to FcRn. In the present invention, preferred examples include molecules that bind to amines in the acidic pH range and the neutral pH range. In this regard, a domain having FcRn-binding activity is preferred. The domain can be suitably used as is as long as it already has FcRn-binding activity in the acidic pH range. If the domain has no or weak FcRn-binding activity in the acidic pH range, it is possible to confer FcRn-binding activity by modifying amino acids in the antigen-binding molecule. Alternatively, the FcRn-binding activity may be enhanced by modifying amino acids in a domain that already has FcRn-binding activity in the acidic pH range. The desired amino acid modification in the FcRn-binding domain can be identified by comparing the FcRn-binding activity in the acidic pH range before and after the amino acid modification.

[0103] The FcRn-binding domain is preferably a region that directly binds to FcRn. A preferred example of an FcRn-binding domain is the Fc region of an antibody. However, a region capable of binding to a polypeptide having FcRn-binding activity, such as albumin or IgG, can indirectly bind to FcRn via albumin, IgG, or the like. Therefore, a region that binds to a polypeptide having FcRn-binding activity can be preferably used as the FcRn-binding region of the present invention. The Fc region comprises an amino acid sequence derived from the constant region of an antibody heavy chain. The Fc region is a region consisting of approximately 216 amino acids, expressed as EU numbering, that are located between the hinge region of the papain cleavage site and the FcRn-binding site. A portion of the heavy chain constant region of an antibody, including the N-terminus of the region, the hinge, CH2 and CH3 domains is.

[0104] The binding activity of the FcRn-binding domain of the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described above in the section on binding activity, and conditions other than pH can be appropriately determined by those skilled in the art. The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate), apparent kd (Apparent dissociation rate), etc. These can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plots, flow cytometers, etc. can be used.

[0105] Conditions other than pH when measuring the FcRn-binding activity of an FcRn-binding domain are not particularly limited and can be appropriately selected by those skilled in the art. The FcRn-binding domains of the present invention can be measured in MES buffer at 37°C. The binding activity of the main FcRn-binding domain to FcRn can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare). The binding activity of the FcRn-binding domain to FcRn can be measured using the FcRn-binding domain or the antigen-binding domain of the present invention containing the FcRn-binding domain. The antigen-binding activity can be evaluated by passing FcRn or an FcRn-binding domain, or an antigen-binding molecule of the present invention containing an FcRn-binding domain, as an analyte, over a chip onto which FcRn or an FcRn-binding domain has been immobilized.

[0106] The acidic pH range, as the condition under which the FcRn-binding domain contained in the antigen-binding molecule of the present invention has binding activity to FcRn, generally means pH 4.0 to pH 6.5, preferably pH 5.5 to pH 6.5, and particularly preferably pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Regarding the temperature used as a measurement condition, the binding affinity between the FcRn-binding domain and FcRn may be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between the FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to evaluate the binding affinity between the FcRn-binding domain and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0107] According to Yeung et al. (J. Immunol. (2009) 182, 7663-7671), natural human IgG1 The binding activity to FcRn was 1.7 μM in the acidic pH range (pH 6.0), but no activity was observed in the neutral pH range. Therefore, in a preferred embodiment, the binding activity to human FcRn under acidic pH conditions is 20 μM or stronger than KD, and the binding activity to human FcRn under neutral pH conditions is 20 μM or stronger than KD. Antigen-binding molecules of the present invention that have binding activity to human FcRn under an acidic pH range condition, including antigen-binding molecules whose binding activity to human FcRn is equivalent to that of native human IgG, can be used. In this embodiment, antigen-binding molecules of the present invention may be used, including antigen-binding molecules with human FcRn-binding activity of 2.0 μM or stronger (KD) under acidic pH conditions. In an even more preferred embodiment, antigen-binding molecules with human FcRn-binding activity of 0.5 μM or stronger (KD) under acidic pH conditions may be used. The KD values ​​described above were determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (in which antigen-binding molecules were immobilized on a chip and human FcRn was allowed to flow as an analyte). is determined by

[0108] In the present invention, an Fc region that has FcRn-binding activity under an acidic pH range is preferred. The domain can be used as is if it already has FcRn-binding activity under an acidic pH range. If the domain has no or weak FcRn-binding activity under an acidic pH range, an Fc region with the desired FcRn-binding activity can be obtained by modifying amino acids in the antigen-binding molecule. Alternatively, an Fc region with the desired or enhanced FcRn-binding activity under an acidic pH range can also be obtained by modifying amino acids in the Fc region. Amino acid modifications in the Fc region that result in such desired binding activity can be identified by comparing the FcRn-binding activity under an acidic pH range before and after the amino acid modifications. Those skilled in the art can appropriately modify amino acids using known techniques described later in the section on amino acid modifications.

[0109] The Fc region contained in the antigen-binding molecule of the present invention and having FcRn-binding activity under acidic pH conditions can be obtained by any method. Specifically, the Fc region can be obtained by modifying the amino acids of a human IgG immunoglobulin used as the starting Fc region to have FcRn-binding activity under acidic pH conditions. An FcRn-binding domain with binding activity or enhanced binding activity can be obtained. Preferred examples of the Fc region of an IgG immunoglobulin for modification include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and variants thereof). Modifications to other amino acids can be made to amino acids at any position, as long as the FcRn-binding activity is maintained under acidic pH conditions or the binding activity to human FcRn under acidic pH conditions is enhanced. When an antigen-binding molecule contains the Fc region of human IgG1 as the Fc region, it is preferred that the FcRn-binding domain contains a modification that enhances the FcRn-binding activity under acidic pH conditions compared to the binding activity of the starting Fc region of human IgG1. Examples of amino acids that can be modified in this way include those described in, for example, WO2000 / 042072 As shown in the table, the EU numbering system is 238th, 252nd, 253rd, 254th, 255th, 256th, 265th, 272nd, 286th, 288th, 303rd, 305th, 307th, 309th, 311th, 312th, 317th. 340th, 356th, 360th, 362nd, 376th, 378th, 380th, 382nd, 386th, 388th, 400th Suitable examples of amino acids that can be modified in this way include the amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and / or 447, as defined by EU numbering, as described in WO2002 / 060919. Possible amino acids are represented by EU numbering as described in WO2004 / 092219. Also included are amino acids at positions 250, 314, and 428, which may be modified. As amino acids, for example, as described in WO2010 / 045193, Preferred examples of such modifications include those at positions 251, 252, 307, 308, 378, 428, 430, 434, and / or 436. These amino acid modifications enhance the FcRn binding of the Fc region of IgG immunoglobulins under acidic pH conditions.

[0110] For example, by using these amino acid modifications alone or in combination, It is possible to enhance the binding of the Fc region of IgG to FcRn in the acidic pH range by using The amino acid modification to be introduced is not particularly limited, and any amino acid modification may be introduced as long as it has the effect of improving plasma retention.

[0111] In a non-limiting embodiment of the present invention, an antigen-binding domain having binding activity to FcRn, particularly human FcRn, can also be used as the FcRn-binding domain. As described above, bispecific antibodies can also be used as the antigen-binding molecules of the present invention. Preferred examples of such bispecific antibodies include those in which one epitope to which the bispecific antibody binds is present in a desired antigen other than FcRn, and the other epitope is present in FcRn. The structure of the bispecific antibody is not limited to a specific structure, as long as it contains a bivalent binding domain having binding valencies to the desired antigen and FcRn, and a carbohydrate receptor-binding domain. For example, antibody structures such as IgG antibodies to which an Fc region is linked can also be used, and "scFv2 (single chain Fv2)," "diabody," or "F(ab')2" can also be preferably used. IgG antibodies When antibody structures such as "scFv2 (single chain Fv2)," "diabody," or "F(ab')2" are used, a carbohydrate-binding domain may be contained in the Fc region, and may also be contained in the FcRn-binding domain and / or the antigen-binding domain. Furthermore, when structures such as "scFv2 (single chain Fv2)," "diabody," or "F(ab')2" are used, a carbohydrate-binding domain may also be contained in the FcRn-binding domain and / or the antigen-binding domain. do.

[0112] When such bispecific antibodies are used, an FcRn-binding domain whose FcRn-binding activity changes depending on ion concentration conditions, as described below in the section "Ion concentration conditions," can be used as the FcRn-binding domain. That is, in a non-limiting embodiment of the present invention, an FcRn-binding domain whose FcRn-binding activity changes depending on metal ion concentration conditions or pH conditions, as described below in the section "Ion concentration conditions," can be used.

[0113] Ion concentration conditions (1) Metal ion concentration conditions In one non-limiting embodiment of the present invention, the ion concentration refers to the metal ion concentration. "Metal ions" include alkali metals excluding hydrogen and Group I metals such as copper group, alkaline earth metals, and zinc ions. Group II elements such as lead, Group III elements excluding boron, Group IV elements excluding carbon and silicon, iron and platinum groups Elements belonging to groups VIII, V, VI and VII, subgroups A, such as antimony, bismuth, and porphyrin Ionization tendency refers to the ions of metallic elements such as rhenium. Metal atoms have the property of releasing valence electrons to become positive ions, a property known as ionization tendency. Metals with a high ionization tendency are considered to be chemically active.

[0114] An example of a suitable metal ion in the present invention is calcium ion. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction such as skeletal muscle, smooth muscle, and cardiac muscle; activation of leukocytes such as movement and phagocytosis; activation of platelets such as deformation and secretion; activation of lymphocytes; activation of mast cells such as histamine secretion; cell responses mediated by catecholamine α receptors and acetylcholine receptors; exocytosis; release of transmitters from neuronal terminals; and axonal flow in neurons. Troponin C, calmodulin, parvalbumin, and myosin light receptors have multiple calcium ion binding sites as intracellular calcium ion receptors, and are thought to have originated from a common origin in molecular evolution. Many binding motifs are known, including cadherin domains, EF hands contained in calmodulin, C2 domains contained in protein kinase C, Gla domains contained in blood coagulation protein Factor IX, and asialoglycoprotein receptors. C-type lectins found in the mannose receptor and the LDL receptor, and A domains found in the LDL receptor , annexin, thrombospondin type 3 domain and EGF-like domain are well known.

[0115] In the present invention, when the metal ion is a calcium ion, calcium ion concentration conditions include low and high calcium ion concentrations. "The binding activity changes depending on the calcium ion concentration" refers to the change in antigen-binding activity of an antigen-binding molecule due to the difference between low and high calcium ion concentrations. For example, this may be the case where the antigen-binding activity of an antigen-binding molecule is higher under high calcium ion concentrations than under low calcium ion concentrations. Another example is where the antigen-binding activity of an antigen-binding molecule is higher under low calcium ion concentrations than under high calcium ion concentrations.

[0116] In this specification, the high calcium ion concentration is not limited to a specific numerical value, but may be a concentration selected from the range of 100 μM to 10 mM. In another embodiment, it may be a concentration selected from the range of 200 μM to 5 mM. The concentration may be selected from between 500 μM and 2.5 mM, and in other embodiments, between 200 μM and The concentration may be selected from the range of 1000 μM to 2 mM. It may also be selected from the range of 400 μM to 1.5 mM. In particular, a concentration selected from the range of 500 μM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood) in vivo, is preferred.

[0117] In this specification, the low calcium ion concentration is not limited to a specific numerical value, but may be a concentration selected from the range of 0.1 μM to 30 μM. In another embodiment, the concentration may be selected from the range of 0.2 μM to 20 μM. In another embodiment, the concentration may be selected from the range of 0.5 μM to 10 μM, and in another embodiment, the concentration may be selected from the range of 1 μM to 5 μM. In another embodiment, the concentration may be selected from the range of 2 μM to 4 μM. In particular, a concentration selected from the range of 1 μM to 5 μM, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0118] In the present invention, the expression "the antigen-binding activity at a low calcium ion concentration is lower than the antigen-binding activity at a high calcium ion concentration" refers to the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 0.1 μM to 30 μM. This means that the antigen-binding activity at a calcium ion concentration selected from the range of 100 μM to 10 mM is weaker than that at a calcium ion concentration selected from the range of 0.5 μM to 10 μM of the antigen-binding molecule. The binding activity to the antigen at the calcium ion concentration is selected from the range of 200 μM to 5 mM. It particularly preferably means that the antigen-binding activity at a calcium ion concentration in an early endosome in a living body is weaker than the antigen-binding activity at a calcium ion concentration in plasma in a living body. Specifically, it means that the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 1 μM to 5 μM is weaker than the antigen-binding activity at a calcium ion concentration selected from the range of 500 μM to 2.5 mM. This means that the binding activity to the antigen is weaker than that of the antibody.

[0119] Whether or not the antigen-binding activity of an antigen-binding domain of the present invention changes depending on metal ion concentration conditions can be determined using known measurement methods, such as those described above in the section on binding activity. For example, to confirm that the antigen-binding activity of an antigen-binding molecule comprising an antigen-binding domain of the present invention changes so that it is higher under high calcium ion concentrations than under low calcium ion concentrations, the antigen-binding activities of the antigen-binding molecule comprising the antigen-binding domain under low and high calcium ion concentrations are compared.

[0120] Furthermore, in the present invention, "antigen binding activity under conditions of low calcium ion concentration" The expression "the antigen-binding activity of an antigen-binding molecule under a high calcium ion concentration is lower than that under a high calcium ion concentration" can also be expressed as "the antigen-binding activity of an antigen-binding molecule under a high calcium ion concentration is higher than that under a low calcium ion concentration." In the present invention, "the antigen-binding activity under a low calcium ion concentration is lower than that under a high calcium ion concentration" can also be expressed as "the antigen-binding ability under a low calcium ion concentration is weaker than that under a high calcium ion concentration," and "the antigen-binding activity under a low calcium ion concentration is made weaker than that under a high calcium ion concentration."

[0121] Conditions other than calcium ion concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, conditions such as HEPES buffer, 37°C, For example, Biacore (GE Healthcare) can be used to measure the When measuring the binding activity between an antigen-binding molecule containing an antigen-binding domain and an antigen, if the antigen is a soluble antigen, the binding activity for the soluble antigen can be evaluated by passing the antigen as an analyte through a chip onto which an antigen-binding molecule containing an antigen-binding domain has been immobilized, whereas when the antigen is a membrane-type antigen, the binding activity for the membrane-type antigen can be evaluated by passing the antigen-binding molecule containing the antigen-binding domain as an analyte through a chip onto which the antigen has been immobilized.

[0122] In the antigen-binding molecules of the present invention, the ratio of antigen-binding activity under low calcium ion concentrations to that under high calcium ion concentrations is not particularly limited, as long as the antigen-binding activity under low calcium ion concentrations is weaker than that under high calcium ion concentrations, but preferably the ratio of the KD (Dissociation constant) for the antigen under low calcium ion concentrations to the KD under high calcium ion concentrations, KD (3 μM Ca) / KD (2 mM Ca), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of KD (3 μM Ca) / KD (2 mM Ca) 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. It can also be specified by the value of KD (3 μM Ca) / KD (1.2 mM Ca). That is, the value of KD (3 μM Ca) / KD (1.2 mM Ca) is 2 or more, more preferably 10 or more, and even more preferably 40 or more. There is no particular upper limit to the value of KD (3 μM Ca) / KD (1.2 mM Ca), and as long as it can be produced by the techniques of a person skilled in the art, it is possible to produce a KD (3 μM Ca) / KD (1.2 mM Ca) above 400. , 1000, 10000, etc., any value is acceptable.

[0123] As the value of binding activity to an antigen, KD (dissociation constant) can be used when the antigen is a soluble antigen, whereas apparent KD (apparent dissociation constant) can be used when the antigen is a membrane antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard plots, flow cytometers, etc. It is possible to do so.

[0124] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an index showing the ratio between the antigen-binding activity of an antigen-binding molecule of the present invention under low calcium concentration conditions and that under high calcium concentration conditions. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an index showing the ratio of binding activities, the kd (dissociation rate constant) for the antigen under low calcium concentration conditions can be used as an index showing the ratio of binding activities. The ratio of kd (low calcium concentration condition) to kd (dissociation rate constant) under high calcium concentration condition, kd (low calcium concentration condition) / kd (high calcium concentration condition), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of the value of (low calcium concentration condition) / kd (high calcium concentration condition) is not particularly limited. The number of the particles may be any number, such as 50, 100, 200, etc., as long as it can be produced within the technical common sense of a person skilled in the art.

[0125] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble antigen, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane-type antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), a flow cytometer, etc. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different calcium ion concentrations, it is preferable to keep the conditions other than the calcium concentration the same.

[0126] For example, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antibodies, comprising the following steps (a) to (c): (a) Obtaining the antigen-binding activity of an antigen-binding domain or antibody under low calcium concentration conditions a process of (b) Obtaining the antigen-binding activity of an antigen-binding domain or antibody under high calcium concentration conditions and (c) The antigen-binding activity under low calcium concentration conditions is higher than that under high calcium concentration conditions. A step of selecting antigen-binding domains or antibodies having lower antigen-binding activity than the target antigen-binding domains or antibodies.

[0127] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies or a library thereof, comprising the following steps (a) to (c): (a) Antigen-binding domains or antibodies or their ligands under high calcium concentration conditions contacting the library with an antigen; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under low calcium concentration conditions; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0128] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under low calcium concentration conditions; a process of (b) selecting antigen-binding domains or antibodies that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to an antigen under high calcium concentration conditions; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0129] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be screened by a screening method comprising the following steps (a) to (c): Thus, it can be obtained. (a) Immobilizing the antigen-binding domain or antibody on a column containing the immobilized antigen under high calcium concentration conditions contacting the library; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under low calcium concentration conditions; and (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0130] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) Immobilizing the antigen-binding domain or antibody on a column containing the immobilized antigen under low calcium concentration conditions passing the library through the (b) recovering the antigen-binding domain or antibody that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antibody recovered in step (b) to bind to an antigen under high calcium concentration conditions; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0131] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under high calcium concentration conditions; a process of (b) obtaining the antigen-binding domain or antibody that bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under low calcium concentration conditions; and (d) selecting an antigen-binding domain in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b); Isolating the antibody or protein.

[0132] The above steps may be repeated two or more times. In the cleaning method, the steps (a) to (c) or (a) to (d) are repeated two or more times. Furthermore, there is provided an antigen-binding domain or antibody having lower antigen-binding activity at low calcium ion concentrations than at high calcium ion concentrations, obtained by a screening method comprising the steps of: (a) detecting a target protein containing a nucleotide sequence corresponding to a target protein;

[0133] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under low calcium concentration conditions is determined by measuring the antigen-binding activity at an ionized calcium concentration between 0.1 μM and 30 μM. Although there are no particular limitations as long as the binding activity is achieved, a preferred ionized calcium concentration is between 0.5 μM and 10 μM for antigen binding activity. The ionized calcium concentration in early endosomes in vivo is an example of the antigen-binding activity, specifically, at 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding domain or antibody under high calcium concentration conditions is not particularly limited as long as the antigen-binding activity is at an ionized calcium concentration of 100 μM to 10 mM, but preferred ionized calcium concentrations include antigen-binding activity at 200 μM to 5 mM. More preferred ionized calcium concentrations are An example of the ionized calcium concentration is the concentration of ionized calcium in plasma in a living body, and specifically, the antigen binding activity at 0.5 mM to 2.5 mM can be mentioned.

[0134] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), which is the dissociation rate. These can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), SciProtein (SciProtein), or other similar methods. It is possible to use a Catchard plot, FACS, or the like.

[0135] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0136] As long as the antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition, the difference between the antigen-binding activity under a high calcium concentration condition and that under a low calcium concentration condition is not particularly limited. Preferably, the antigen-binding activity under a high calcium concentration condition is at least twice as high as that under a low calcium concentration condition. It is more preferably 10 times or more, and even more preferably 40 times or more.

[0137] The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains or antibodies, and it is possible to screen, for example, the antigen-binding domains or antibodies described above. For example, antigen-binding domains or antibodies having native sequences may be screened, or antigen-binding domains or antibodies with substituted amino acid sequences may be screened.

[0138] (2) Amino acids whose binding activity to antigen-binding domains changes depending on calcium ion concentration The antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when the metal ion is calcium ion concentration, they may be prepared from pre-existing antibodies, pre-existing libraries (phage libraries, etc.), hybridomas obtained by immunizing animals, or B cells from immunized animals. It is possible to use antibodies or libraries prepared from these antibodies or libraries, or antibodies or libraries into which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced (libraries with a high content of amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acids, libraries into which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced at specific sites, etc.).

[0139] As described above, when the metal ion is a calcium ion, examples of amino acids that change the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions include any amino acid that forms a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490), Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J. (1990) 265, 261-265), Bairoch and Cox (FEBS Lett. (1990) 269, 454-456), Davis (New Biol. (1990) 2, 410-419), Schaefer et al. (Genomics (1995) 25, 638-643), Economou et al. (EMBO J. (1990) 9, 349-354), Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). Any known calcium-binding motif, such as that of a lectin, may be included in the antigen-binding molecules of the present invention. In addition to the above, a suitable example of such a calcium-binding motif may also be the calcium-binding motif contained in the antigen-binding domain of SEQ ID NO: 6.

[0140] Furthermore, amino acids with metal chelating activity can also be suitably used as amino acids whose antigen-binding activity changes depending on calcium ion concentration conditions, such as serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

[0141] The position of the antigen-binding domain containing the amino acid is not limited to a specific position, and can be any position in the heavy chain variable region or light chain variable region that forms the antigen-binding domain, as long as the antigen-binding activity of the antigen-binding molecule is changed depending on the calcium ion concentration. That is, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain antigen-binding domain contains an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration. In another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain CDR3 contains the amino acid. In another non-limiting embodiment, the antigen-binding domain of the present invention contains the amino acid at positions 95, 96, 100a, and / or 101 according to the Kabat numbering in the heavy chain CDR3. It can be obtained from a library that mainly contains antigen-binding molecules whose sequences differ from each other.

[0142] Furthermore, in one non-limiting embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains an amino acid in its light chain antigen-binding domain that changes the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration. In another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid in its light chain CDR1. In another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid at positions 30, 31, and / or 32 according to the Kabat numbering system in light chain CDR1.

[0143] In another non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in the light chain CDR2. A library is provided that is mainly composed of antigen-binding molecules whose sequences differ from one another and are contained within the 50 positions represented by the group.

[0144] In yet another non-limiting embodiment, the antigen-binding domains of the present invention can be obtained from a library mainly consisting of antigen-binding molecules with different sequences, each of which contains the amino acid residue at position 92 according to the Kabat numbering system in the light chain CDR3. The binding molecules may be obtained from a library consisting primarily of binding molecules.

[0145] Furthermore, the antigen-binding domain of the present invention may be a domain in which the amino acid residues are contained in two or three CDRs selected from the light chain CDR1, CDR2, and CDR3 described above and have different sequences. In another embodiment of the present invention, the antigen-binding domain can be obtained from a library mainly composed of antigen-binding molecules in which the amino acid residues are identical to those in the light chain Kabat numbering system. Included in one or more of the 30th, 31st, 32nd, 50th and / or 92nd positions, The antigen-binding molecules can be obtained from a library mainly consisting of antigen-binding molecules having sequences different from each other.

[0146] These amino acid residues also form a calcium-binding motif, and / or As long as the binding activity of the antigen-binding molecule to an antigen changes depending on the calcium ion concentration, these amino acid residues may be contained alone or in combination of two or more. In addition, troponin C, calmodulin, parvalbumin, myocardin, and the like, which have multiple calcium ion-binding sites and are thought to have originated from a common origin in molecular evolution, are also included. The light chain is known to contain CDR1, CDR2 and / or CDR3. Alternatively, CDR3 can be engineered. For example, for the above purpose, a cadherin domain, an EF hand contained in calmodulin, a C2 domain contained in protein kinase C, a Gla domain contained in blood coagulation protein Factor IX, an asialoglycoprotein receptor, or C-type lectins contained in the mannose receptor, A domains contained in the LDL receptor, and annexins Syn, thrombospondin type 3 domain and EGF-like domain can be used as appropriate.

[0147] In one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library containing multiple antigen-binding molecules of the present invention with different sequences by combining a heavy chain variable region selected as a framework sequence that previously contains "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions" with a light chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, in which the ion concentration is calcium ion concentration, is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 7 (6RL#9-IgG1) or SEQ ID NO: 8 (6KC4-1#85-IgG1) with a light chain variable region prepared as a randomized variable region sequence library. Alternatively, instead of the light chain variable region prepared as a randomized variable region sequence library, a library can be prepared by appropriately selecting from light chain variable regions with germline sequences. A suitable example is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 7 (6RL#9-IgG1) or SEQ ID NO: 8 (6KC4-1#85-IgG1) with a light chain variable region having a germline sequence.

[0148] As used herein, the term "library" refers to multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. The sequences of the multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules contained in the library are not a single sequence, but rather are antigen-binding molecules or fusion polypeptides containing antigen-binding molecules with different sequences. Methods for displaying fusion polypeptides containing antibody fragments on the surface of bacteriophage to produce such libraries are known in the art and are described, for example, in WO1992001047 and herein. Related methods are also described in WO1992020791, WO1993006213, WO1993011236, and WO1993019172, and those skilled in the art can use these methods as appropriate. Other known documents (HR Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236) , identification of antibodies against various antigens displayed on the phage surface using artificially rearranged variable region gene repertoires has been demonstrated.

[0149] (3) Hydrogen ion concentration conditions In one embodiment of the present invention, the ion concentration condition refers to the hydrogen ion concentration condition or the pH condition. In the present invention, the concentration condition of protons, i.e., the atomic nuclei of hydrogen atoms, is also treated as the hydrogen exponent (pH) condition. When the activity of hydrogen ions in an aqueous solution is expressed as aH+, pH is defined as -log10aH+. When the ionic strength in an aqueous solution is (for example, 10- 3 If it is lower than aH+ is approximately equal to the hydrogen ion strength. For example, the ionic product of water at 25°C and 1 atmosphere is Kw = aH + aOH = 10-14, so for pure water it is aH+ = aOH = 10-7. In this case, pH = 7 is neutral, and pH below 7 is Aqueous solutions with a pH lower than 7 are acidic, and solutions with a pH higher than 7 are alkaline.

[0150] In the present invention, when pH conditions are used as the ion concentration conditions, the pH conditions include high hydrogen ion concentration or low pH, i.e., acidic pH range conditions, and low hydrogen ion concentration or high Examples of such conditions include pH, i.e., conditions in the neutral pH range. "The change in binding activity depending on pH conditions" refers to the change in the antigen-binding activity of an antigen-binding molecule due to the difference between high hydrogen ion concentration or low pH (acidic pH range) and low hydrogen ion concentration or high pH (neutral pH range). For example, this may be the case when the antigen-binding activity of an antigen-binding molecule is higher in the neutral pH range than in the acidic pH range. Another example may be the case when the antigen-binding activity of an antigen-binding molecule is higher in the acidic pH range than in the neutral pH range.

[0151] In the present specification, the neutral pH range is not limited to a specific value, but can be suitably selected from the range of pH 6.7 to pH 10.0. In another embodiment, the neutral pH range can be selected from the range of pH 6.7 to pH 9.5. In another embodiment, the pH may be selected from the range of pH 7.0 to pH 9.0, and in another embodiment, the pH may be selected from the range of pH 7.0 to pH 8.0. In particular, a pH of 7.4, which is close to the pH in plasma (blood) in vivo, is preferred.

[0152] As used herein, the acidic pH range is not limited to a specific numerical value, but may preferably be selected from the range of pH 4.0 to pH 6.5. In another embodiment, it may be selected from the range of pH 4.5 to pH 6.5. In a different embodiment, it may be selected from the range of pH 5.0 to pH 6.5, and in another embodiment, it may be selected from the range of pH 5.5 to pH 6.5. In particular, a pH of 5.8, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0153] In the present invention, the antigen-binding activity of an antigen-binding molecule at a high proton concentration or low pH (acidic pH range) is lower than that at a low proton concentration or high pH (neutral pH range) means that the antigen-binding activity of the antigen-binding molecule at a pH selected from the range of pH 4.0 to pH 6.5 is lower than that at a pH selected from the range of pH 6.7 to pH 10.0. It means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 4.5 and pH 6.5 is weaker than that at a pH selected from between pH 6.7 and pH 9.5. It more preferably means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 5.0 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 9.0. It also preferably means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 5.5 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 8.0. It particularly preferably means that the antigen-binding activity at a pH in early endosomes in vivo is weaker than that at the pH of plasma in vivo. Specifically, it means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than that at pH 5.8. This means that the binding activity to the antigen at pH 7.4 is weaker than the binding activity to the antigen at pH 7.4.

[0154] Whether the antigen-binding activity of the antigen-binding domain of the present invention changes depending on pH conditions can be determined, for example, by using known measurement methods such as those described above in the section on binding activity. That is, the binding activity is measured under different pH conditions in the measurement method. For example, to confirm that the antigen-binding activity of an antigen-binding molecule comprising an antigen-binding domain of the present invention under a neutral pH condition is higher than that under an acidic pH condition, the antigen-binding activities of the antigen-binding molecule under acidic and neutral pH conditions are compared.

[0155] Furthermore, in the present invention, the expression "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" can also be expressed as meaning that the antigen-binding activity of an antigen-binding molecule at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range. "The antigen-binding activity at a high proton concentration or low pH, i.e., in the acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in the neutral pH range" can also be written as "The antigen-binding activity at a high proton concentration or low pH, i.e., in the acidic pH range, is weaker than that at a low proton concentration or high pH, ​​i.e., in the neutral pH range." Similarly, "The antigen-binding activity at a high proton concentration or low pH, i.e., in the acidic pH range is reduced to be lower than that at a low proton concentration or high pH, ​​i.e., in the neutral pH range" can also be written as "The antigen-binding activity at a high proton concentration or low pH, i.e., in the acidic pH range, is weaker than that at a low proton concentration or high pH, ​​i.e., in the neutral pH range."

[0156] Conditions other than hydrogen ion concentration or pH when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, HEPES buffer, 37°C For example, Biacore (GE Healthcare) can be used. When measuring the binding activity between an antigen-binding molecule containing an antigen-binding domain and an antigen, if the antigen is a soluble antigen, the binding activity for the soluble antigen can be evaluated by passing the antigen as an analyte through a chip onto which an antigen-binding molecule containing an antigen-binding domain has been immobilized. When the antigen is a membrane-type antigen, the binding activity for the membrane-type antigen can be evaluated by passing the antigen-binding molecule containing the antigen-binding domain as an analyte through a chip onto which the antigen has been immobilized.

[0157] In the antigen-binding molecules of the present invention, as long as the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weaker than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, the ratio of the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, to the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is not particularly limited, but is preferably KD (Dissociation Density) for the antigen at a high proton concentration or low pH, i.e., in an acidic pH range. The ratio of KD(pH5.8) / KD(pH7.4), which is the ratio of the dissociation constant (KD) to the KD at low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, is 2 or more, more preferably 10 or more, and even more preferably 40 or more. There is no particular upper limit to the KD(pH5.8) / KD(pH7.4) value, and it may be any value, such as 400, 1000, or 10,000, as long as it can be produced by those skilled in the art.

[0158] As the value of binding activity to an antigen, KD (dissociation constant) can be used when the antigen is a soluble antigen, whereas apparent KD (apparent dissociation constant) can be used when the antigen is a membrane antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard plots, flow cytometers, etc. It is possible to do so.

[0159] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention at a high proton concentration or low pH, i.e., an acidic pH range, to that at a low proton concentration or high pH, ​​i.e., a neutral pH range. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of the binding activity ratio, the ratio of kd (dissociation rate constant) at a high proton concentration or low pH, i.e., an acidic pH range, to kd (dissociation rate constant) at a low proton concentration or high pH, ​​i.e., a neutral pH range, i.e., kd (at an acidic pH range) / kd (at a neutral pH range), is preferably is 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. Kd (in an acidic pH range) / kd (in a neutral pH range) The upper limit of the value is not particularly limited, and may be 50, 100, 200, or the like as long as it can be produced within the technical common sense of a person skilled in the art. Any value is acceptable.

[0160] As the value of antigen-binding activity, kd (dissociation rate constant) can be used when the antigen is a soluble antigen, and apparent kd (apparent dissociation rate constant) can be used when the antigen is a membrane-type antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), a flow cytometer, etc. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different hydrogen ion concentrations, i.e., pH, it is preferable to keep the conditions other than the hydrogen ion concentration, i.e., pH, the same.

[0161] For example, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antibodies, comprising the following steps (a) to (c): (a) Obtaining the antigen-binding activity of an antigen-binding domain or antibody under an acidic pH range condition , (b) Obtaining the antigen-binding activity of the antigen-binding domain or antibody under neutral pH conditions , and (c) The antigen-binding activity in the acidic pH range is higher than that in the neutral pH range. Selecting for lower antigen-binding domains or antibodies.

[0162] Furthermore, an embodiment of the present invention, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, can be obtained by screening antigen-binding domains or antibodies or a library thereof, comprising the following steps (a) to (c): (a) Antigen-binding domains or antibodies or libraries thereof under neutral pH conditions contacting the antibody with an antigen; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under an acidic pH range; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0163] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under an acidic pH range; The course, (b) selecting antigen-binding domains or antibodies that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to an antigen in a neutral pH range; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0164] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) Immobilizing a library of antigen-binding domains or antibodies on a column containing immobilized antigens at a neutral pH. contacting the (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under an acidic pH condition; and (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0165] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) Immobilizing a library of antigen-binding domains or antibodies on a column immobilized with an antigen under acidic pH conditions passing the (b) recovering the antigen-binding domain or antibody that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antibody recovered in step (b) to bind to an antigen in a neutral pH range; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0166] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under a neutral pH range; The course, (b) obtaining the antigen-binding domain or antibody that bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under an acidic pH condition; and (d) selecting an antigen-binding domain in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b); Isolating the antibody or protein.

[0167] The above steps may be repeated two or more times. In the cleaning method, the steps (a) to (c) or (a) to (d) are repeated two or more times. Furthermore, there is provided an antigen-binding domain or antibody having lower antigen-binding activity in an acidic pH range than in a neutral pH range, obtained by a screening method comprising the steps of: (a) repeating steps (a) to (c) or (a) to (d) the number of times steps (a) to (c) or (a) to (d) are repeated, but is generally not more than 10 times.

[0168] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under high hydrogen ion concentration conditions or low pH, i.e., in the acidic pH range, is not particularly limited as long as it is antigen-binding activity at a pH between 4.0 and 6.5, but a preferred pH includes antigen-binding activity at a pH between 4.5 and 6.6. Another preferred pH includes antigen-binding activity at a pH between 5.0 and 6.5, and even more preferred pH includes antigen-binding activity at a pH between 5.5 and 6.5. A more preferred pH includes the pH in early endosomes in vivo, specifically, antigen-binding activity at pH 5.8. Furthermore, the antigen-binding activity of an antigen-binding domain or antibody under conditions of low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, can be particularly high if the antigen-binding activity is between pH 6.7 and 10. Although not limited thereto, preferred examples of the antigen-binding activity include a pH between 6.7 and 9.5. Other preferred examples of the antigen-binding activity include a pH between 7.0 and 9.5, and even more preferred examples of the antigen-binding activity between 7.0 and 8.0. A more preferred pH is the pH in plasma in vivo, specifically, antigen-binding activity at a pH of 7.4.

[0169] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), which is the dissociation rate. These can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scan Chard plots, FACS, etc. can be used.

[0170] In the present invention, the step of selecting antigen-binding domains or antibodies whose antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range, means the same as the step of selecting antigen-binding domains or antibodies whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range.

[0171] As long as the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in the neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in the acidic pH range, the difference between the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in the neutral pH range, and that at a high proton concentration or low pH, i.e., in the acidic pH range, is not particularly limited, but preferably the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in the neutral pH range, is at least twice as high as that at a high proton concentration or low pH, i.e., in the acidic pH range. It is more preferably 10 times or more, and even more preferably 40 times or more.

[0172] The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains or antibodies, and it is possible to screen, for example, the antigen-binding domains or antibodies described above. For example, antigen-binding domains or antibodies having native sequences may be screened, or antigen-binding domains or antibodies with substituted amino acid sequences may be screened.

[0173] The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be prepared in any manner, and may be prepared, for example, from pre-existing antibodies, pre-existing libraries (phage libraries, etc.), hybridomas obtained by immunizing animals, antibodies or libraries prepared from B cells from immunized animals, or from these antibodies or libraries. It is possible to use antibodies or libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced into the library (libraries with a high content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, or libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites).

[0174] Antigens produced from hybridomas obtained by immunizing animals or B cells from immunized animals As a method for obtaining an antigen-binding domain or antibody from a binding domain or antibody, the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in the neutral pH range, is higher than the antigen-binding activity at a high proton concentration or low pH, i.e., in the acidic pH range, for example, a method for obtaining an antigen-binding domain or antibody from a binding domain or antibody, wherein at least one of the amino acids in the antigen-binding domain or antibody is a nucleotide sequence corresponding to a nucleotide sequence at a side Preferred examples of such antigen-binding molecules or antibodies include those in which the amino acid sequence has been substituted with an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid mutation, or in which an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid has been inserted into the antigen-binding domain or antibody.

[0175] The position at which the mutation of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is introduced is not particularly limited, and any site may be used as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range compared to before the substitution or insertion (the value of KD(acidic pH range) / KD(neutral pH range) is increased, or the value of kd(acidic pH range) / kd(neutral pH range) is increased). For example, when the antigen-binding molecule is an antibody, the variable region or CDR of the antibody may be introduced. The number of amino acids to be substituted with or inserted into amino acids (e.g., histidine or glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 can be appropriately determined by those skilled in the art. A single amino acid (e.g., histidine or glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 can be substituted, a single amino acid (e.g., histidine or glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 can be inserted, or two or more amino acids (e.g., histidine or glutamic acid) having a side chain pKa of 4.0-8.0 can be inserted. These amino acids can be substituted with glutamic acid or unnatural amino acids, and the side chain pKa of these amino acids is between 4.0 and 8.0. The above amino acids (e.g., histidine and glutamic acid) or unnatural amino acids can be inserted. In addition to substitution with or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, deletion, addition, insertion, and / or substitution of other amino acids can also be performed simultaneously. Substitution with or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids can be carried out randomly by methods known to those skilled in the art, such as histidine scanning, in which alanine in alanine scanning is replaced with histidine, and antigen-binding molecules with increased KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / kd (neutral pH range) values ​​compared to before mutation can be selected from antigen-binding domains or antibodies into which mutations such as substitution or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been randomly introduced.

[0176] Preferred examples of antigen-binding molecules that have been mutated to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids and have lower antigen-binding activity in the acidic pH range than in the neutral pH range include antigen-binding molecules whose antigen-binding activity in the neutral pH range after mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids is equivalent to the antigen-binding activity in the neutral pH range before mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids. In the present invention, an antigen-binding molecule after mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid has antigen-binding activity equivalent to that of an antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid means that, when the antigen-binding activity of the antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is taken as 100%, This means that the antigen-binding activity of an antigen-binding molecule after mutation of an amino acid (e.g., histidine or glutamic acid) or unnatural amino acid is at least 10%, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. The antigen binding activity at pH 7.4 after mutation of unnatural amino acids (e.g., histidine and glutamic acid) was The antigen-binding activity at pH 7.4 may be higher than that before the mutation of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid. If the antigen-binding activity of an antigen-binding molecule is reduced by the insertion, the antigen-binding activity can be made equivalent to the antigen-binding activity before the substitution or insertion of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid by substitution, deletion, addition, and / or insertion of one or more amino acids in the antigen-binding molecule. In the present invention, the substitution, deletion, addition, and / or insertion of one or more amino acids after the substitution or insertion of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is performed. Also included are antigen-binding molecules whose binding activity is equivalent to that of the original antigen-binding molecules.

[0177] Furthermore, when the antigen-binding molecule is a substance comprising an antibody constant region, another preferred embodiment of the antigen-binding molecule has lower antigen-binding activity in an acidic pH range than in a neutral pH range, for example, by modifying the antibody constant region contained in the antigen-binding molecule. Specific examples of the modified antibody constant region include the constant regions set forth in SEQ ID NOs: 9, 10, 11, and 12.

[0178] (4) Amino acids that change the binding activity of the antigen-binding domain to the antigen depending on the hydrogen ion concentration. The antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when the ion concentration conditions are hydrogen ion concentration conditions or pH conditions, they may be prepared from pre-existing antibodies, pre-existing libraries (phage libraries, etc.), hybridomas obtained by immunizing animals, antibodies or libraries prepared from B cells from immunized animals, or antibodies or libraries derived from these antibodies or libraries. It is possible to use antibodies or libraries into which mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been introduced (libraries with a high content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, or libraries into which mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been introduced at specific sites). Suitable examples of such electron-donating amino acids include natural amino acids such as histidine and glutamic acid, as well as histidine analogs (US20090035836) and unnatural amino acids such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768). Unnatural amino acids are artificially modified to have a pKa (Angew. Chem. Int. Ed. (2005) 44, 34, Chem Soc Rev. (2004) 33 (7), 422-430, Amino Acids. (1999) 16 (3-4), 345-379).

[0179] In one non-limiting embodiment of the present invention, a library comprising a plurality of antigen-binding molecules of the present invention having different sequences can also be prepared by combining a light chain variable region into which "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library.

[0180] Non-limiting examples of such amino acid residues include those contained in light chain CDR1. Other non-limiting examples of such amino acid residues include those contained in light chain CDR2. Further non-limiting examples of such amino acid residues include those contained in light chain CDR3.

[0181] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include the amino acid residues at positions 24, 27, 28, 31, 32, and / or 34 according to the Kabat numbering in the CDR1 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR2 include the amino acid residues at positions 24, 27, 28, 31, 32, and / or 34 according to the Kabat numbering in the CDR2 of the light chain variable region. amino acid residues at positions 50, 51, 52, 53, 54, 55 and / or 56, as represented by the Further, non-limiting examples of the amino acid residues contained in the CDR3 of the light chain include those at positions 89, 90, 91, 92, and 93 according to the Kabat numbering in the CDR3 of the light chain variable region. Examples of such amino acid residues include those at positions 91, 92, 93, 94, and / or 95A. These amino acid residues may be contained alone or in combination of two or more, as long as the antigen-binding activity of the antigen-binding molecule changes depending on the hydrogen ion concentration.

[0182] Non-limiting examples of positions to be substituted with histidine or unnatural amino acids in the present invention include the positions described in WO2009 / 125825 shown below. are shown in Kabat numbering. Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, H102 Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, L94

[0183] Among these alterations, H32, H61, L53, L90, and L94 are considered to be highly universal alterations. However, the present invention is not limited to this and can be appropriately designed depending on the purpose.

[0184] Furthermore, although not particularly limited, preferred alteration sites when the antigen is an IL-6 receptor (for example, human IL-6 receptor) include the following: Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, H102 Light chain: L24, L27, L28, L32, L53, L56, L90, L92, L94

[0185] Specific preferred combinations of substitutions of histidine or unnatural amino acids at multiple sites in combination include, for example, the combination of H27, H31, and H35; the combination of H27, H31, H32, H35, H58, H62, and H102; the combination of L32 and L53; and the combination of L28, L32, and L53. Furthermore, preferred combinations of substitution sites in the heavy chain and light chain include the combination of H27, H31, L32, and L53. Only one of these sites may be substituted with histidine or an unnatural amino acid, or multiple sites may be substituted with histidine or an unnatural amino acid.

[0186] Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, another method for changing the antigen binding of the antigen-binding molecule depending on ion concentration conditions includes a method of modifying amino acids in the antibody constant region. Specific examples of such antibody constant regions include the antibody constant regions described in the Examples of WO2009 / 125825 (SEQ ID NO: 13, SEQ ID NO: 14) , SEQ ID NO: 15, SEQ ID NO: 16). Another method for modifying an antibody constant region includes, for example, examining multiple constant region isotypes (IgG1, IgG2, IgG3, IgG4) and selecting an isotype that has reduced antigen-binding activity in the acidic pH range (faster dissociation rate in the acidic pH range). Another method includes 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 in the acidic pH range (faster dissociation rate in the acidic pH range). The hinge region sequence of an antibody constant region varies 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 that has reduced antigen-binding activity in the acidic pH range (faster dissociation rate in the acidic pH range) under ion concentration conditions, for example, an isotype that has reduced antigen-binding activity in the acidic pH range (faster dissociation rate in the acidic pH range). Furthermore, because differences in the amino acid sequence of the hinge region have a significant effect on antigen-binding activity, the hinge region is considered to be a desirable location for amino acid substitution in the amino acid sequence of the wild-type isotype.

[0187] Antigen-binding molecules whose antigen-binding activity changes depending on ion concentration conditions can be prepared by using the above-mentioned methods to perform amino acid substitution or insertion into antigen-binding molecules that do not have such properties. However, 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 (mice, rats, hamsters, rabbits, human immunoglobulin transgenic mice, human immunoglobulin transgenic rats, human immunoglobulin transgenic rabbits, llamas, camels, etc.) with an antigen and screening the resulting antibodies using ion concentration-dependent antigen binding as an index. Alternatively, antibodies with the desired properties can be directly obtained from an in vitro presented antibody library by screening antibodies that bind to ion concentration-dependent antigens. Screening can be performed using the binding between the antibody and the target antibody as an index, and antibodies having the desired properties can be directly obtained, and the method is not particularly limited.

[0188] Amino acid modification To modify the amino acids in the antigen-binding domain, site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR (overlap extension PCR) can be used. Known methods such as the above can be appropriately employed. Furthermore, several known methods can also be employed as methods for modifying amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is a type of stop codon, can also be suitably used.

[0189] Neutralizing activity In one non-limiting embodiment, the present invention provides a pharmaceutical composition comprising an antigen-binding molecule comprising an FcRn-binding domain, an antigen-binding domain whose antigen-binding activity changes depending on ion concentration, and one or more carbohydrate receptor-binding domains whose carbohydrate receptor-binding activity changes depending on ion concentration. Generally, neutralizing activity refers to the activity of inhibiting the biological activity of a ligand, such as a virus or toxin, that has biological activity against cells. Specifically, a substance with neutralizing activity refers to a substance that binds to the ligand or a receptor to which the ligand binds, and inhibits the binding of the ligand to the receptor. A receptor whose binding to a ligand is blocked by neutralizing activity is no longer able to exert biological activity mediated by the receptor. When the antigen-binding molecule is an antibody, an antibody with such neutralizing activity is generally called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand with that in the presence or absence of the test substance.

[0190] For example, the main ligand of IL-6R is represented by SEQ ID NO: 17. A suitable example is IL-6, which is a type I membrane protein whose amino terminus forms the extracellular domain. IL-6R, a protein, forms a heterotetramer with the gp130 receptor, the dimerization of which is induced by IL-6 (HEINRICH et al. (Biochem. J. (1998) 334, 297-314)). Tetramer formation activates Jaks associated with the gp130 receptor. The phosphorylation sites of the receptor and Jak act as binding sites for SH2-containing Stat family members such as Stat3, MAP kinases, PI3 / Akt, and other SH2-containing proteins and adaptors. Stat bound to Stat is phosphorylated by Jak. The phosphorylated Stat forms a dimer and enters the nucleus. and regulate the transcription of target genes. Jak or Stat receptors are translocated via other classes of receptors. Deregulated IL-6 signaling cascades are observed in the pathology of autoimmune diseases, inflammation, and cancers such as multiple myeloma and prostate cancer. Stat3, which can act as an oncogene, is constitutively activated in many cancers. In multiple myeloma, the signaling cascade from IL-6R and epidermal growth factor receptor (EGFR) There is crosstalk between signaling cascades from family members (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

[0191] Since these intracellular signal cascades differ for each cell type, target molecules can be set appropriately for each target cell of interest and are not limited to the factors mentioned above. Neutralizing activity can be evaluated by measuring the activation of in vivo signals. In addition, activation of in vivo signals can also be detected using the transcription induction effect on target genes located downstream of the in vivo signal cascade as an indicator. Changes in the transcription activity of target genes can be detected using the principle of reporter assay. Specifically, the transcription factor of the target gene Or GFP (Green Fluorescence Protein) or luciferase downstream of the promoter region By placing a reporter gene such as a reporter gene and measuring its reporter activity, changes in transcription activity can be measured as reporter activity. Commercially available kits for measuring activation of in vivo signaling can be used as appropriate (e.g., Mercury Pathway Profiling Luciferase System (Clontech)).

[0192] Furthermore, EGF receptors, which normally act on signal cascades that promote cell proliferation, The neutralizing activity of a neutralizing antibody can be evaluated by measuring the proliferation activity of target cells as a method for measuring the neutralizing activity of receptor ligands such as those of the EGF family. For example, the proliferation of cells whose proliferation is promoted by growth factors of the EGF family, such as HB-EGF, can be evaluated. The following methods are preferably used to evaluate or measure the inhibitory effect on HB-EGF proliferation based on the neutralizing activity of anti-HB-EGF antibodies. A method for evaluating or measuring the cell proliferation inhibitory activity in vitro is to measure the incorporation of [3H]-labeled thymidine added to the medium by living cells as an index of DNA replication ability. A simpler method is to use trypan blue. The dye exclusion method, which measures the ability of cells to exclude dyes such as β-glucan, and the MTT method are used. The latter method utilizes the ability of living cells to convert the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue formazan product. More specifically, a test antibody is added to the culture medium of test cells together with a ligand, and after a certain period of time has passed, an MTT solution is added to the culture medium and allowed to stand for a certain period of time, allowing MTT to be taken up by the cells. As a result, the yellow compound MTT is absorbed into the mitochondria within the cells. The blue product is converted into a blue compound by succinate dehydrogenase. After dissolving and coloring this blue product, the absorbance is measured and used as an indicator of the number of living cells. Reagents such as MTS, XTT, WST-1, and WST-8 are also commercially available (Nacalai Tesque, etc.) and are suitable. When measuring the activity, a control antibody having the same isotype as the anti-HB-EGF antibody but lacking the cell growth inhibitory activity can be used in the same manner as the anti-HB-EGF antibody, and the activity can be determined by determining whether the anti-HB-EGF antibody exhibits stronger cell growth inhibitory activity than the control antibody.

[0193] Examples of cells for evaluating the activity include the ovarian cancer cell line RMG-1, whose proliferation is promoted by HB-EGF, and the extracellular domain of human EGFR and mouse GCSF receptor. hEGFR / mG-CSFR is a fusion protein in which the intracellular domains of the human EGFR and mG-CSFR are fused in-frame. Mouse Ba / F3 cells transformed with a vector ligated to express the coding gene. Thus, those skilled in the art can use cells for measuring the cell proliferation activity by appropriately selecting cells for evaluating the activity.

[0194] The antigen-binding molecules provided by the present invention can eliminate antigens from plasma, and therefore the antigen-binding molecules themselves do not necessarily need to have neutralizing activity. However, it is more preferable that the antigen-binding molecules exert neutralizing activity against the antigen during the period until the antigen is taken up into cells expressing Fcγ receptors via Fcγ receptor-mediated endocytosis.

[0195] Furthermore, the antigen-binding molecules provided by the present invention can promote the intracellular dissociation of antigens bound to the antigen-binding molecules extracellularly, and therefore, antigens dissociated from the antigen-binding molecules intracellularly are degraded in lysosomes. Therefore, it is not necessary for the antigen-binding molecules themselves to have neutralizing activity. However, it is more preferable that the antigen-binding molecules exert neutralizing activity against the antigen during the period until the antigen is taken up together with the antigen-binding molecules into cells expressing the glycosyl receptor by glycosyl receptor-mediated endocytosis, thereby blocking the function of the antigen present in plasma.

[0196] Furthermore, the antigen-binding molecules provided by the present invention can reduce the total antigen concentration or free antigen concentration in plasma. It is not necessarily required that the antigen-binding molecule itself has neutralizing activity, since the neutralizing activity can be reduced. However, it is more preferable that the antigen-binding molecule exerts neutralizing activity against the antigen during the period until the antigen is taken up into cells expressing the sugar chain receptor via sugar chain receptor-mediated endocytosis.

[0197] Carbohydrate receptors Glycans are a group of compounds in which various sugars are linked by glycosidic bonds. Many sugar chains in living organisms exist as complex molecules bound to proteins or lipids, and are collectively called glycoconjugates. Among these, glycoconjugates in which sugars are bound to proteins are called glycoproteins.

[0198] Examples of sugar chains recognized by the sugar chain receptor to which the sugar chain receptor-binding domain used in the present invention binds include sugar chains that constitute glycoproteins. Examples of sugar chains on glycoproteins include O-linked sugar chains and N-linked sugar chains. More preferably, examples of sugar chains on glycoproteins include N-linked sugar chains.

[0199] O-linked glycans of glycoproteins are formed by bonding the hydroxyl group of a serine or threonine residue to the O-glycan. The sugar directly linked to a serine or threonine residue is often N-acetylgalactosamine (GalNAc), and its core structure consists of (1) galactose (Gal) linked to N-acetylgalactosamine (GalNAc) via a β1-3 bond, (2) galactose linked to GalNAc via a β1-3 bond, and (1) GalNAc bound to GalNAc via a β1-6 bond and N-acetylglucosamine (GlcNAc) bound to GalNAc via a β1-3 bond, (2) GlcNAc bound to GalNAc via a β1-3 bond, (3) GlcNAc bound to GalNAc via a β1-3 bond, (4) GlcNAc bound to GalNAc via a β1-3 bond and GlcNAc bound to GalNAc via a β1-6 bond, (5) GlcNAc bound to GalNAc via a β1-6 bond, (6) GalNAc bound to GalNAc via a β1-6 bond, and (7) GalNAc bound to GalNAc via a β1-3 bond.

[0200] All N-linked glycans of glycoproteins share a common core structure, Man6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, called the trimannosyl core. Based on the structure and location of the sugar residues attached to the trimannosyl core, N-linked glycans can be classified into three subgroups: .

[0201] Complex glycans contain no mannose residues other than the trimannosyl core, and a GlcNAc residue is present at the reducing end of the side chain, linking it to two α-mannosyl residues on the trimannosyl core.

[0202] High-mannose glycans contain only α-mannose residues in addition to the trimannosyl core. The sugar chains of this group contain a common heptasaccharide core: Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

[0203] Complex glycans contain one or two α-mannosyl groups, similar to the high mannose type. The side chains of the complex-type glycans are linked to the Manα1-6 arm of the rimannosyl core, and the side chains of the complex-type glycans are linked to the Manα1-3 arm of the core. The presence or absence of a bisecting GlcNAc bond at the C-4 position of the β-mannosyl residue contributes to the structural diversity of complex-type glycans. Among the type glycan subgroups, the complex type contains the most diverse structures.

[0204] A carbohydrate receptor is a receptor that recognizes and binds to the above-mentioned carbohydrate chains. Any molecule can be used as a carbohydrate receptor as long as it binds to the recognized carbohydrate chain, but preferred receptors are receptors expressed on cells. For example, O-linked carbohydrate chains on CD99 bind to PILR (paired Ig-like type 2 receptor). It has been shown that ATP plays an important role in the binding of ATP to ATP (The Journal of Immunology gy (2008) vol.180 (3), 1686-1693). In addition, the N-linked galactose It is known that asialoglycoprotein receptors bind to N-linked glycans, and mannose receptors bind to N-linked glycans whose terminal is mannose. Such receptors are preferably used as sugar chain receptors in the present invention. That is, the antigen-binding molecules of the present invention have a domain that binds to such receptors (sugar chain receptor-binding domain). Therefore, when the sugar chain receptor is an asialoglycoprotein receptor, an N-linked sugar chain having a galactose terminal is preferably used in the present invention. When the sugar chain receptor is a mannose receptor, the sugar chain receptor can be suitably used as a sugar chain receptor-binding domain contained in an antigen-binding molecule in the present invention. Such a chain can be suitably used as the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention.

[0205] Carbohydrate receptor binding domain The antigen-binding molecules of the present invention have one or more binding domains to glycoreceptors, particularly human glycoreceptors. The type and number of binding domains to glycoreceptors, particularly human glycoreceptors, are not particularly limited, as long as the antigen-binding molecule has binding activity to glycoreceptors, particularly human glycoreceptors, in the neutral pH range and its binding activity to glycoreceptors in the acidic pH range is lower than that in the neutral pH range. Furthermore, domains that have binding activity to glycoreceptors, particularly human glycoreceptors, directly or indirectly can be used. Examples of such domains include glycans that directly have binding activity to glycoreceptors, particularly human glycoreceptors; Fc domains of IgG immunoglobulins; and domains that bind to glycoreceptors, particularly human glycoreceptors. Examples include antibodies against glycoconjugates; anti-glycosylated glycoconjugate receptors, particularly binding peptides against human glycoconjugate receptors; and scaffold molecules against glycoconjugate receptors, particularly human glycoconjugate receptors. In the present invention, a glycoconjugate receptor-binding domain that has binding activity to glycoconjugate receptors, particularly human glycoconjugate receptors, in the neutral pH range and whose binding activity to glycoconjugate receptors in the acidic pH range is lower than that in the neutral pH range is preferred. Such a domain can be used as is if it is a glycoconjugate receptor-binding domain that has binding activity to glycoconjugate receptors, particularly human glycoconjugate receptors, in the neutral pH range and whose binding activity to glycoconjugate receptors in the acidic pH range is lower than that in the neutral pH range. A glycoconjugate receptor-binding domain having an N-linked glycoconjugate terminally consisting of galactose. The binding activity of the glycan receptor-binding domain of the asialoglycoprotein receptor, which is a glycan receptor that binds to the glycan, in the acidic pH range is lower than the binding activity in the neutral pH range. A suitable example is one in which the binding activity to the mannose receptor, which is a sugar chain receptor that binds to the sugar chain, in an acidic pH range is lower than the binding activity in a neutral pH range.

[0206] When a glycan receptor-binding domain has no or weak binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range, the binding activity to a glycan receptor, particularly a human glycan receptor, can be obtained by modifying amino acids in the antigen-binding molecule. Furthermore, the binding activity to a glycan receptor, particularly a human glycan receptor, can be enhanced by modifying amino acids in a domain that already has binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range. The desired amino acid modification of a glycan receptor-binding domain, particularly a human glycan receptor, can be identified by comparing the binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range before and after the amino acid modification.

[0207] When the binding activity of a glycan receptor-binding domain to a glycan receptor, particularly a human glycan receptor, in the acidic pH range is not lower than the binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range, by modifying amino acids in the antigen-binding molecule, it is possible to obtain a binding activity that makes the binding activity to a glycan receptor, particularly a human glycan receptor, in the acidic pH range lower than the binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range. Amino acid modifications in the binding domain to a glycan receptor, particularly a human glycan receptor, can be performed by comparing the binding activity in the acidic pH range before and after the amino acid modification. The desired modification can be found by comparing the binding activity to a sugar chain receptor, particularly a human sugar chain receptor, with the binding activity to a sugar chain receptor, particularly a human sugar chain receptor, in the neutral range.

[0208] In the present invention, a carbohydrate receptor-binding domain can be introduced into a site other than the antigen-binding domain or FcRn-binding domain constituting an antigen-binding molecule. Furthermore, the carbohydrate receptor-binding domain can be introduced into any site in the structure of the antigen-binding molecule, as long as it does not inhibit antigen binding by the antigen-binding domain. The site can be introduced into the antigen-binding domain, or into any other site, as long as it does not inhibit antigen binding by the antigen-binding domain. In another embodiment, the carbohydrate receptor-binding domain can be introduced into any site in the structure of the antigen-binding molecule, as long as it does not inhibit binding between the FcRn-binding domain of the antigen-binding molecule and FcRn, particularly human FcRn. For example, the hinge region of an IgA antibody can be a candidate amino acid sequence for a carbohydrate receptor-binding domain for binding an O-linked carbohydrate chain, and a motif sequence for adding an N-linked carbohydrate chain, Asn-X-Ser / Thr, where X is an amino acid other than Pro, can be a candidate amino acid sequence for a carbohydrate receptor-binding domain for binding an N-linked carbohydrate chain. The antigen-binding molecules of the present invention containing a sugar chain receptor-binding domain to which a desired sugar chain is bound can be produced from the culture medium of host cells, as described below, into which a gene encoding an antigen-binding molecule containing such an amino acid sequence has been introduced.

[0209] In the present invention, the sugar chain receptor binding domain may be chemically prepared. For example, a galactose derivative mimicking an N-glycan having a galactose terminal or a high mannose type sugar chain may be used. Chemical ligands such as mannose derivatives mimicking sialic acid chains and derivatives mimicking sialic acid may be covalently conjugated to antigen-binding molecules. Examples of such chemical ligands include those described in Bioorg. Med. Chem. (2011) 19 (8), 2494-2500 and Bioorg. Med. Chem. (2009) 17 (20), 7254-7264, Bioorg. Med. Chem. (2008) 16 (9), 5216-5231, J. Am. Chem. Soc. (2004) 126 (33), 10355-10363, J. Pept. Sci. (2003) 9 (6), 375-385, Methods Enzymol. (2010) 478, 343-363, etc. Examples of conjugated amino acids at the antigen-binding site include, but are not limited to, lysine and cysteine. Conjugation to a specific site on an antigen-binding molecule can be achieved by methods known to those skilled in the art, such as substituting an amino acid at the conjugation site with cysteine, or substituting lysine at a site where conjugation is not desired with another amino acid. Conjugation can be achieved by methods known to those skilled in the art, such as reacting maleimide with the thiol of cysteine ​​or reacting an activated ester with lysine.

[0210] Conditions other than pH when measuring the binding activity to an antigen or FcRn, particularly human FcRn, can be appropriately selected by those skilled in the art and are not limited to a specific method. Measurement can be performed in MES buffer at 37°C so that the antibody titer of an antigen-binding molecule can be measured. The original binding activity and the binding activity to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art. For example, they can be measured using Biacore (GE Healthcare) or the like. When the antigen is a soluble antigen, the binding activity of the antigen-binding molecule to the 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. The binding activity of the antigen-binding molecule to FcRn, particularly human FcRn, can be measured by passing FcRn, particularly human FcRn, or the antigen-binding molecule as an analyte through a chip on which the antigen-binding molecule or FcRn, particularly human FcRn, is immobilized.

[0211] In the present invention, the binding activity to FcRn, particularly human FcRn, in an acidic pH range means the binding activity to FcRn, particularly human FcRn, at a pH of 4.0 to 6.5. The binding activity to FcRn, particularly human FcRn, in an acidic pH range is preferably the binding activity to FcRn at any pH between 5.5 and 6.5, for example, at a pH selected from pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. The term "antigen-binding activity" particularly preferably refers to the antigen-binding activity at any pH between 5.8 and 6.0, which is close to the pH in early endosomes in vivo, for example, at a pH selected from pH 5.80, 5.81, 5.82, 5.83, 5.84, 5.85, 5.86, 5.87, 5.88, 5.89, 5.90, 5.91, 5.92, 5.93, 5.94, 5.95, 5.96, 5.97, 5.98, 5.99, and 6.00.

[0212] The binding activity of an antigen-binding molecule of the present invention comprising an FcRn-binding domain to FcRn, particularly human FcRn, can be evaluated at any temperature between 10°C and 50°C when measuring binding activity. Preferably, any temperature between 15°C and 40°C can be used to measure the binding activity of an antigen-binding molecule of the present invention comprising an FcRn-binding domain to FcRn, particularly human FcRn. More preferably, any temperature between 20°C and 35°C, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, can be used to measure the binding activity of an antigen-binding molecule of the present invention comprising an FcRn-binding domain to FcRn, particularly human FcRn.

[0213] In the present invention, "not inhibiting antigen binding by an antigen-binding domain" means that the antigen-binding activity of an antigen-binding molecule, upon introduction of a glycan receptor-binding domain, maintains 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the antigen-binding activity of the antigen-binding molecule before introduction of the glycan receptor-binding domain. When the antigen-binding activity of an antigen-binding molecule decreases due to the introduction of a glycan receptor-binding domain, it can be changed to the same level as the antigen-binding activity before the introduction of the glycan receptor-binding domain by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after the introduction of such a glycan receptor-binding domain. Furthermore, "not inhibiting the binding of the glycan receptor-binding domain to FcRn, particularly human FcRn," means that the FcRn-binding activity of the antigen-binding molecule to FcRn, particularly human FcRn, maintained by the introduction of the glycan receptor-binding domain is 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the FcRn-binding activity of the antigen-binding molecule before the introduction of the glycan receptor-binding domain. When the binding activity of a molecule to FcRn, particularly human FcRn, is reduced, it can be changed to the same level as the binding activity to FcRn, particularly human FcRn, before the introduction of the sugar chain receptor-binding domain by, for example, substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after the introduction of such a sugar chain receptor-binding domain. Methods for measuring and determining the binding activity to an antigen or FcRn, particularly human FcRn, are described below.

[0214] Glycan Preferred examples of the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention include sugar chain receptor-binding domains to which a desired sugar chain is bound. Preferred examples include N-linked glycans and N-linked glycans, but For example, a series of enzymatic methods in a cell-free system, such as a method for producing a post-translationally modified antibody in a cell-free protein synthesis system (JP 2006-141241 A), which is characterized by preparing a cell extract from cultured cells of an immortalized mammalian cell line having protein secretion ability and adding mRNA encoding the antibody to the extract, can be used to produce a desired sugar chain-bound antibody. Furthermore, one such known method involves introducing into host cells a gene encoding a sugar chain receptor-binding domain contained in a naturally or artificially produced antigen-binding molecule into which a motif sequence for adding a desired sugar chain has been introduced by recombinant gene techniques or the like, thereby producing the antigen-binding molecule of the present invention containing a sugar chain receptor-binding domain to which a desired sugar chain is bound from the culture medium of the host cell.

[0215] When the glycan is an O-linked glycan, it will be possible to design the motif sequence for the addition of the O-linked glycan by using a publicly known database. O-linked glycans are added to the hinge region of IgA antibodies, and gene sequences encoding glycan receptor binding domains with such known O-linked glycans added will be candidates for the motif sequence. When the glycan is an N-linked glycan, it is known that the motif sequence for the addition of the N-linked glycan is a motif of three consecutive amino acids, Asn-X-Ser / Thr. For this reason, it is possible to design the motif sequence for the addition of the N-linked glycan by using recombinant By introducing a gene encoding an antigen-binding molecule containing a glycan receptor-binding domain designed to encode the motif sequence Asn-X-Ser / Thr for N-linked glycan addition by genetic techniques or the like into host cells as described below, the antigen-binding molecule of the present invention containing a glycan receptor-binding domain to which a desired glycan is bound can be produced from the culture medium of the host cells.

[0216] The glycan structure of the antigen-binding molecule produced as described above may be a single one, or it may be produced as a mixture with multiple glycans linked thereto. Such mixtures can also be preferably used in the present invention. Furthermore, antigen-binding molecules in which a specific glycan is linked to its glycan-binding domain can also be preferably used in the present invention.

[0217] Several known methods can be used to link specific sugar chains to the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention. One such known method is a method for purifying antigen-binding molecules having specific sugar chains by utilizing the properties of sugar chains possessed by natural or artificially produced antigen-binding molecules using recombinant genetic techniques, etc. It is known that antibodies having high-mannose sugar chains can be purified using affinity chromatography using ConA-sepharose (Millward (Biologicals (2008) 36, 49-60)). Such purification methods can be used in the present invention to prepare antigen-binding molecules having N-linked sugar chains in which the non-reducing end is mannose.

[0218] In another embodiment, enzyme treatment can be appropriately employed to obtain antigen-binding molecules having specific sugar chains. As described later in the Examples, N-glycosylation can be performed using an N-glycosylation method in which the non-reducing end is galactose. Antigen-binding molecules with conjugated sugar chains can be prepared by sialidase treatment of antigen-binding molecules with complex sugar chains in which the non-reducing end is sialic acid. It is also known that antibodies with high-mannose sugar chains can be prepared by removing galactose from the sugar chains by treatment with sialidase and β-galactosidase (Newkirk, Clin. Exp. Immunol. (1996) 106, 259-264). These preparations include sialidase and β-galactosidase treatments. The method of the present invention is to provide an antigen-binding molecule having an N-linked sugar chain in which the non-reducing end is mannose. can be used to make

[0219] In a different aspect, for the purpose of obtaining an antigen-binding molecule of the present invention having a specific sugar chain, a method of recovering the antigen-binding molecule from the culture medium of host cells into which a recombinant gene encoding the antigen-binding molecule has been transfected and whose glycosidase activity has been altered (by, including but not limited to, genetic or recombinant gene techniques) so that the specific sugar chain accumulates can also be appropriately employed. It is known that antibodies having N-linked sugar chains can be recovered from the culture medium of a Lec1 mutant strain derived from CHO cells lacking N-acetylglucosaminyltransferase I activity, into which a recombinant gene encoding the antibody has been transfected (Wright and Morrison (J. Exp. Med. (1994) 180, 1087-1096)). In the present invention, antibodies having N-linked sugar chains with mannose at the non-reducing end are Antigen-binding molecules can be recovered from the culture medium of a Lec1 mutant strain transduced with a recombinant gene encoding the molecule.

[0220] In yet another embodiment, for the purpose of obtaining an antigen-binding molecule of the present invention having a specific sugar chain, a method can be appropriately employed in which an inhibitor that inhibits a specific glycosidase reaction is added during the culture of cells that produce the antigen-binding molecule, and the antigen-binding molecule having a specific sugar chain that has accumulated in the culture medium is recovered. Antibodies with glycan-type glycosylation are produced by adding kifunesine to the CHO cells that produce the antibodies. It is known that the antigen-binding molecule can be recovered from the culture medium of the cells by the above-mentioned method (Zhou (Biotechnol. Bioeng. (2008) 99, 652-665)). In the present invention, an antigen-binding molecule having an N-linked sugar chain that does not have fucose at its reducing end and has mannose at its non-reducing end can be recovered from the culture medium of the cells by the above-mentioned method (Zhou (Biotechnol. Bioeng. (2008) 99, 652-665)). CHO cells transfected with a recombinant gene encoding the molecule were cultured with kifunesine. Furthermore, antigen-binding molecules of the present invention having a specific sugar chain can be obtained by, for example, combining a method in which such inhibitors are added during the culture of the host cells in which glycosidase activity has been altered. It is known that antibodies having a specific sugar chain can be recovered by such a combination (Kanda et al., 2004). (Glycobiology (2007) 17, 104-118)), similar combinations can be appropriately employed in the present invention for the purpose of obtaining antigen-binding molecules having specific sugar chains.

[0221] In addition, methods for expressing proteins with specific glycan structures by genetically modifying hosts such as Pichia pastoris are known (Biochemistry. 2008 Sep 30;47(39):10294-304., J Biotechnol. 2009 Feb 23;139(4):318-25., Nat Biotechnol. 2006 Feb;24(2):210-5.), and by using such methods, antigen-binding molecules can be prepared in which specific glycan structures (e.g., N-glycans with terminal galactose) are uniformly linked to N-glycosylation sequences.

[0222] An antigen-binding molecule comprising a glycan receptor-binding domain of the present invention, particularly an antigen-binding molecule comprising a human-derived glycan receptor-binding domain, binds to a glycan receptor in a pH-dependent manner and, alternatively, has glycan receptor-binding activity, particularly human-derived glycan receptor-binding activity, in the neutral pH range. If the binding activity to a glycan receptor in an acidic pH range can be made lower than the binding activity to a glycan receptor in the neutral pH range, it is possible to promote the uptake of antigens into cells by the antigen-binding molecule, promote the decrease in plasma antigen concentration by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by a single antigen-binding molecule.

[0223] Binding activity to carbohydrate receptors In the present invention, the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in an acidic pH range means the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, at a pH of 4.0 to 6.5, preferably at any pH of 5.5 to 6.5, for example, at a pH selected from pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. It means the binding activity to a sugar chain receptor of human origin, and particularly preferably means the binding activity to a sugar chain receptor, particularly a sugar chain receptor of human origin, at any pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, for example, a pH selected from pH 5.80, 5.81, 5.82, 5.83, 5.84, 5.85, 5.86, 5.87, 5.88, 5.89, 5.90, 5.91, 5.92, 5.93, 5.94, 5.95, 5.96, 5.97, 5.98, 5.99, and 6.00. Furthermore, in the present invention, the binding activity to a sugar chain receptor in a neutral pH range, particularly a sugar chain receptor of human origin, means the binding activity to a sugar chain receptor at pH 6.7 to pH 10.0, In particular, it means the binding activity to a sugar chain receptor of human origin. Preferably, it means the binding activity to a sugar chain receptor, in particular to a sugar chain receptor of human origin, at any pH between pH 7.0 and pH 8.0, for example, at a pH selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. It means a sugar chain receptor at pH 7.4, which is close to the pH of plasma in vivo, particularly a sugar chain receptor derived from a human. This refers to the binding activity to the original sugar chain receptor.

[0224] Conditions other than pH when measuring the binding activity to a glycosyl receptor, particularly a glycosyl receptor of human origin, can be appropriately selected by those skilled in the art and are not particularly limited to specific conditions. For example, as described in WO2009 / 125825, measurements can be performed in MES buffer at 37°C. Furthermore, the binding activity of antigen-binding molecules to glycosyl receptors, particularly glycosyl receptors of human origin, can be measured by methods known to those skilled in the art. For example, measurements can be performed using Biacore (GE Healthcare) or the like. The binding activity of an antigen-binding molecule to a glycosyl receptor, particularly a human-derived glycosyl receptor, can be measured, for example, by passing the antigen-binding molecule as an analyte over a chip on which a glycosyl receptor, particularly a human-derived glycosyl receptor molecule, is immobilized, thereby evaluating the binding activity of the antigen-binding molecule to a glycosyl receptor, particularly a human-derived glycosyl receptor. Conversely, the binding activity to a glycosyl receptor, particularly a human-derived glycosyl receptor, can be evaluated by passing a solubilized glycosyl receptor, particularly a human-derived glycosyl receptor, over a chip on which an antigen-binding molecule is immobilized as an analyte.

[0225] In the present invention, the ratio of the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in the acidic pH range to the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in the neutral pH range is not particularly limited, as long as the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in the acidic pH range is weaker than the antigen-binding activity in the neutral pH range. Preferably, the KD (Dissociation constant) at pH 5.8 for a sugar chain receptor, particularly a human-derived sugar chain receptor, is The ratio of KD (pH 5.8) to KD (pH 7.4), KD (pH 5.8) / KD (pH 7.4), is preferably 2 or more. Preferably, the KD (pH 5.8) / KD (pH 7.4) value is 10 or more, and more preferably, the KD (pH 5.8) / KD (pH 7.4) value is 40 or more. There are no particular upper limits to the KD (pH 5.8) / KD (pH 7.4) value, and any value such as 400, 1000, or 10000 may be used as long as it can be produced by those skilled in the art.

[0226] The KD (dissociation constant) can be used as a value of binding activity to a sugar chain receptor, particularly a human sugar chain receptor. The KD (dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, and flow cytometers. etc. can be suitably used.

[0227] In the present invention, other indicators that indicate the ratio of the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in the acidic pH range to the binding activity to a sugar chain receptor, particularly a human-derived sugar chain receptor, in the neutral pH range include, for example, the dissociation rate constant k d (Dissociation rate constant) can also be suitably used as an index showing the ratio of binding activity instead of KD (dissociation constant). d When using the dissociation rate constant, the k d (dissociation rate constant) and k in the neutral pH range d (dissociation rate constant) ratio k d (pH acidic range) / k d The value of (neutral pH range) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. d (pH acidic range) / k d The upper limit of the value (neutral pH range) is not particularly limited, and can be set to 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.

[0228] For example, the interaction between galactose and the asialoglycoprotein receptor, a type of sugar chain receptor, is pH-dependent, exhibiting high binding activity in the neutral pH range and low binding activity in the acidic pH range (J Biol Chem Vol. 274, No. 50, pp. 35400-35406, 1999). Similarly, the interaction between mannose and the mannose receptor, a type of sugar chain receptor, is pH-dependent, exhibiting high binding activity in the neutral pH range and low binding activity in the acidic pH range (J Biol Chem. 1994 Nov 11;269(45):28405-13.). Therefore, in the present invention, the galactose / asialoglycoprotein receptor and the mannose / mannose receptor can be preferably used as the sugar chain / sugar chain receptor.

[0229] Furthermore, the binding activity of the galactose / asialoglycoprotein receptor and the mannose / mannose receptor is not only pH-dependent but also calcium ion concentration-dependent. Since most sugar chain receptors are C-type lectins, the binding between sugar chain receptors and sugar chains is The binding between a sugar chain receptor and a sugar chain may be calcium ion concentration dependent, similar to the binding between an antigen-binding molecule and an antigen, provided that the binding at a high calcium ion concentration is stronger than the binding at a low calcium ion concentration.

[0230] In the present invention, when measuring the binding activity of an antigen-binding molecule to a sugar chain receptor, particularly a human sugar chain receptor, at different pH levels, it is preferable to keep the conditions other than pH the same.

[0231] antigen binding molecule The present invention provides antigen-binding molecules comprising an antigen-binding domain, an FcRn-binding domain, and one or more glycoreceptor-binding domains, wherein the number of glycoreceptor-binding domains is increased, and the antigen-binding molecules further comprise antigen-binding domains whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range. The present invention also provides methods for producing the antigen-binding molecules, and pharmaceutical compositions comprising the antigen-binding molecules. Furthermore, the present invention provides methods for intracellular uptake of the antigen-binding molecules and / or antigens bound by the antigen-binding molecules, methods for increasing the number of antigens bound by the antigen-binding molecules per molecule, methods for reducing extracellular antigens, methods for improving the pharmacokinetics of the antigen-binding molecules, and methods for promoting dissociation of antigens from the antigen-binding molecules, all of which comprise contacting the antigen-binding molecules with cells expressing glycoreceptors in vivo or ex vivo. The present invention also provides methods for promoting in vivo or ex vivo cellular uptake of an antigen-binding molecule and / or an antigen to which the antigen-binding molecule binds, comprising increasing the number of binding domains for the antigen-binding domain, an FcRn-binding domain, and two or more binding domains for the carbohydrate receptor in an antigen-binding molecule; methods for increasing the number of antigens to which the antigen-binding molecule binds per molecule in vivo or ex vivo; methods for increasing the ability of the antigen-binding molecule to eliminate antigens in vivo or ex vivo; methods for improving the pharmacokinetics of the antigen-binding molecule; and methods for promoting dissociation of an antigen from the antigen-binding molecule.

[0232] Method for producing antigen-binding molecules The present invention provides methods for producing antigen-binding molecules that comprise an antigen-binding domain, an FcRn-binding domain in particular a human FcRn-binding domain, and one or more carbohydrate receptor-binding domains, and that have carbohydrate receptor-binding activity in a neutral pH range, lower carbohydrate receptor-binding activity in an acidic pH range than that in a neutral pH range, and lower antigen-binding activity in an acidic pH range than that in a neutral pH range. The present invention also provides methods for producing antigen-binding molecules that have an excellent effect of promoting the reduction of plasma antigen concentration upon administration of the antigen-binding molecule and have excellent pharmacokinetics. Furthermore, the present invention provides methods for producing antigen-binding molecules that are particularly useful when used as pharmaceutical compositions.

[0233] Specifically, the present invention provides a method for producing an antigen-binding molecule, comprising the steps of: (a) The polypeptide sequence of an antigen-binding molecule containing an antigen-binding domain and an FcRn-binding domain is provided. providing a (b) A candidate motif for the carbohydrate receptor-binding domain in the polypeptide sequence identifying the amino acid sequence; (c) designing a motif for a carbohydrate receptor binding domain comprising an amino acid sequence that differs from the amino acid sequence identified in (b) by at least one amino acid; (d) constructing a gene encoding a polypeptide of an antigen-binding molecule comprising the motif of the sugar chain receptor-binding domain designed in (c); (e) recovering the antigen-binding molecule from the culture medium of host cells transformed with the gene obtained in (d).

[0234] The steps (c) and (d) may be repeated two or more times. The number of times is not particularly limited, but is usually within 10 times. The production method of the present invention may also include a step of further treating the isolated antigen-binding molecule with an enzyme.

[0235] The antigen-binding domain contained in the antigen-binding molecule produced by the production method provided by the present invention can be provided by the method described above in the section "Antigen-binding domain."

[0236] Furthermore, an FcRn-binding domain having binding activity to FcRn, particularly human FcRn, contained in an antigen-binding molecule produced by the production method provided by the present invention can be provided by the method described above in the section "FcRn-binding domain." That is, the FcRn-binding domain is not particularly limited as long as it has binding activity to FcRn, particularly human FcRn, in the acidic pH range, and any domain that has binding activity to FcRn, particularly human FcRn, directly or indirectly can be used. Examples of such domains include IgG-type FcRn domains that have direct binding activity to FcRn, particularly human FcRn. Binding activity to the Fc region of immunoglobulin, albumin, albumin domain 3, anti-human FcRn antibody, anti-human FcRn peptide, anti-human FcRn scaffold molecule, etc., or indirectly to human FcRn Examples of such molecules include IgG having the above structure and molecules that bind to albumin. A polypeptide sequence of a domain that directly or indirectly has binding activity to FcRn, particularly human FcRn, can be provided as the polypeptide sequence of the antigen-binding domain.

[0237] Preferred examples of glycan receptor-binding domains contained in antigen-binding molecules produced by the production methods provided by the present invention include glycan receptor-binding domains to which a desired glycan is bound. Preferred examples of the desired glycan include O-linked glycans and N-linked glycans, and known methods can be used to bind a glycan to a glycan receptor-binding domain. One such known method involves introducing into host cells a gene encoding the glycan receptor-binding domain contained in a naturally or artificially produced antigen-binding molecule into which a motif sequence for adding a desired glycan (i.e., a motif for a glycan receptor-binding domain) has been introduced by recombinant gene techniques, etc., thereby producing antigen-binding molecules of the present invention containing a glycan receptor-binding domain to which a desired glycan is bound from the culture medium of the host cells.

[0238] In the polypeptide sequence provided as described above, the amino acid sequence that is a candidate for the motif of the sugar chain receptor binding domain can be identified by, for example, the following method. The motif sequence for O-linked sugar chain addition can be identified by using a known database, etc. Therefore, it can be identified. Furthermore, when the hinge portion of an antibody is contained in an antigen-binding molecule of the present invention, it can be identified from which antibody isotype the hinge portion is derived. Such hinge portions can be identified as candidate motifs for the O-glycan receptor-binding domain. Furthermore, when the glycan is an N-linked glycan, the motif sequence for N-linked glycan addition (i.e., the motif of the N-glycan receptor binding domain) is known to be a motif of three consecutive amino acids, Asn-X-Ser / Thr. Designed to encode the Asn-X-Ser / Thr motif of the glycan receptor-binding domain For the purpose of designing a specific carbohydrate receptor binding domain, the presence or absence of a sequence identical or similar to Asn-X-Ser / Thr in the provided polypeptide sequence can be identified.

[0239] A motif for a sugar chain receptor-binding domain containing an amino acid sequence that differs from the amino acid sequence identified above by at least one amino acid can be designed. For example, by using a known database, etc., the motif sequence for adding an O-linked sugar chain identified above can be designed. By substituting amino acids in the candidate polypeptide sequence, O-linked glycan motifs Furthermore, when the hinge portion of an antibody contains an antigen-binding molecule of the present invention, if the hinge portion does not contain a hinge portion derived from an IgA antibody, the hinge sequence can be designed. An O-linked glycan motif sequence can be designed by substituting the sequence of the hinge region derived from an IgA antibody for the sequence of the O-linked glycan motif. If no sequence identical or similar to Asn-X-Ser / Thr is identified in the peptide sequence, by inserting an Asn-X-Ser / Thr sequence at an appropriate position in the provided polypeptide sequence. By doing so, a new motif for an N-linked sugar chain receptor binding domain can be added. Substitute amino acid residues of similar sequences of Asn-X-Ser / Thr sequences found in the polypeptide sequence By this, the analogous sequence can be modified to an Asn-X-Ser / Thr sequence, which is the motif of the N-linked sugar chain receptor binding domain.

[0240] The antigen-binding molecules of the present invention have one or more binding domains to glycoreceptors, particularly human glycoreceptors. The type and number of binding domains to glycoreceptors, particularly human glycoreceptors, are not particularly limited, as long as the antigen-binding molecule has binding activity to glycoreceptors, particularly human glycoreceptors, in the neutral pH range and its binding activity to glycoreceptors in the acidic pH range is lower than that in the neutral pH range. Furthermore, domains that have binding activity to glycoreceptors, particularly human glycoreceptors, directly or indirectly can be used. Examples of such domains include glycans that directly have binding activity to glycoreceptors, particularly human glycoreceptors; the Fc domain of IgG immunoglobulin; and antibodies to glycoreceptors, particularly human glycoreceptors. Examples include anti-glycosylated receptors, particularly binding peptides to human glycan receptors; and scaffold molecules to glycan receptors, particularly human glycan receptors. In the present invention, preferred glycan receptor-binding domains have binding activity to glycan receptors, particularly human glycan receptors, in the neutral pH range and have lower binding activity to glycan receptors in the acidic pH range than that in the neutral pH range. Such domains can be used as they are, provided that they have binding activity to glycan receptors, particularly human glycan receptors, in the neutral pH range and have lower binding activity to glycan receptors in the acidic pH range than that in the neutral pH range. A glycan receptor-binding domain having an N-linked glycan with a terminal galactose and a glycan receptor-binding domain having the glycan receptor in the acidic pH range can be used as is. A preferred example is a glycan receptor-binding domain having an N-linked glycan with a mannose terminal and a glycan receptor-binding domain having a binding activity to the asialoglycoprotein receptor, which is a glycan receptor that binds to the glycan, in an acidic pH range, which is lower than the binding activity in a neutral pH range. The binding activity to the mannose receptor, a sugar chain receptor, in the acidic pH range is also a preferred example of a lower binding activity than in the neutral pH range.

[0241] When a glycan receptor-binding domain has no or weak binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range, the binding activity to a glycan receptor, particularly a human glycan receptor, can be obtained by modifying amino acids in the antigen-binding molecule. Furthermore, the binding activity to a glycan receptor, particularly a human glycan receptor, can be enhanced by modifying amino acids in a domain that already has binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range. The desired amino acid modification of a glycan receptor-binding domain, particularly a human glycan receptor, can be identified by comparing the binding activity to a glycan receptor, particularly a human glycan receptor, in the neutral pH range before and after the amino acid modification.

[0242] When the binding activity of a glycan receptor-binding domain to a glycan receptor, particularly a human glycan receptor, in an acidic pH range is not lower than the binding activity to a glycan receptor, particularly a human glycan receptor, in a neutral pH range, it is possible to obtain a binding activity that is lower in an acidic pH range than in a neutral pH range by modifying amino acids in the antigen-binding molecule. The desired amino acid modification of the binding domain to a glycan receptor, particularly a human glycan receptor, can be identified by comparing the binding activity to a glycan receptor, particularly a human glycan receptor, in an acidic pH range with the binding activity to a glycan receptor, particularly a human glycan receptor, in a neutral pH range before and after the amino acid modification.

[0243] In the present invention, a carbohydrate receptor-binding domain can be introduced into any site in the structure of an antigen-binding molecule, as long as it does not inhibit antigen binding by the antigen-binding domain. The site can be introduced into the antigen-binding domain, or into any other site, as long as it does not inhibit antigen binding by the antigen-binding domain. In another embodiment, a carbohydrate receptor-binding domain can be introduced into any site in the structure of an antigen-binding molecule, as long as it does not inhibit binding between the FcRn-binding domain of the antigen-binding molecule and FcRn, particularly human FcRn. For example, the hinge region of an IgA antibody contains an O-linked It can be a candidate amino acid sequence for the glycan receptor binding domain for glycan binding, and the motif sequence for N-linked glycan addition, Asn-X-Ser / Thr, is a potential candidate for the N-linked glycan binding domain. These can be candidate amino acid sequences for the sugar chain receptor-binding domain. Antigen-binding molecules of the present invention containing a sugar chain receptor-binding domain to which a desired sugar chain is bound can be produced from the culture medium of host cells, as described below, into which a gene encoding an antigen-binding molecule containing such an amino acid sequence has been introduced.

[0244] In the present invention, "not inhibiting antigen binding by an antigen-binding domain" means that the antigen-binding activity of an antigen-binding molecule, upon introduction of a glycan receptor-binding domain, maintains 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the antigen-binding activity of the antigen-binding molecule before introduction of the glycan receptor-binding domain. When the antigen-binding activity of an antigen-binding molecule decreases due to the introduction of a glycan receptor-binding domain, it can be changed to the same level as the antigen-binding activity before the introduction of the glycan receptor-binding domain by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after the introduction of such a glycan receptor-binding domain. Furthermore, "not inhibiting the binding of the glycan receptor-binding domain to FcRn, particularly human FcRn," means that the FcRn-binding activity of the antigen-binding molecule to FcRn, particularly human FcRn, maintained by the introduction of the glycan receptor-binding domain is 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the FcRn-binding activity of the antigen-binding molecule before the introduction of the glycan receptor-binding domain. When the binding activity of a molecule to FcRn, particularly human FcRn, is reduced, it can be changed to the same level as the binding activity to FcRn, particularly human FcRn, before the introduction of the sugar chain receptor-binding domain by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been made equivalent by substituting, deleting, adding, and / or inserting one or more amino acids after the introduction of such a sugar chain receptor-binding domain.

[0245] Furthermore, the sugar chain receptor-binding domain can also be introduced into sites other than the antigen-binding domain or FcRn-binding domain that constitute the antigen-binding molecule.

[0246] The structure of the antigen-binding molecule of interest in the present invention is not particularly limited, and antigen-binding molecules of any structure can be suitably used. However, preferred examples of antigen-binding molecules having an antigen-binding domain, an FcRn-binding domain, particularly a human FcRn-binding domain, and two or more sugar chain receptor domains include antibodies. Preferred examples of antibodies of the present invention include IgG antibodies. When an IgG antibody is used, the type is not limited, and isotypes such as IgG1, IgG2, IgG3, and IgG4 are also usable. IgG of any type (subclass) can be used. The constant region may be included, and amino acid mutations may be introduced into the constant region. Examples of amino acid mutations that may be introduced include, but are not limited to, those that increase or decrease binding to Fcγ receptors (Proc Natl Acad Sci USA. 1 (2006) 103 (11), 4005-10). Furthermore, by selecting an appropriate constant region, such as an IgG2 constant region, it is also possible to alter pH-dependent binding.

[0247] 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, modified antibodies with substituted amino acid sequences, such as chimeric antibodies, particularly humanized antibodies, are also preferably used. Bispecific antibodies, modified antibodies bound to various molecules, and polypeptides containing antibody fragments may also be used.

[0248] A "chimeric antibody" is an antibody that is made by combining sequences from different animals. Specific examples of the polymeric antibody include those obtained by combining the variable (V) regions of the heavy and light chains of a mouse antibody with those of a human antibody. Examples of antibodies include antibodies consisting of the constant (C) regions of the heavy and light chains of a human antibody.

[0249] The term "humanized antibody" refers to an antibody derived from a mammal other than a human, such as a mouse antibody, which is also called a reshaped human antibody, and which has complementarity determining regions (CDRs) of the antibody. The CDRs are 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 Institutes of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342, 877). General genetic recombination techniques are also known (EP125023 , WO1996 / 002576).

[0250] A bispecific antibody is an antibody that has variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody may recognize two or more different antigens, or it may recognize two or more different epitopes on the same antigen.

[0251] Examples of polypeptides containing antibody fragments include Fab fragments, F(ab')2 fragments, scFv (Nat Biotechnol. (2005) 23(9), 1126-36), domain antibodies (dAbs) (WO2004 / 058821, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, and Fc fusion proteins. Molecules containing an Fc region can use the Fc region as a binding domain to FcRn, particularly human FcRn. Molecules in which a human FcRn-binding domain is fused to these molecules can also be used.

[0252] Genes encoding the antigen-binding domain, FcRn-binding domain, and sugar chain receptor-binding domain designed as described above can be constructed. Gene construction methods are known, and they can be constructed by chemical synthesis or by dividing the nucleotides that make up the polynucleotides using PCR or other methods. They can also be prepared by linking them through an enzymatic reaction in the presence of a template primer or the like. The antigen-binding domain, FcRn-binding domain, and carbohydrate receptor-binding domain designed as described above can be linked by expressing each domain separately in a host cell described below, and then recovering the polypeptides from the culture medium or the like through a chemical reaction in the presence of a crosslinking agent. Synthetic chemical linkers (chemical crosslinking agents) can be crosslinked using crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl subtilisin (DSB), and cyclohexyl 1,2-diol (C1H2O). Dithiobis(succinimidyl)propionate (DSP), dithiobis(sulfosuccinimidyl)propionate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(sulfosuccinimidyl)propionate (DSP), dithiobis(sulfosuccinimidyl)propionate Dimethylsuccinimidyl succinate (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), Dimethylsuccinimidyl succinate (DTSSP ... Succinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinkers are commercially available.

[0253] Alternatively, the antigen-binding domain, FcRn-binding domain, and sugar chain receptor-binding domain designed as described above can be expressed in a host cell as described below, and then recovered from the culture medium or the like. When linking the domains in frame by peptide bonds, the domains can be linked directly or via a linker having a specific peptide sequence. The linker linking the domains can be any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (see, for example, Protein Engineering (1996) 9 (3), 299-305), but peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be selected appropriately by those skilled in the art depending on the purpose. However, a preferred length is 5 amino acids or more (the upper limit is not particularly limited, but typically 3 or more). 0 amino acids or less, preferably 20 amino acids or less, and particularly preferably 15 amino acids or less .

[0254] For example, for a peptide linker: Ser Gly·Ser Gly Gly Ser Ser Gly Gly Gly·Gly·Gly·Ser (SEQ ID NO: 18) Ser·Gly·Gly·Gly (SEQ ID NO: 19) Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 20) Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 21) Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 22) Ser·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 23) Gly·Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 24) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 25) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 20))n (Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 21))n [n is an integer of 1 or more]. However, the length and sequence of the peptide linker are Linkers that can be appropriately selected by those skilled in the art depending on the purpose can also be used in the present invention.

[0255] The gene obtained in the production method of the present invention is usually carried (inserted) into a suitable vector and introduced into a host cell. The vector is not particularly limited as long as it can stably retain the inserted nucleic acid. For example, if Escherichia coli is used as the host, a cloning vector such as pBluescript vector (Stratagene) is preferred, but various commercially available vectors are also suitable. Vectors such as vectors can be used. When vectors are used to produce the antigen-binding molecules of the present invention, expression vectors are particularly useful. Expression vectors are not particularly limited as long as they express antigen-binding molecules in test tubes, E. coli, cultured cells, or individual organisms. Suitable expression vectors include, but are not limited to, the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for E. 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. Insertion of the genes of the present invention into vectors can be carried out by standard methods, for example, by ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology, edited by Ausubel et al. (1987) Published by John Wiley & Sons, Sections 11.4-11.11).

[0256] 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 Examples of methods for introducing a vector into a host cell include calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 9.1-9.9), and lipofection. This can be done by known methods such as the microinjection method or the microinjection method.

[0257] 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, culture can also be performed using media supplemented with serum such as FBS or fetal bovine serum (FCS). The cells can also be cultured in serum-free medium. The pH during culture is about 6 to 8. However, the pH is not limited to this range and different pHs can be selected for each culture or depending on the culture period. Culture is usually carried out at about 30 to 40°C for about 15 to 200 hours. The medium is exchanged, aerated, and stirred as necessary.

[0258] 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 may be incorporated into the desired polypeptide. These signals may be endogenous or heterologous to the antigen-binding molecule of interest.

[0259] On the other hand, systems for producing polypeptides in vivo include, for example, production systems using animals and production systems using plants. A vector containing a gene of interest has been introduced into the cells of an animal or plant, and the polypeptide encoded by the gene is produced in the cells and recovered from the body fluids or living body of the animal or plant. In the present invention, the "host" includes these animals and plants.

[0260] In the case of a production system in which an animal is used as a host, mammals and insects are used. Mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Alternatively, transgenic animals may be used as appropriate.

[0261] 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 recovered from the milk produced by the transgenic goat born to the goat that received the embryo, or its offspring. To increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat, appropriate hormones can be administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12, 699-702).

[0262] Furthermore, silkworms, for example, can be used as insects to produce the antigen-binding molecules of the present invention. When silkworms are used, the antigen-binding molecules of interest can be collected 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.

[0263] Furthermore, when using plants to produce the antigen-binding molecules of the present invention, tobacco can be used, for example. When using tobacco, a plant expression vector, such as pMON 530, into which a polynucleotide encoding the antigen-binding molecule of interest has been inserted is transformed into Agrobacterium tumefaciens. The desired antigen-binding molecule can be recovered from tobacco leaves, such as Nicotiana tabacum, infected with the bacteria (Ma et al., Eur. J. Immunol. (1994) 24, 131-8). In addition, clones of duckweed (Lemna minor) infected with the same bacteria The desired antigen-binding molecule can be recovered from the cells (Cox KM et al. Nat. Biotechnol. (2006) (12), 1591-1597).

[0264] When a sugar chain receptor-binding domain to which a desired sugar chain is bound is used as an example of the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention, a known method can be used to bind a sugar chain to the sugar chain receptor-binding domain. For example, a series of enzymatic cell-free methods, such as a method for producing a post-translationally modified antibody in a cell-free protein synthesis system (JP 2006-141241 A), which comprises preparing a cell extract from cultured cells of an immortalized mammalian cell line capable of secreting proteins and adding mRNA encoding the antibody to the extract, can be used to bind the desired sugar chain. The method of the present invention can be employed to produce a sugar chain receptor-binding domain having a sugar chain receptor-binding domain. The method of the present invention can also include a step of further treating such an antigen-binding molecule with an enzyme.

[0265] The glycan structure of the antigen-binding molecule produced as described above may be a single one, or it may be produced as a mixture with multiple glycans linked thereto. Such mixtures can also be preferably used in the present invention. Furthermore, antigen-binding molecules in which a specific glycan is linked to its glycan-binding domain can also be preferably used in the present invention.

[0266] Several known methods can be used to link specific sugar chains to the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention. One such known method is a method for purifying antigen-binding molecules having specific sugar chains by utilizing the properties of sugar chains possessed by natural or artificially produced antigen-binding molecules by recombinant gene techniques, etc. It is known that antibodies having high-mannose sugar chains can be purified using affinity chromatography using ConA-sepharose (Millward (Biologicals (2008) 36, 49-60)). Such a purification method can be used in the present invention to prepare antigen-binding molecules having an N-linked sugar chain in which the non-reducing end is mannose.

[0267] If necessary, an appropriate protein-modifying enzyme can be used before or after purification of the antigen-binding molecule to optionally modify the antigen-binding molecule and partially or comprehensively remove modified molecules such as peptides. Examples of protein-modifying enzymes that can be used include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase. The production methods of the present invention can also include a step of further treating such antigen-binding molecules with an enzyme.

[0268] Although several known methods can be used to link specific sugar chains to the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention, enzyme treatment can also be used as appropriate to obtain antigen-binding molecules having specific sugar chains. As described later in the Examples, antigen-binding molecules having N-linked sugar chains with terminal galactose can be synthesized by conjugating them to complex sugar chains with terminal sialic acid. Antigen-binding molecules having high-mannose sugar chains can be prepared by sialidase treatment. It is also known that antibodies having high-mannose sugar chains can be prepared by removing galactose from the sugar chains by treatment with sialidase and β-galactosidase (Newkirk (Clin. Exp. Immunol. (1996) 106, 259-264)). The method for producing the antigen-binding protein having an N-linked sugar chain with a terminal mannose in the present invention is The production method of the present invention may also include a step of further treating such antigen-binding molecules with an enzyme.

[0269] By introducing the antigen-binding molecule of the present invention into host cells as described below, the antigen-binding molecule containing a sugar chain receptor-binding domain to which a desired sugar chain is bound can be produced from the culture medium of the host cells.

[0270] In a different aspect, for the purpose of obtaining an antigen-binding molecule of the present invention having a specific sugar chain, a method of recovering the antigen-binding molecule from the culture medium of host cells into which a recombinant gene encoding the antigen-binding molecule has been transfected and whose glycosidase activity has been altered (by, including but not limited to, genetic or recombinant gene techniques) so that the specific sugar chain accumulates can also be appropriately employed. It is known that antibodies having N-linked sugar chains can be recovered from the culture medium of a Lec1 mutant strain derived from CHO cells lacking N-acetylglucosaminyltransferase I activity, into which a recombinant gene encoding the antibody has been transfected (Wright and Morrison (J. Exp. Med. (1994) 180, 1087-1096)). In the present invention, antibodies having N-linked sugar chains with mannose at the non-reducing end are Antigen-binding molecules can be recovered from the culture medium of a Lec1 mutant strain transduced with a recombinant gene encoding the molecule.

[0271] In yet another embodiment, for the purpose of obtaining an antigen-binding molecule of the present invention having a specific sugar chain, a method can be appropriately employed in which an inhibitor that inhibits a specific glycosidase reaction is added during the culture of cells that produce the antigen-binding molecule, and the antigen-binding molecule having a specific sugar chain that has accumulated in the culture medium is then recovered. The antibody was produced by adding kifunesine to the CHO cells that produce the antibody. It is known that it can be recovered from the culture medium of the cells (Zhou (Biotechnol. Bioeng. (2008) 99, 652-665)). In the present invention, N-type mannose-terminated fucose-free mannose is used. The antigen-binding molecule having a conjugated sugar chain can be recovered from the culture medium by, for example, culturing CHO cells transfected with a recombinant gene encoding the molecule in the presence of kifunesine. Furthermore, it is also possible to obtain antigen-binding molecules of the present invention having a specific sugar chain by combining, for example, the method of adding such inhibitors during the culture of the host cells with altered glycosidase activity described above. It is known that antibodies having a specific sugar chain can be recovered by such a combination (Kanda et al. (Glycobiology (2007) 17, 104-118)). In the present invention, a similar combination can be used to obtain antigen-binding molecules having a specific sugar chain. A suitable combination may be adopted.

[0272] The glycan structure of the antigen-binding molecule produced as described above may be a single one, or it may be produced as a mixture with multiple glycans linked thereto. Such mixtures can also be preferably used in the present invention. Furthermore, antigen-binding molecules in which a specific glycan is linked to its glycan-binding domain can also be preferably used in the present invention.

[0273] The antigen-binding molecules thus obtained can be isolated from inside or outside host cells (culture medium, milk, etc.) and purified as substantially pure and homogeneous antigen-binding molecules. The separation and purification methods used for separating and purifying antigen-binding molecules can be appropriately selected and combined depending on the purpose from separation and purification methods commonly used in polypeptide purification, but are not limited to any particular method. For example, 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.

[0274] 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 (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be carried out using liquid phase chromatography, such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. For example, a column using protein A is Examples of suitable polymers include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0275] The antigen-binding molecules prepared as described above are evaluated using the above-mentioned methods for measuring antigen-binding activity, FcRn-binding activity, or carbohydrate receptor-binding activity to determine whether they have the desired properties in terms of antigen-binding activity, FcRn-binding activity, particularly human FcRn-binding activity, carbohydrate receptor-binding activity, etc. Based on the results of such evaluation, the step of designing a carbohydrate receptor-binding domain motif and the step of constructing a gene encoding an antigen-binding molecule polypeptide containing the carbohydrate receptor-binding domain motif are further repeated one or more times to prepare antigen-binding molecules with the desired properties.

[0276] The antigen-binding molecules of the present invention comprising a sugar chain receptor-binding domain, particularly antigen-binding molecules comprising a human-derived sugar chain receptor-binding domain, bind to sugar chain receptors in a pH-dependent manner, and / or Alternatively, if an antigen-binding molecule has glycan receptor-binding activity in the neutral pH range, particularly human-derived glycan receptor-binding activity, and its glycan receptor-binding activity in the acidic pH range can be made lower than its glycan receptor-binding activity in the neutral pH range, it will be possible to promote the intracellular uptake of antigens by the antigen-binding molecule, promote the decrease in plasma antigen concentration by administration of the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by a single antigen-binding molecule.

[0277] The antigen-binding molecules produced by the production methods of the present invention are antigen-binding molecules that promote a decrease in plasma antigen concentration upon administration. Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules that promote a decrease in plasma antigen concentration upon administration.

[0278] Furthermore, the antigen-binding molecules produced by the production methods of the present invention have improved pharmacokinetics. Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules with improved pharmacokinetics.

[0279] Furthermore, it is believed that antigen-binding molecules produced by the production methods can increase the number of antigens that can be bound by a single antigen-binding molecule when administered to animals such as humans, mice, and monkeys. Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules that increase the number of antigens that can be bound by a single antigen-binding molecule.

[0280] Furthermore, when administered to animals such as humans, mice, and monkeys, antigen-binding molecules produced by the production methods of the present invention are thought to be able to intracellularly dissociate antigens bound to the antigen-binding molecules. Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules that can intracellularly dissociate antigens bound to the antigen-binding molecules.

[0281] Furthermore, when administered to animals such as humans, mice, and monkeys, antigen-binding molecules produced by the production methods of the present invention are thought to be able to cause antigen-binding molecules that have been taken up into cells in an antigen-bound state to be released extracellularly in an unbound state. Thus, the production methods of the present invention can be used as a method for producing antigen-binding molecules that are taken up into cells in an antigen-bound state and released extracellularly in an unbound state.

[0282] Furthermore, these antigen-binding molecules are considered to be particularly excellent as pharmaceuticals because, compared with conventional antigen-binding molecules, their administration has a stronger effect of lowering antigen concentrations in plasma. Therefore, the production method of the present invention can be used as a method for producing antigen-binding molecules for use as pharmaceutical compositions.

[0283] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising the antigen-binding molecules 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 or antigen-binding molecules produced by the production methods of the present invention are useful as pharmaceutical compositions because their administration has a greater effect of lowering plasma antigen concentrations than conventional antigen-binding molecules. The pharmaceutical compositions of the present invention can also contain a pharmaceutically acceptable carrier.

[0284] In the present invention, the pharmaceutical composition generally refers to an agent for treating or preventing a disease, or for testing or diagnosing a disease.

[0285] The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. For example, the pharmaceutical composition of the present invention can be used parenterally in the form of a sterile solution or suspension containing water or other pharmaceutically acceptable liquid for injection. For example, the pharmaceutical composition of the present invention can be used parenterally in the form of a sterile solution or suspension containing water or other pharmaceutically acceptable liquid for injection. The active ingredient is formulated by appropriately combining it with a carrier or vehicle, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing it in a unit dose form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set so that an appropriate volume within the indicated range is obtained.

[0286] Sterile compositions for injection can be formulated according to standard pharmaceutical practice using a vehicle such as distilled water for injection. 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). Suitable solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), can also be used in combination.

[0287] Oily liquids include sesame oil and soybean oil, and may contain benzyl benzoate and / or benzyl alcohol as a solubilizing agent. They may also contain buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into suitable ampoules.

[0288] The pharmaceutical composition of the present invention is preferably administered parenterally.For example, the pharmaceutical composition of the present invention can be prepared as an injection, a nasal administration, a pulmonary administration, or a transdermal administration composition.For example, the pharmaceutical composition of the present invention can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0289] The method of administration of the pharmaceutical composition of the present invention can be appropriately selected based on the patient's age and symptoms. The dosage of a pharmaceutical composition containing an antigen-binding molecule can be set, for example, within the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Furthermore, the dosage can be set, for example, within the range of 0.001 to 100,000 mg per patient, although the dosage of the pharmaceutical composition of the present invention is not necessarily limited to these values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but a person skilled in the art can select an appropriate dosage and administration method taking these conditions into consideration.

[0290] It should be noted that the amino acids contained in the amino acid sequences described in the present invention may be post-translationally modified (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation) by those skilled in the art. These post-translationally modified amino acids may also be included in the scope of antigen-binding molecules specified by the amino acid sequences described in the present invention.

[0291] A method for promoting cellular uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule, comprising contacting the molecule with cells that express a sugar chain receptor in vivo or ex vivo. The present invention further provides methods for promoting the intracellular uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule, comprising contacting an antigen-binding molecule and an antigen that binds to the antigen-binding molecule with a cell that expresses a carbohydrate receptor that binds to a carbohydrate receptor-binding domain contained in the antigen-binding molecule in vivo or ex vivo.

[0292] In the present invention, "uptake into cells" means that an antigen-binding molecule or an antigen that binds to the antigen-binding molecule is taken up into cells by endocytosis. In addition, in the present invention, "promoting uptake into cells" means accelerating the rate at which an antigen-binding molecule that binds to an antigen extracellularly is taken up into cells. Therefore, in the present invention, the uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule into cells is promoted. Whether or not a glycoconjugate has been incorporated into a glycoconjugate receptor-expressing cell can be determined by determining whether or not there is an increase in the rate of uptake of the antigen-binding molecule or the antigen that binds to the antigen-binding molecule into the cell. The rate of antigen uptake into the cell can be calculated, for example, by adding the antigen-binding molecule and the antigen to a culture medium containing glycoconjugate receptor-expressing cells and measuring the decrease in the concentration of the antigen-binding molecule or the antigen that binds to the antigen-binding molecule in the culture medium over time, or by measuring the amount of the antigen-binding molecule that has been incorporated into the glycoconjugate receptor-expressing cell or the antigen that binds to the antigen-binding molecule over time. Contact of the glycoconjugate receptor-expressing cell with the antigen-binding molecule and the antigen that binds to the antigen-binding molecule can be performed in vitro as described above, or can be performed in vivo by administering the antigen-binding molecule.

[0293] The present invention provides methods for accelerating the rate of intracellular uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule, which methods involve contacting an antigen-binding molecule and an antigen that binds to the antigen-binding molecule with cells that express a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule in vivo or ex vivo. For example, the rate of antigen elimination in plasma can be accelerated by (1) a so-called ex vivo method in which plasma containing the antigen-binding molecule and the antigen that binds to the antigen-binding molecule is temporarily removed from the body, contacted with cells that express a glycan receptor, and after a certain period of time, the plasma containing the antigen-binding molecule that does not bind to the antigen is recycled (also referred to as resecretion or recycling) to the outside of the cells, and then returned to the body; or (2) a method in which the antigen-binding molecule is administered in vivo. Furthermore, in method (1), a method in which plasma containing the antigen that binds to the antigen-binding molecule is temporarily removed from the body, contacted with cells that express the antigen-binding molecule and a glycan receptor, and after a certain period of time, the plasma is returned to the body can also be used. Therefore, whether or not the cellular uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule has been promoted can also be confirmed by, for example, measuring whether the rate of elimination of the antigen present in plasma is accelerated compared to when the antigen-binding molecule is not administered, or whether the antigen concentration in plasma is reduced by an ex vivo method or administration of the antigen-binding molecule.

[0294] Furthermore, whether or not uptake is promoted can be determined by the fact that the rate of antigen elimination in plasma confirmed in (1) and (2) above is determined by the presence of human natural IgG, especially human natural IgG1, instead of the antigen-binding molecule. This can also be confirmed by confirming whether the rate of elimination of the antigen in plasma is accelerated compared to that confirmed by the method using ...

[0295] The present invention also provides a method for promoting the cellular uptake of an antigen bound to an antigen-binding molecule, comprising contacting the cell in vivo or ex vivo with a cell expressing a glycan receptor that binds to a glycan receptor-binding domain contained in an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions. The present invention also provides a method for promoting the cellular uptake of an antigen bound to an antigen-binding molecule, comprising contacting the cell in vivo or ex vivo with a cell expressing a glycan receptor that binds to a glycan receptor-binding domain contained in an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions. The present invention also provides a method for promoting the cellular uptake of an antigen bound to an antigen-binding molecule, comprising contacting the cell in vivo or ex vivo with a cell expressing a glycan receptor that binds to a glycan receptor-binding domain contained in an antigen-binding molecule, wherein at least one amino acid in the antigen-binding domain is an amino acid whose antigen-binding activity changes depending on calcium ion concentration conditions or pH conditions.

[0296] In the present invention, the method for "changing the antigen-binding activity of an antigen-binding domain by changing ion concentration conditions" may be one or a combination of any of the methods described herein as methods for producing antigen-binding molecules.

[0297] In the present invention, any cells can be used as the cells expressing a sugar chain receptor that binds to the sugar chain receptor-binding domain contained in the antigen-binding molecule, as long as they express the desired sugar chain receptor. To identify cells expressing the desired sugar chain receptor, publicly known databases such as the Human Protein Atlas (http: / / www.proteinatlas.org / ) can be used. Alternatively, whether or not cells used for contacting with an antigen-binding molecule of the present invention express a desired glycosyl receptor can be confirmed by a method for confirming the expression of a gene encoding the desired glycosyl receptor or by an immunological method using an antibody that binds to the desired glycosyl receptor. These methods are also known. Contact of glycosyl receptor-expressing cells with an antigen-binding molecule and an antigen that binds to the antigen-binding molecule can be carried out in vivo as well as in vitro. Therefore, in the present invention, contacting a glycosyl receptor-expressing cell with an antigen-binding molecule also includes administering the antigen-binding molecule to a living body. The contact time can be appropriately selected, for example, from 1 minute to several weeks, 30 minutes to 1 week, 1 hour to 3 days, or 2 hours to 1 day, i.e., the time required for the antigen-binding molecule or the antigen that binds to the antigen-binding molecule to be taken up into cells by endocytosis.

[0298] For example, hepatocytes can be used as cells that express an asialoglycoprotein receptor as a sugar chain receptor, and a wide variety of cells, including blood cells, can be used as cells that express a mannose receptor as a sugar chain receptor.

[0299] A method for increasing the number of antigens bound to a single antigen-binding molecule, comprising contacting the antigen-binding molecule with cells expressing a carbohydrate receptor in vivo or ex vivo. The present invention also provides methods for increasing the number of antigens bound by a single antigen-binding molecule, comprising contacting an antigen-binding molecule having an antigen-binding domain, an FcRn-binding domain, and one or more carbohydrate receptor-binding domains with cells in vivo or ex vivo expressing a carbohydrate receptor that binds to the carbohydrate receptor-binding domain contained in the antigen-binding molecule.

[0300] In the present invention, "the number of antigens bound by an antigen-binding molecule per molecule" refers to the number of antigens that can be bound by an antigen-binding molecule before it is degraded and disappears. In the present invention, "increasing the number of antigens that can be bound by an antigen-binding molecule per molecule" refers to increasing the number of times that an antigen molecule bound to an antigen-binding molecule dissociates and re-binds to an antigen molecule. The antigen molecules that bind to an antigen-binding molecule may be the same antigen molecule present in a reaction system in which both molecules are present, or they may be different molecules. In other words, it refers to the total number of times that an antigen-binding molecule binds to an antigen in the reaction system. In other words, when one cycle is assumed to be the uptake of an antigen-bound antigen-binding molecule into a cell, dissociation of the antigen in an endosome, and then return to the extracellular space, this refers to an increase in the number of cycles that the antigen-binding molecule can go through before it is degraded and disappears. The antigen-binding molecules of the present invention, which have binding activity to a glycosyl receptor in the neutral pH range, are taken up into the cells of cells expressing the glycosyl receptor by endocytosis after binding to the glycosyl receptor. The antigen-binding molecules of the present invention, which are released from their sugar chain receptors in an acidic range, bind to FcRn, particularly human FcRn, in the acidic range and are recycled back to the extracellular space. After the antigen dissociates from the antigen-binding molecule in an acidic range, the antigen-binding molecules of the present invention are recycled back to the extracellular space and are then able to bind to the antigen again. Therefore, whether the number of cycles has increased can be determined by whether the "intracellular uptake" described above is promoted or whether the "pharmacokinetics" described below is improved.

[0301] The present invention provides a method for increasing the number of antigens bound by an antigen-binding molecule per molecule, which comprises contacting an antigen-binding molecule and an antigen that binds to the antigen-binding molecule with cells that express a glycan receptor that binds to a glycan receptor-binding domain contained in the antigen-binding molecule in vivo or ex vivo. For example, (1) a so-called ex vivo method in which plasma containing the antigen-binding molecule and the antigen that binds to the antigen-binding molecule is temporarily removed from the body, contacted with cells that express a glycan receptor, and recycled (also referred to as re-secretion or recirculation) to the outside of the cells after a certain period of time, and the plasma containing the antigen-binding molecule that does not bind to the antigen is then returned to the body; or (2) an ex vivo method in which the antigen is The rate of antigen elimination from plasma can be accelerated by administering a binding molecule to the body. Alternatively, in method (1), plasma containing an antigen that binds to the antigen-binding molecule can be removed from the body, contacted with cells expressing the antigen-binding molecule and a glycoconjugate receptor, and then returned to the body after a certain period of time. Therefore, whether the number of antigens bound to each antigen-binding molecule has increased can be confirmed, for example, by measuring whether the rate of antigen elimination from plasma is accelerated compared to when the antigen-binding molecule is not administered, or by measuring whether the antigen concentration in plasma is reduced by an ex vivo method or administration of the antigen-binding molecule.

[0302] Furthermore, whether the number of antigens bound to one antigen-binding molecule is increased can be determined by comparing the rate of antigen elimination in plasma confirmed in (1) and (2) above with the rate of antigen elimination in plasma confirmed by a method using human native IgG, particularly human native IgG1, instead of antigen-binding molecules. This can also be confirmed by checking whether the rate of elimination of the compound is accelerated compared to that of the compound.

[0303] The present invention also provides a method for increasing the number of antigens that can be bound by a single antigen-binding molecule, which comprises contacting an antigen-binding molecule whose antigen-binding activity changes depending on ion concentration conditions and the number of antigens that can be bound by a single antigen-binding molecule is increased in vivo or ex vivo with a cell expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule.The present invention also provides a method for increasing the number of antigens that can be bound by a single antigen-binding molecule, which comprises contacting an antigen-binding molecule whose antigen-binding activity changes depending on ion concentration conditions and the number of antigens that can be bound by a single antigen-binding molecule is increased in vivo or ex vivo with a cell expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule. Furthermore, the present invention provides methods for increasing the number of antigens bound by a single antigen-binding molecule, comprising contacting an antigen-binding molecule, in vivo or ex vivo, with a cell expressing a sugar chain receptor that binds to the sugar chain receptor-binding domain contained in the antigen-binding molecule, wherein at least one amino acid in the antigen-binding domain is an amino acid whose antigen-binding activity changes depending on calcium ion concentration or pH, and the number of antigens to which the single antigen-binding molecule can bind is increased.

[0304] In the present invention, the method for "changing the antigen-binding activity of an antigen-binding domain by changing ion concentration conditions" may be one or a combination of any of the methods described herein as methods for producing antigen-binding molecules.

[0305] In the present invention, cells expressing a sugar chain receptor that binds to the sugar chain receptor-binding domain contained in an antigen-binding molecule can be any cells that express the desired sugar chain receptor, and are not limited to specific cells. To identify cells that express the desired sugar chain receptor, known databases such as the Human Protein Atlas (http: / / www.proteinatlas.org / ) can be used. Alternatively, whether or not cells used for contacting with an antigen-binding molecule of the present invention express a desired glycosyl receptor can be confirmed by a method for confirming the expression of a gene encoding the desired glycosyl receptor or by an immunological method using an antibody that binds to the desired glycosyl receptor. These methods are also known. Contact of glycosyl receptor-expressing cells with an antigen-binding molecule and an antigen that binds to the antigen-binding molecule can be carried out in vivo as well as in vitro. Therefore, in the present invention, contacting a glycosyl receptor-expressing cell with an antigen-binding molecule also includes administering the antigen-binding molecule to a living body. The contact time can be appropriately selected, for example, from 1 minute to several weeks, 30 minutes to 1 week, 1 hour to 3 days, or 2 hours to 1 day, i.e., the time required for the antigen-binding molecule or the antigen that binds to the antigen-binding molecule to be taken up into cells by endocytosis.

[0306] For example, hepatocytes can be used as cells that express an asialoglycoprotein receptor as a sugar chain receptor. A wide range of cells can be used, including blood cells.

[0307] A method for reducing extracellular antigens comprising contacting cells expressing carbohydrate receptors in vivo or ex vivo. The present invention also provides methods for reducing extracellular antigens, which comprise contacting an antigen-binding molecule comprising an antigen-binding domain, an FcRn-binding domain, and one or more carbohydrate receptor-binding domains with cells in vivo or ex vivo expressing a carbohydrate receptor that binds to the carbohydrate receptor-binding domain contained in the antigen-binding molecule.

[0308] An extracellular antigen refers to an antigen present outside of a cell that expresses a glycosyl receptor. When glycosyl receptor-expressing cells are present in a living body, examples of extracellular antigens include antigens present outside of the cell in body fluids such as blood, plasma, serum, urine, lymph, saliva, and tears. However, as long as the antigen-binding molecule of the present invention administered to a living body can bind to the extracellular part of a cell that expresses a glycosyl receptor, the antigen is not limited to antigens present in these body fluids. A particularly preferred example of an extracellular antigen is an antigen present in plasma. When glycosyl receptor-expressing cells are present outside of a living body, examples of extracellular antigens include antigens present in body fluids such as blood, plasma, serum, urine, lymph, saliva, and tears extracted from the living body, as well as in the culture medium in which the cells are cultured.

[0309] Antigen-binding molecules of the present invention that have binding activity to a glycosyl receptor in a neutral pH range bind to the glycosyl receptor and are then taken up into cells expressing the glycosyl receptor by endocytosis. The antigen-binding molecules of the present invention that are released from the glycosyl receptor in an acidic pH range are recycled to the extracellular space by binding to FcRn, particularly human FcRn, in the acidic pH range. After dissociating the antigen from the antigen-binding molecule in an acidic pH range, the recycled antigen-binding molecules of the present invention are able to bind to the antigen again. In this case, the dissociated antigen is degraded in intracellular lysosomes. Therefore, each time the antigen-binding molecule is recycled, the antigen present outside the cells expressing the glycosyl receptor bound to the antigen-binding molecule of the present invention is degraded in the lysosomes in the cells expressing the glycosyl receptor. It is believed that this degradation of the antigen results in a decrease in the amount of antigen present outside the cells. Whether or not the antigen present outside the cells has decreased can be evaluated by measuring the amount of antigen present outside the cells in body fluids such as blood, plasma, serum, urine, lymph, saliva, and tears when the cells expressing the sugar chain receptor are present in vivo, or by measuring the amount of antigen present in body fluids such as blood, plasma, serum, urine, lymph, saliva, and tears extracted from the body, as well as the culture medium in which the cells are cultured when the cells expressing the sugar chain receptor are present outside the body.

[0310] The present invention provides methods for reducing extracellular antigens, which comprise contacting an antigen-binding molecule and an antigen that binds to the antigen-binding molecule with cells expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule in vivo or ex vivo. For example, (1) a so-called ex vivo method in which plasma containing the antigen-binding molecule and the antigen that binds to the antigen-binding molecule is temporarily removed from the body, contacted with cells that express a glycan receptor, and after a certain period of time, the plasma containing the antigen-binding molecule that does not bind to the antigen is recycled (also referred to as resecretion or recycling) to the outside of the cell and returned to the body; or (2) a method in which the antigen-binding molecule is administered in vivo. Furthermore, in method (1), a method in which plasma containing the antigen that binds to the antigen-binding molecule is temporarily removed from the body, contacted with cells that express the antigen-binding molecule and a glycan receptor, and after a certain period of time, the plasma is returned to the body can also be used. Therefore, whether or not the amount of antigen present outside the cells has decreased can also be confirmed by, for example, measuring whether or not the amount of antigen present in plasma has decreased compared to when the antigen-binding molecule is not administered, or whether or not the antigen concentration in plasma has been reduced by an ex vivo method or administration of the antigen-binding molecule.

[0311] In addition, whether the amount of antigen in plasma is reduced or not can be determined by checking the rate of antigen elimination in plasma confirmed in (1) and (2) above, using human natural IgG, especially human IgG, instead of antigen-binding molecules. This can also be confirmed by checking whether the rate of antigen elimination from plasma is accelerated compared to that confirmed by a method using natural IgG1.

[0312] The present invention also provides a method for reducing extracellular antigens of cells, which comprises contacting an antigen-binding molecule of the present invention, comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, with cells expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule, in vivo or ex vivo. The present invention also provides a method for reducing extracellular antigens of cells, which comprises contacting an antigen-binding molecule of the present invention, comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, with cells expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule, in vivo or ex vivo. The present invention also provides a method for reducing extracellular antigens of cells, which comprises contacting an antigen-binding molecule of the present invention, comprising an antigen-binding domain whose antigen-binding activity changes depending on calcium ion concentration conditions or pH conditions, with cells expressing a glycan receptor that binds to the glycan receptor-binding domain contained in the antigen-binding molecule, in vivo or ex vivo.

[0313] In the present invention, the method for "changing the antigen-binding activity of an antigen-binding domain by changing ion concentration conditions" may be one or a combination of any of the methods described herein as methods for producing antigen-binding molecules.

[0314] In the present invention, cells expressing a sugar chain receptor that binds to the sugar chain receptor-binding domain contained in an antigen-binding molecule can be any cells that express the desired sugar chain receptor, and are not limited to specific cells. To identify cells that express the desired sugar chain receptor, known databases such as the Human Protein Atlas (http: / / www.proteinatlas.org / ) can be used. Alternatively, whether or not cells used for contacting with an antigen-binding molecule of the present invention express a desired glycosyl receptor can be confirmed by a method for confirming the expression of a gene encoding the desired glycosyl receptor or by an immunological method using an antibody that binds to the desired glycosyl receptor. These methods are also known. Contact of glycosyl receptor-expressing cells with an antigen-binding molecule and an antigen that binds to the antigen-binding molecule can be carried out in vivo as well as in vitro. Therefore, in the present invention, contacting a glycosyl receptor-expressing cell with an antigen-binding molecule also includes administering the antigen-binding molecule to a living body. The contact time can be appropriately selected, for example, from 1 minute to several weeks, 30 minutes to 1 week, 1 hour to 3 days, or 2 hours to 1 day, i.e., the time required for the antigen-binding molecule or the antigen that binds to the antigen-binding molecule to be taken up into cells by endocytosis.

[0315] For example, hepatocytes can be used as cells that express an asialoglycoprotein receptor as a sugar chain receptor, and a wide variety of cells, including blood cells, can be used as cells that express a mannose receptor as a sugar chain receptor.

[0316] A method for improving the pharmacokinetics of an antigen-binding molecule, comprising contacting the molecule with cells expressing a carbohydrate receptor in vivo. The present invention also provides methods for improving t...

Claims

1. A method for increasing the in vivo or in vitro antigen elimination ability of an antigen-binding molecule, the method comprising increasing the number of binding domains for a carbohydrate receptor in an antigen-binding molecule that contains an antigen-binding domain, an FcRn-binding domain, and one or more binding domains for the carbohydrate receptor, the antigen-binding domain has a higher antigen-binding activity at pH 7.4 than at pH 5.8; the glycan receptor binding domain is a glycan, the sugar chain receptor is an asialoglycoprotein receptor or a mannose receptor; The method, wherein the antigen-binding molecule is an antibody.

2. The method of claim 1, wherein at least one amino acid in the antigen-binding domain includes at least one amino acid having a side chain pKa of 4.0-8.

0.

3. The method of claim 1, wherein at least one amino acid of the antigen-binding domain comprises a calcium-binding motif.

4. The method of any one of claims 1 to 3, wherein the antigen-binding domain comprises a variable region of an antibody.

5. The method according to any one of claims 1 to 4, wherein the FcRn binding domain comprises an Fc region of an antibody.

6. The method of claim 5 , wherein the antibody is an IgG antibody.

7. The method of claim 6, wherein the IgG antibody is any of IgG1, IgG2, IgG3 or IgG4.

8. The method according to any one of claims 1 to 7, characterized in that the binding activity of the glycan receptor-binding domain to a glycan receptor varies depending on ion concentration conditions, and the ion concentration conditions are pH conditions.

9. The method according to claim 8, wherein the binding activity of the glycan receptor-binding domain to the glycan receptor at pH 7.4 is higher than that at pH 5.

8.

10. The method according to claim 1, wherein the glycan is an O-linked glycan.

11. The method according to claim 1, wherein the glycan is an N-linked glycan.

12. The method according to claim 11, wherein the glycan receptor binding domain contains a motif to which an N-linked glycan binds.

13. The method according to claim 11 or 12, wherein the terminal end of the N-linked glycan contains galactose.

14. The method according to claim 13, wherein the N-linked glycan has three or more terminal galactose residues.

15. The method according to claim 11 or 12, wherein the terminus of the N-linked glycan contains mannose.

16. The method according to any one of claims 1 to 15, wherein the carbohydrate receptor binding domain is contained in an antigen binding domain.

17. The method according to any one of claims 1 to 15, wherein the carbohydrate receptor binding domain is contained in an FcRn binding domain.

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