Antigen-binding molecule for promoting clearance from plasma of antigen comprising saccharide chain receptor-binding domain

Abstract

To provide an antigen-binding molecule for promoting uptake of an antigen into a cell, to provide an antigen-binding molecule capable of promoting a decrease of the antigen concentration in plasma, to provide an antigen-binding molecule capable of binding to an antigen a plurality of times, to provide an antigen-binding molecule improved in pharmacokinetics, to provide a pharmaceutical composition containing the antigen-binding molecule, and to provide a method for producing the same.SOLUTION: Provided are an antigen-binding molecule containing a sugar chain receptor-binding domain and having a weak antigen-binding activity in the pH of early-stage endosome compared to the antigen-binding activity in the pH of plasma; as well as a pharmaceutical composition containing the antigen-binding molecule; and to provide a method for producing the same. Use of the inventive antigen-binding molecule can promote the uptake of an antigen into a cell and increase the number of antigens that a single antibody molecule can bind. Furthermore, administration of the antibody can promote the reduction of antigens in plasma and prolong the pharmacokinetics of the antibody.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 an antigen-binding molecule that promotes antigen uptake into cells, a method for reducing antigen concentrations in plasma, and a method for treating inflammatory bowel diseases. antigen-binding molecules that can promote pharmacokinetics, antigen-binding molecules that can bind to antigens multiple times, -binding molecule with improved activity, pharmaceutical composition containing said antigen-binding molecule, and production thereof - Patents.com Regarding the method. [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 put on the market, and many antibody drugs are currently being developed. On the other hand, technologies applicable to second-generation antibody drugs include Various technologies have been developed to improve effector function, antigen binding ability, pharmacokinetics, and stability. There have been reports of techniques to increase the immunogenicity or reduce the risk of immunogenicity (Non-Patent Documents 3) The dosage of antibody drugs is generally very high, making it difficult to prepare subcutaneous formulations. However, the high manufacturing costs are considered to be issues. As a result, we have developed methods to improve the pharmacokinetics of antibodies and methods to improve the affinity between antibodies and antigens. There is a way to improve it.

[0004] Artificial amino acid substitutions in the constant region have been reported as a method for improving the pharmacokinetics of antibodies. (Non-patent Documents 4 and 5) Affinity immunoreaction is a technique for enhancing antigen binding ability and antigen neutralization ability. A continuity maturation technique (Non-Patent Document 6) has been reported, and the CDR regions of the variable regions It is possible to enhance the binding activity to antigens by introducing mutations into amino acids such as: Enhanced antigen-binding ability improves in vitro biological activity or reduces dosage It is possible to improve the efficacy of the drug in vivo (Non-Patent Document 7).

[0005] On the other hand, the amount of antigen that can be neutralized by one antibody molecule depends on the affinity. By strengthening the antibody, it is possible to neutralize the antigen with a small amount of antibody. Furthermore, it is possible to enhance the affinity of the antibody to the antigen (Non-Patent Document 6). If the affinity can be made infinite, one antibody molecule can bind to one antigen molecule. However, the previous method only neutralized the antigens of the two antigens. The limit of neutralization is the stoichiometric neutralization reaction of one antigen molecule (two antigens in the case of a bivalent antibody). It was impossible to completely neutralize the antigen with an antibody amount less than the antigen amount. However, there was a limit to the effect of neutralizing antibodies in strengthening the immune system (Non-Patent Document 9). In order to maintain the anti-inflammatory effect for a certain period of time, the amount of antibodies must be greater than the amount of antigen produced in the body during that period. The amount of antibody to be administered must be sufficient to improve the pharmacokinetics of the antibody or to enhance affinity. However, there was a limit to how much antibody dosage could be reduced using only the modulation technology. 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 be able to neutralize multiple antigens. A new method to achieve this has recently been developed that neutralizes the antigens by using pH-dependent An antibody that binds specifically to an antigen under neutral conditions in plasma has been reported (Patent Document 1). pH-dependent antigens that bind strongly to the antigen and dissociate from the antigen under the acidic conditions in the endosome The bound antibody can dissociate 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 binds to the antigen again. This allows a single antibody to bind to multiple antigens repeatedly. do.

[0006] In addition, the plasma retention of antigens is much longer than that of antibodies, which are recycled by binding to FcRn. When such an antibody with a long plasma retention time binds to its antigen, the antibody-antigen complex is released into the blood. The plasma retention time is similar to that of antibodies. Therefore, antigens bind to antibodies and are released into the bloodstream. In such cases, the antibody's antigen binding activity increases. Increasing affinity for antigens does not accelerate their clearance from plasma. The pH-dependent antigen-binding antibody described above accelerates the elimination of antigens from plasma compared to conventional antibodies. It has also been reported that this method is effective (Patent Document 1).

[0007] In this way, pH-dependent antigen-binding antibodies bind to multiple antigens with a single antibody, and are more sensitive than conventional antibodies. This allows for the antigen to be eliminated from the plasma more rapidly than conventional antibodies. However, the antigen of this pH-dependent antigen-binding antibody has not been repeatedly tested. This further improves the ability to bind to and promote the elimination of antigens from plasma. No antibody engineering techniques have been reported.

[0008] On the other hand, glycoproteins present in plasma bind to their sugar chain-specific receptors and are absorbed into the plasma. At this time, glycoproteins are absorbed into the glycan receptors present on the cell surface and disappear from the cellular surface (Non-Patent Document 10). It binds to the receptor and is taken up into the cell, where it dissociates from the sugar chain receptor and is then transported by the lysosome. However, it is known that the sugar chain receptor is recycled back to the cell surface. In other words, this glycoprotein and the sugar chain receptor bind strongly under the neutral conditions in plasma. It has a pH-dependent binding ability that dissociates under the acidic conditions in the endosome (non-specific (Patent Literature 11, Non-Patent Literature 12) The pH-dependent binding of such glycoproteins to sugar chain receptors The sugar chains attached to glycoproteins are involved in the process, and examples of such sugar chains and sugar chain receptors are Among N-linked glycans, glycans with terminal galacrose and asialoglycoproteins are Mannose receptor (Non-patent document 11), sugar chains with mannose at the terminal and mannose receptor (Non-Patent Document 12) and the like.

[0009] Due to the properties of N-linked glycans, functionally unnecessary N-links are not found in biopharmaceuticals, including antibodies. Adding conjugated sugar chains is not desirable from the viewpoint of its efficacy or maintaining plasma retention. It is believed that this has the effect of promoting the elimination of antigens from plasma (Non-patent Documents 13 and 14). No examples have been implemented with the aim of further improving it.

[0010] The references cited herein are as follows: The entire contents of these documents are incorporated herein by reference. This is not an admission that the invention is prior art. [Prior art documents] [Patent documents]

[0011]

Patent Document 1

Non-licensed literature

[0012]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0013] The present inventors have demonstrated that antigen-binding molecules with antigen-binding activity can promote the uptake of antigens into cells. a method for causing an antigen-binding molecule to bind to an antigen multiple times; a method for increasing plasma and a method for promoting the reduction of antigen concentration in plasma and a method for improving the plasma retention of antigen-binding molecules. As a result, the present inventors have found that the antigen-binding domain, FcRn, particularly human FcRn It has a binding domain for the sugar chain receptor and a domain that binds to the sugar chain receptor, However, the ion concentration in the endosome is weaker than that in plasma. The antigen-binding molecule having binding activity can promote the uptake of antigens into cells and can also increase the concentration of antigens in plasma. The ion concentration in early endosomes was compared with that in the control ion concentration. An antigen-binding molecule having an antigen-binding domain with weak antigen-binding activity under certain conditions is The antigen-binding molecule can further enhance the uptake of the antigen into cells. The number of antigens that can bind to each other can be increased, and the antigen concentration in plasma can be increased by administering the antigen-binding molecule. We have found that promoting the reduction of IL-16 can improve the pharmacokinetics of antigen-binding molecules.

[0014] That is, the present invention provides a method for promoting the uptake of an antigen into a cell by an antigen-binding molecule, a method for increasing the number of antigens that can be bound by antigen-binding molecules of the same, and a method for increasing the number of antigens that can be bound by antigen-binding molecules of the same, A method for promoting reduction of antigen concentration, a method for improving the pharmacokinetics of antigen-binding molecules, and a method for enhancing the cellular activity of antigens. antigen-binding molecules with enhanced uptake into cells, antigen-binding molecules with an increased number of antigens that can bind; An antigen-binding molecule, a drug kinetics molecule, and a method for the treatment of rheumatoid arthritis, the administration of which can promote a decrease in the antigen concentration in plasma. -binding molecule with improved activity, pharmaceutical composition containing said antigen-binding molecule, and methods for producing the same - Patents.com More specifically, regarding the law, [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) an amino acid sequence that differs from the amino acid sequence identified in (b) by at least one amino acid; a step of designing a motif for a sugar chain receptor binding domain containing the (d) A polypeptide of an antigen-binding molecule containing the motif of the carbohydrate receptor-binding domain designed in (c). A step of producing a gene encoding the peptide (e) Extracting antigen-binding molecules from the culture medium of host cells transformed with the gene obtained in (d). Recovery process [2] A method comprising further treating the antigen-binding molecules obtained in (e) with an enzyme. a method for producing [3] The antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions. The manufacturing method according to [1] or [2], [4] The production method according to [3], wherein the ion concentration conditions are pH conditions. [5] The antigen-binding domain has antigen-binding activity in the medium pH range rather than in the acidic pH range. The production method according to [4], wherein the antigen-binding activity is high under conditions in a serotonin-containing range. [6] At least one amino acid in the antigen-binding domain is an amino acid having a side chain pKa of 4.0 to 8.0. or at least one amino acid having a side chain pKa of 4.0-8.0 in the antigen-binding domain. By inserting amino acids, the binding activity to the antigen in the acidic pH range is improved compared to that in the medium pH range. and providing an antigen-binding domain that has high binding activity to the antigen under conditions of a physiological range. The method according to [5], [7] The method according to [3], wherein the ion concentration conditions are calcium ion concentration conditions. law, [8] The antigen-binding domain binds to an antigen under a low calcium ion concentration condition. Its binding activity to the antigen under high calcium ion concentrations is higher than that under high calcium ion concentrations [7]. The manufacturing method according to [9] At least one amino acid in the antigen-binding domain is substituted with a calcium-binding motif. low calcium binding motifs can be obtained by substituting calcium-binding motifs into the antigen-binding domain. The binding activity to the antigen under high calcium ion concentration was higher than that under high sodium ion concentration. providing an antigen-binding domain that has high binding activity to the antigen under various conditions,

[0033]

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

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

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

[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 sugar chain receptor-binding domain has a binding activity to a sugar chain receptor under ion concentration conditions. The method according to any one of [1] to

[12] , wherein the temperature is changed by

[15] The method according to

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

[16] Binding of the sugar chain receptor-binding domain to a sugar chain receptor under acidic pH conditions. The binding activity to the sugar chain receptor under neutral pH conditions is higher than the activity [1] to [1 5. The method according to any one of

[17] The method according to

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

[18] The sugar chain receptor binding domain of the present invention is capable of accepting sugar chains under conditions of low calcium ion concentration. The binding activity of the glycoconjugate to the glycoconjugate receptor under conditions of high calcium ion concentration is higher than that of the glycoconjugate to the human body. The method according to

[17] , wherein the binding activity is high.

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

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

[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 sugar chain receptor is an asialoglycoprotein receptor

[21] to [ 24],

[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] FcRn-binding domain: antigen-binding activity changes depending on ionic concentration The binding activity of the antigen-binding domain and the carbohydrate receptor changes depending on the ion concentration. an antigen-binding molecule comprising one or more carbohydrate receptor-binding domains;

[30] The antigen-binding activity of the antigen-binding domain changes depending on pH conditions. [2 9. The antigen-binding molecule according to

[31] The antigen-binding activity of the antigen-binding domain is higher under an acidic pH range than under an acidic pH range. The antigen-binding molecule of

[30] , which has high antigen-binding activity under neutral pH conditions.

[32] At least one amino acid in the antigen-binding domain has at least one side chain pK The antigen-binding molecule of

[31] , which contains an amino acid in which a is 4.0-8.0.

[33] A method for determining whether the antigen-binding activity of the antigen-binding domain changes depending on calcium ion concentration. The antigen-binding molecule of

[29] , wherein the antigen-binding molecule is changed by the

[34] The binding of the antigen-binding domain to an antigen under a low calcium ion concentration condition. The binding activity to the antigen under high calcium ion concentration is higher than the combined activity [3 3). The antigen-binding molecule according to

[35] At least one amino acid in the antigen-binding domain contains a calcium-binding motif. The antigen-binding molecule of

[34] ,

[36] Any of

[29] to

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

[37] Any of

[29] to

[36] , wherein the FcRn-binding domain comprises an Fc region of an antibody. an antigen-binding molecule according to the present invention;

[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 sugar chain receptor-binding domain has a binding activity to a sugar chain receptor under ion concentration conditions. The antigen-binding molecule of any one of

[29] to

[39] , wherein the binding site varies depending on the child,

[41] The antigen-binding molecule of

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

[42] Binding of the sugar chain receptor-binding domain to a sugar chain receptor under acidic pH conditions. It has higher binding activity to the sugar chain receptor under neutral pH conditions than the activity described in

[41] . an antigen-binding molecule of

[43] The antibody according to

[40] , wherein the ion concentration conditions are calcium ion concentrations. original binding molecule,

[44] The sugar chain receptor binding domain of the present invention is capable of accepting sugar chains under conditions of low calcium ion concentration. The binding activity of the glycoconjugate to the glycoconjugate receptor under conditions of high calcium ion concentration is higher than that of the glycoconjugate to the human body. The antigen-binding molecule of

[43] , which has high binding activity.

[45] The method according to any one of

[29] to

[44] , wherein the sugar chain receptor binding domain is a sugar chain. The antigen-binding molecule described above,

[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 N-linked sugar chain according to

[48] , wherein the terminals of the N-linked sugar chain contain three or more terminal galactoses. antigen-binding molecules,

[50] The sugar chain receptor is an asialoglycoprotein receptor

[47] to [ 49] The antigen-binding molecule according to any one of

[51] The method according to

[46] or

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

[52] The method according to

[46] or

[47] , wherein the sugar chain receptor is a mannose receptor. antigen-binding molecules,

[53] The method according to any one of

[28] to

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

[54] The carbohydrate receptor-binding domain is included in the antigen-binding domain.

[0023] The antigen-binding molecule according to any one of the preceding

[0024]

[55] The carbohydrate receptor-binding domain is contained in an FcRn-binding domain, 3). The antigen-binding molecule according to any one of [1] to [3].

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

[28] to

[55] .

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

[28] to

[55] , The method comprises contacting the antigen-binding molecule with a cell containing the antigen in vivo or ex vivo. How to import it into

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

[28] to

[55] , The antigen-binding molecule-bound antigen is then contacted with a target cell in vivo or ex vivo. a method for incorporating the antibody into the cells;

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

[28] to

[55] , The method further comprises contacting the cells in vivo or ex vivo with the antigen-binding molecules. a method for increasing the number of antigens present in a given antibody;

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

[28] to

[55] , The method involves contacting cells containing the antigen in vivo or ex vivo to reduce the amount of antigen present outside the cell. How to do this

[61] The method according to

[58] , wherein the extracellular space is plasma.

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

[28] to

[55] , a method for improving the pharmacokinetics of an antigen-binding molecule, comprising contacting the molecule in vivo with a cell containing the antigen-binding molecule;

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

[28] to

[55] , The method comprises contacting the cells in vivo or ex vivo with the cells that bind to the antigen-binding molecule outside the cells. a method for promoting dissociation of a bound antigen from an antigen-binding molecule;

[64] An antigen-binding domain, an FcRn-binding domain, and one or more binding domains for carbohydrate receptors. Increasing the number of binding domains for the sugar chain receptor in an antigen-binding molecule containing the a method selected from any of the following, comprising: (i) Intracellular antigen binding of cells expressing the sugar chain receptor in vivo or ex vivo Methods for promoting uptake of molecules (ii) Intracellular antigen binding of cells expressing the sugar chain receptor in vivo or ex vivo Methods for enhancing uptake of antigens bound to molecules - Patents.com (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 ability of an antigen-binding molecule to eliminate an antigen in vivo or in vitro. (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 antigen-binding activity of the antigen-binding molecule varies depending on ion concentration conditions. The method according to

[64] ,

[66] The antigen-binding activity of the antigen-binding domain changes depending on pH conditions. [6 4).

[67] The antigen-binding activity of the antigen-binding domain is higher under an acidic pH range than under an acidic pH range. The method according to

[66] , wherein the antibody has high antigen-binding activity under neutral pH conditions.

[68] At least one amino acid in the antigen-binding domain has a side chain pKa of 4.0-8.0. The method according to claim 67, wherein the amino acid sequence contains at least one amino acid.

[69] A method for determining whether the antigen-binding activity of the antigen-binding domain changes depending on calcium ion concentration. The method according to

[64] , characterized in that the change is due to

[70] The binding of the antigen-binding domain to an antigen under conditions of low calcium ion concentration. The binding activity to the antigen under high calcium ion concentration is higher than the combined activity [6 9).

[71] At least one amino acid in the antigen-binding domain contains a calcium-binding motif. The method according to

[70] ,

[72] Any of

[64] to

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

[73] Any of

[64] to

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

[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 sugar chain receptor-binding domain has a binding activity to a sugar chain receptor under ion concentration conditions. The method according to any one of

[64] to

[75] , wherein the temperature is changed by

[77] The method according to

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

[78] Binding of the sugar chain receptor-binding domain to a sugar chain receptor under acidic pH conditions. It has higher binding activity to the sugar chain receptor under neutral pH conditions than the activity described in

[76] . The method of

[79] The method according to

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

[80] The sugar chain receptor binding domain of the present invention is capable of accepting sugar chains under conditions of low calcium ion concentration. The binding activity of the glycoconjugate to the glycoconjugate receptor under conditions of high calcium ion concentration is higher than that of the glycoconjugate to the human body. The method according to

[79] , which has high binding activity;

[81] The method according to any one of

[64] to

[80] , wherein the sugar chain receptor binding domain is a sugar chain. The method of posting,

[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 N-linked sugar chain according to

[85] , wherein the terminals of the N-linked sugar chain contain three or more terminal galactoses. method,

[87] The sugar chain receptor is an asialoglycoprotein receptor

[84] to [ 86],

[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. method,

[90] The method according to any one of

[64] to

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

[91] The carbohydrate receptor-binding domain is included in the antigen-binding domain.

[0033]

[92] The Fc region contains the carbohydrate receptor-binding domain

[64] , or the FcRn-binding domain

[64] .

[90] The method according to any one of 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 will facilitate understanding of the invention described herein. is provided for.

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

[0018] and / or As used herein, the term "and / or" means that "and" and "or" are appropriate. Specifically, for example, "position 33, position 55, and / or "The amino acid at position 96 or 106 has been substituted" includes the following amino acid alteration variations: be wrapped in; (a) 33rd place, (b) 55th place, (c) 96th place, (d) 33rd and 55th place, (e) 33rd and 96th place, (f) 55th place and and 96th place, (g) 33rd place, 55th place and 96th place.

[0019] antigen As used herein, the term "antigen" refers to any structure that includes an epitope to which an antigen-binding domain binds. In another sense, antigens can be inorganic or organic. Examples of antigen-binding molecules whose pharmacokinetics can be improved by the method of the present invention include, for example, For example, membrane antigens such as receptor proteins (membrane-bound receptors, soluble receptors) and cell surface markers Antigen-binding molecules that recognize soluble antigens such as cytokines are preferred. Suitable 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, adenosine Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADA M12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aF GF, 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, B 7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, B AK, 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, B MP-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, C1 0, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen 35 (CEA), cancer-associated antigen, cathepsin 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, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL 5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, C CR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a , CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25 , CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40 , CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD7 4, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, C D152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens 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, CX CR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC- SIGN, 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, enkephalin Enzyme kinase, eNOS, Eot, Eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast Cell-activating protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF 3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, Lactalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G25 0, 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), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GF R-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-EG F, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, 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, ICA M, 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, I L-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 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 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta Ta1, integrin beta2, 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), latency Potential 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-selenium Cutin, 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-ALF A, 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 , Mullerian inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NCadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, neuronal growth factor receptors (NGR), Long-acting 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 (PS MA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, Laxin 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, S IGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TA CE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP Alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Spe cific, TGF-link RI(ALK-5), TGF-link RII, TGF-link RIIb, TGF-link RIII, TGF -Gen 1, TGF-Gen 2, TGF-Gen 3, TGF-Gen 4, TGF-Gen 5, Ck -1, Tie, Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TN F-linker, TNF-linker 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(T RAIL R1 Apo-2、DR4)、TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、 TNFRSF10C(TRAILR3 DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNF RSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK RFN14) TNFRSF13B(TACI) TNFRSF13C(BAFF R) TNFRSF14 (HVEM 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(LTb). R TNF RIII, TNFC R, TNFRSF4(OX40ACT35, TXGP1 R), TNFRSF5(CD40p50), T NFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7 (CD27), T.S NFRSF8(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 ligand 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-a Connectin, DIF, TNFSF2), TNFSF1B (TNF-b 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), TNFSF 9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRA IL-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, uro キナーゼ, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEG F. VEGFR, VEGFR-3(flt-4), VEGI, VIM, ウイルス antigen, VLA, VLA-1, VLA-4, VNRイン テグリン, フォン・ヴィレブランド factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A , WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT 10A, 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-2 0R, acidified LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, macromolecular kinin, 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, C 1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7 , C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinog en, 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 Tapi, tPA, plasminog and, 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、 alphabeta1 integrin、Angiopoietin ligand-2、Angptl2、Anthrax、Cadherin、Carbo nic 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 e peptide 1 receptor、Glutamate carboxypeptidase II、GMCSFR、Hepatitis C virus E2 glycoprotein、Hepcidin、IL-17 receptor、IL-22 receptor、IL-23 rece ptor、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、P LXNB3 、PLXNC1 、PLXND1 、Programmed cell death ligand 1、Proprotein con vertase 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、Staphylococc al lipoteichoic acid、Tenascin、TG2、Thymic stromal lymphoprotein receptor , TNF superfamily receptor 12A, Transmembrane glycoprotein NMB, TREM-1, TRE M-2, trophoblast glycoprotein, TSH receptor, TTR, tubulin, ULBP2 and hormone Examples include receptors for steroids and growth factors.

[0020] An epitope, which means an antigenic determinant present in an antigen, is an antigen disclosed herein. It refers to the site on the antigen to which the antigen-binding domain in the original binding molecule binds. An epitope can be defined by its structure. The epitope can also be defined by the binding activity to the antigen in the compound molecule. When the epitope is a peptide or polypeptide, the epitope is determined by the amino acid residues that make up the epitope. In addition, if the epitope is a glycan, it is possible to identify a specific glycan. It is also possible to identify epitopes by their chain structure.

[0021] A linear epitope is an epitope that contains a recognized primary amino acid sequence. Linear epitopes typically have at least three, and most usually at least five. For example, about 8 to about 10, 6 to 20 amino acids are included in the unique sequence.

[0022] Conformational epitopes, in contrast to linear epitopes, are those that are formed by one or more amino acids that comprise the epitope. epitopes whose sequence is not a single defined component of the recognized epitope (e.g., amino acid sequence The primary sequence of the amino acid is not necessarily recognized by the antibody that defines the epitope. Conformational epitopes encompass an increased number of amino acids relative to linear epitopes. With regard to the recognition of conformational epitopes, antibodies may be used to recognize peptides or proteins. Recognize the three-dimensional structure of a protein. For example, recognize how a protein molecule folds to form a three-dimensional structure. In some cases, certain amino acids and / or polypeptides that form conformational epitopes 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. Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66 ,See 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 be confirmed by the following method. do.

[0024] For example, a test antigen-binding molecule containing an antigen-binding domain for IL-6R is present in the IL-6R molecule. The recognition of a linear epitope present in the antibody can be confirmed, for example, as follows. For the above purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R was prepared. The peptide can be synthesized chemically. Alternatively, the cDNA of IL-6R can be synthesized. The region encoding the amino acid sequence corresponding to the extracellular domain is used for genetic engineering. Next, a linear peptide consisting of the amino acid sequence that constitutes the extracellular domain is obtained. The binding activity of the peptide to a test antigen-binding molecule containing an antigen-binding domain for IL-6R was 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. Based on the level of inhibition by the linear peptide of the binding of the antigen-binding molecule to the linear peptide, These tests can reveal the binding activity of linear peptides. The binding activity of the antigen-binding molecule against the target antigen can be determined.

[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. Cells expressing IL-6R are prepared. When the antigen-binding molecule contacts the cells, it binds strongly to the cells while immobilizing the antigen-binding molecule. Substantially, 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 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% The following binding activity is referred to:

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

[0027] In an ELISA format, a test antigen-binding molecule containing an antigen-binding domain for IL-6R The binding activity of IL-6R-expressing cells was compared with the signal level generated by the enzyme reaction. Specifically, IL-6R expression is quantitatively evaluated by immobilizing IL-6R-expressing cells on an ELISA plate. A test antigen-binding molecule is added to the cell, and the test antigen-binding molecule bound to the cell is detected as the test antigen-binding molecule. Alternatively, in FACS, the test antigen binding is detected using an enzyme-labeled antibody that recognizes the Prepare a dilution series of the molecule and determine the antibody binding titer to IL-6R-expressing cells. The binding activity of test antigen-binding molecules for IL-6R-expressing cells can be compared by this method.

[0028] The binding of the test antigen-binding molecule to the antigen expressed on the surface of cells suspended in a buffer solution or the like is The detection can be performed by a flow cytometer. For example, the following device is known. 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 antigen-binding molecule is diluted appropriately with a suitable buffer solution to obtain the desired antigen-binding molecule. For example, the concentration can be adjusted to any value 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). This is reflected in the fluorescence intensity obtained by the G The chromatographic mean value is obtained to obtain the test antibody titer expressed by the amount of binding of the test antigen-binding molecule. The binding activity of the 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 for the same epitope. Competition between the original binding molecules can be detected by cross-blocking assays. The ELISA assay is a preferred cross-blocking assay.

[0031] Specifically, in the cross-blocking assay, the wells of a microtiter plate The IL-6R protein coated on the membrane was detected in the presence or absence of a candidate competing antigen-binding molecule. After pre-incubation in the presence of IL-1, the test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to the -6R protein is determined by the amount of antigen-binding molecule that competes for binding to the same epitope. This indirectly correlates with the binding ability of the competing antigen-binding molecule that is a candidate for binding to the same epitope. The greater the affinity of the competing antigen-binding molecule for the target antigen, the greater the IL-1 binding affinity of the test antigen-binding molecule. The binding activity to wells coated with -6R protein is reduced.

[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. For example, biotin-labeled antibodies can be used. The original binding molecule was synthesized by using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assay using enzyme labels such as peroxidase This is particularly called a competitive ELISA assay. Antigen-binding molecules can be detected or measured in other ways. Specifically, radiolabeling, fluorescent labeling, and the like are well known.

[0033] Results obtained in control tests performed in the absence of candidate competing antigen-binding molecules The competitive antigen-binding molecule is compared with the combined activity of the test antigen containing the antigen-binding domain for IL-6R. The binding of the original binding molecule is reduced to at least 20%, preferably at least 20-50%, more preferably If the test antigen-binding molecule can block at least 50%, it is considered to be substantially different from the competing antigen-binding molecule. antigen binding proteins that bind to the same epitope or that compete for binding to the same epitope. It is a compound molecule.

[0034] Structure of the epitope to which a test antigen-binding molecule containing an antigen-binding domain for IL-6R binds If an epitope is identified, the test antigen-binding molecule and the control antigen-binding molecule share an epitope. The purpose is to detect peptides in which amino acid mutations have been introduced into the peptide that constitutes the epitope. The binding activity of both antigen-binding molecules can be evaluated by comparing the binding activity of both antigen-binding molecules.

[0035] As a method for measuring such binding activity, for example, the above-mentioned ELISA format Binding of test antigen-binding molecules and control antigen-binding molecules to linear peptides containing mutations Alternatively, the activity can be measured by binding to a column. The binding activity of the mutant peptide was assessed by loading the test antigen-binding molecule and the control antigen-binding molecule onto the column. It can also be measured by quantifying the antigen-binding molecules eluted in the eluate after the molecules are allowed to flow down. The method of adsorbing the mutant peptide onto a column as a fusion peptide with GST, for example, is It is publicly known.

[0036] In addition, if the identified epitope is a conformational epitope, the test antigen-binding molecule and the control antigen Whether or not the original binding molecule shares an epitope can be evaluated by the following method. 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, the cell suspension is washed with an appropriate buffer solution and the target antigen-binding molecule is added. An FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule was added. The fluorescence intensity and cell number of the cells stained with the labeled antibody were measured using a FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are adjusted appropriately using a suitable buffer. The desired concentration can be prepared by diluting appropriately. For example, 10 μg / ml to 10 ng The amount of labeled antibody bound to the cells is measured using a concentration of 0.01% or less. The fluorescence intensity obtained by analysis using LQUEST Software (BD), i.e., Geom In other words, by obtaining the value of the geometric mean, The binding activity of the test antigen-binding molecule and the control antigen-binding molecule, represented by the amount of bound labeled antibody, is measured. It can be determined.

[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. The test and control antigen-binding molecules are stained with labeled antibodies. The fluorescence intensity of the cells is then measured. When FACSCalibur was used as a flow cytometer for fluorescence detection, 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 binding of the antigen-binding molecule is calculated based on the above formula. It is possible. 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 Geometric Mean comparison value (ΔGeo-Mean value of mutant IL-6R molecule) was compared with the IL-6 of the test antigen-binding molecule. The value is compared with the ΔGeo-Mean comparison value, which reflects the amount of binding to R-expressing cells. , used to calculate the ΔGeo-Mean comparison value for mutant IL-6R expressing cells and IL-6R expressing cells It is particularly preferred that the concentrations of the test antigen-binding molecules be prepared at the same or substantially the same concentrations. It is preferable that an antigen-binding molecule that has been confirmed to recognize an epitope in IL-6R is used as a target. 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). If the results are substantially comparable by comparison, the test antigen-binding molecule and the control antigen-binding molecule The epitopes of the original binding molecules can be assessed to be identical.

[0040] antigen-binding domain As used herein, an "antigen-binding domain" refers to any domain that binds to an antigen of interest. Domains of a different structure can also be used. Examples of such domains include the heavy chain of an antibody. The variable region of the light chain and the 35 amino acid sequence contained in Avimer, a cell membrane protein present in living organisms, A module called an A domain (WO2004 / 044011, WO2005 / 040229), cell membrane The 10F protein-binding domain in fibronectin, a glycoprotein expressed in the mouse Adnectin (WO2002 / 032925) containing the n3 domain, three helical domains consisting of 58 amino acids of Protein A Affibody (WO1995 / 00) with an IgG binding domain as a scaffold, which constitutes a bundle of IgG 1937), a subunit of a turn containing 33 amino acid residues and 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, neutrophil gelatinase-associated lipocalin Eight antiparallel strands are highly conserved in lipocalin molecules such as NGAL. The four loop regions support one side of the barrel structure twisted toward the center. WO2003 / 029462), and the immune system as an acquired immune system of jawless fish such as lampreys and hagfish. Variable lymphocyte receptors without globulin structure Leucine-rich repeat (LRR) modules of VLRs The parallel sheet structure inside the horseshoe-shaped structure is a hollow area (WO20 Preferred examples of the antigen-binding domain of the present invention include antibody domains. The antigen-binding domain includes the variable regions of the heavy and light chains. Examples of antibodies include "scFv (single chain Fv)" and "single chain antibody (single chain anti body)”, “Fv”, “scFv2(single chain Fv 2)”, “diabody”, “Fab”, “F(a b')2'', domain antibody (dAb) (WO2004 / 058821, WO2003 / 002609), scFv-sc (WO2005 Suitable examples of molecules containing an Fc region include Fc fusion proteins. The c region can be used as a binding domain for FcRn, particularly human FcRn. Molecules in which a human FcRn-binding domain is fused to the antibody can also be used.

[0041] The antigen-binding domains of the antigen-binding molecules of the present invention bind to the same epitope. The same epitope can be, for example, SEQ ID NO: 1 (IL-6R_PP; NP_000556. 1) can be present in a protein consisting of the amino acid sequence described in SEQ ID NO: 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 molecules of the present invention may be Each of the antibodies can bind to a different epitope, where the different epitopes can be, for example, It can be present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. A protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in SEQ ID NO: 1 For such purposes, the antigen-binding domains contained in the bispecific antibody may be present in the A bispecific antibody can be made up of variable regions that recognize different epitopes. Bispecific antibodies are antibodies that recognize two or more different antigens. It may be an antibody that recognizes two or more different epitopes on the same antigen. It is possible.

[0042] The antigen-binding domain of the present invention may also be a receptor (receptor) that binds to a target (antigen). The domains involved in binding to their targets (antigens) in the target (protein) are preferably used. That is, the antigen-binding molecule binds to a receptor protein that binds to a target (antigen). Proteins that combine FcRn, especially the FcRn-binding domain, with a carbohydrate receptor-binding domain 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). The antigen-binding molecules have binding domains for these receptor proteins and FcRn, especially human FcRn. Even if the protein is a fusion protein with a phosphodiesterase, the binding activity to the target molecule may change depending on the ion concentration. The binding activity to carbohydrate receptors and FcRn, especially human FcRn, has been changed. The antigen-binding molecules promote the uptake of antigens into cells, and administration of the antigen-binding molecules increases plasma It is possible to promote the reduction of antigen concentration in the blood. The pharmacokinetics have been improved, which increases the number of antigens that can be bound by a single antigen-binding molecule. It is possible.

[0043] The antigen-binding domain of the present invention also includes a natural or artificial ligand that binds to a target. The domain involved in binding to its target in the antibody can be preferably used. Genotypic binding molecules are natural or artificial targets that bind to targets and have antagonistic or neutralizing activity. The protein ligand contains FcRn, especially the FcRn-binding domain and the carbohydrate receptor-binding domain. Examples of such antigen-binding domains that can be used as artificial ligands include: For example, artificial ligands such as mutant IL-6 (EMBO J. (1994) 13 (24), 5863-70) are mentioned. Even if the antigen-binding molecule of the present invention is an artificial ligand fusion molecule, it is possible to obtain a stable binding affinity under various ion concentration conditions. The binding to target molecules changes depending on the conditions, and the binding activity to carbohydrate receptors and FcRn specificity are also affected. Antigen-binding molecules that specifically bind to human FcRn promote the uptake of antigens into cells. Administration of antigen-binding molecules can promote a decrease in the antigen concentration in plasma. In addition, the pharmacokinetics of such antigen-binding molecules is improved, and one antigen-binding molecule can bind to It is possible to increase the number of antigens that can be detected.

[0044] specific Specificity means that one molecule of a molecule that specifically binds to one or more of its binding partners. This refers to a state in which the antigen-binding domain does not show any significant binding to any molecules other than the antigen-binding domain. The antibody is specific to a particular epitope among multiple epitopes contained in an antigen. It is also used when the antigen-binding domain binds to multiple different epitopes. When the antigen is contained in a target antigen, an antigen-binding molecule having the antigen-binding domain can bind to the target epitope. It can bind to a variety of antigens, including ribonucleotides.

[0045] antibody As used herein, antibodies are antibodies that may be natural or partially or wholly synthetic. Antibodies are immunoglobulins produced from natural sources such as plasma and serum in which they occur. The antibody can be isolated from the culture supernatant of hybridoma cells that produce the antibody, or can be isolated from a genetically engineered Antibodies can be partially or completely synthesized using techniques such as recombinant DNA technology. Immunoglobulin isotypes and their isotype subclasses are preferably mentioned. Human immunoglobulins include IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, and IgA1. The antibodies of the present invention contain nine classes (isotypes): IgE, IgM, and IgM. Isotypes 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 include producing antibodies that bind to IL-6R (anti-IL-6R antibodies). 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. Monoclonal antibodies of mammalian origin include those produced by hybridomas. and host cells transformed by genetic engineering techniques with an expression vector containing an antibody gene. The monoclonal antibody of the present invention includes those produced by human cells. These include "antibody" and "chimeric antibody."

[0048] Monoclonal antibody-producing hybridomas can be isolated by known techniques, e.g. It can be produced as follows: IL-6R protein is used as a sensitizing antigen. A mammal is immunized according to a conventional immunization method. The resulting immune cells are then fused using a conventional cell fusion method. The clones are then fused with known parent cells by conventional screening methods. We identified hybrids that produce anti-IL-6R antibodies by screening for local antibody-producing cells. A ridoma may be selected.

[0049] Specifically, monoclonal antibodies are produced, for example, as follows. The IL-6R gene, the nucleotide sequence of which is disclosed in sequence number 2 (IL-6R_PN; NM_000565.3), By expressing the gene, the nucleotide sequence shown in SEQ ID NO: 1 can be used as a sensitizing antigen for antibody production. In other words, the gene sequence encoding IL-6R can be obtained by cloning the gene sequence of a known gene. A suitable host cell is transformed by inserting the vector into the host cell. The desired human IL-6R protein is purified from the culture supernatant by a known method. To obtain a soluble form of IL-6R from IL-6R, for example, the method described by Mullberg et al. (J. Immunol. (1994) 1 52 (10), 4958-4968), 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 it into an expression vector and expressing it, but The region and size of the IL-6R peptide used as the target peptide are not particularly limited to a particular embodiment. The region corresponds to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 1. Any sequence can be selected from the sequences. It is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, it is preferably 8 to A peptide of 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 protein can be used as a sensitizing antigen. For example, Fc fragments of antibodies and peptide tags are preferably used to produce proteins. The vector expressing the fusion protein can contain two or more desired polypeptides. The genes encoding the fragments are fused in frame, and the fused gene is expressed as described above. Fusion proteins can be produced by inserting the protein into a vector. r Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.4 7-9.58 (1989) Cold Spring Harbor Lab. Press). The method for obtaining IL-6R used in the present invention and the immunization method using the same are described in WO2003 / 000883 and WO2004 / 022 754, WO2006 / 006693, etc.

[0052] Mammals to be immunized with the sensitizing antigen are not limited to specific animals, but include: It is preferable to select the cells in consideration of their compatibility with the parent cells used in cell fusion. Rodents, such as mice, rats, hamsters, rabbits, monkeys, etc., are preferred. Used.

[0053] The above animals are immunized with a sensitizing antigen according to a known method. As a method, a sensitizing antigen is administered to a mammal by intraperitoneal or subcutaneous injection. Immunization is carried out. Specifically, PBS (Phosphate-Buffered Saline) or physiological saline is used. The sensitizing antigen diluted at an appropriate dilution ratio is mixed with a conventional adjuvant, such as Freund's adjuvant, if desired. After mixing and emulsifying with the complete adjuvant, the sensitizing antigen is administered to the mammal for 4 to 21 days. In addition, a suitable carrier can be used when immunizing with a sensitizing antigen. When a small amount of a partial peptide is used as a sensitizing antigen, albumin, keyhole Immunization with the sensitizing antigen peptide bound to a carrier protein such as limpet hemocyanin In some cases, it may be desirable to

[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 The method of immunization is characterized in that the expression of the antibody in vivo provides immune stimulation to the immunized animal. Compared with the general immunization method in which protein antigens are administered to immunized animals, 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, IL-6R protein is The DNA encoding IL-6R is then administered to the immunized animal. The resulting DNA is inserted into an appropriate expression vector and administered to an animal to be immunized. As the expression vector, 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 the living body. For example, gold particles carrying an expression vector can be used to target cells of an immunized animal using a gene gun. DNA immunization is achieved by introducing the DNA into the body. Furthermore, the production of antibodies that recognize IL-6R is It 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 cells, splenocytes in particular can be used.

[0057] Mammalian myeloma cells are used as the cells to be fused with the immune cells. Preferably, the roma cells are equipped with an appropriate selectable marker for screening. A selectable marker is a trait that allows a plant to survive (or not survive) under specific culture conditions. The selectable marker is a hypoxanthine-guanine-phosphoribosyltransferase deficiency gene. deficiency (hereafter abbreviated as HGPRT deficiency), or thymidine kinase deficiency (hereafter 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 selective medium. You will start to do this.

[0058] HGPRT-deficient and TK-deficient cells express 6-thioguanine and 8-azaguanine (hereafter abbreviated as 8AG), respectively. These pyrimidines can be selected on a medium containing 5' bromodeoxyuridine or 5' bromodeoxyuridine. Normal cells that incorporate pyrimidine analogs into their DNA die. Cells lacking these enzymes, which cannot incorporate the log, are unable to survive in selective media. Another selectable marker called G418 resistance is the neomycin resistance gene. Conferring resistance to oxystreptamine antibiotics (gentamicin analogues). Various suitable myeloma cells are known.

[0059] Such myeloma cells include, for example, 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. J. Immunol.(1976)6 (7), 511-51 9), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO(J. Immunol. Methods(1980)35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med.(1978)148 (1), 313-323), R210(Nature(1979)277 (5692), 131-133) and the like can be suitably used.

[0060] Basically, known methods, such as the method of Kohler and Milstein et al. (Methods Enzyme ol. (1981) 73, 3-46), etc., cell fusion between the immune cells and myeloma cells is carried out. More specifically, for example, the cells are cultured in a normal nutrient medium in the presence of a cell fusion promoter. Cell fusion can be performed. Fusion promoters include, for example, 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 can be set arbitrarily. The ratio of immune cells to cells is preferably 1 to 10. 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] The cell fusion is carried out by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium; PEG solution (average molecular weight: 1000 to 6000) preheated to about 37°C is usually added in 30 to 6 The mixture is gently mixed to obtain the desired fused cells. Then, the appropriate culture medium listed above is added sequentially to form hybridoma cells. By repeating the procedure of centrifugation and removal of the supernatant, the cells that are not favorable for hybridoma growth can be isolated. Unwanted cell fusion agents and the like can be removed.

[0063] The hybridomas thus obtained are cultured in a conventional selective culture medium, such as HAT medium (hypothelial growth factor AT). Selected by culturing in a medium containing xanthine, aminopterin, and thymidine The hybridoma may be incubated for a period of time sufficient to kill cells other than the desired hybridoma (non-fused cells) (usually 10 min). The culture can be continued using the HAT culture medium (usually, a sufficient period of time is several days to several weeks). Next, hybridomas producing the desired antibodies are screened by the conventional limiting dilution method. Sequencing and single cloning are performed.

[0064] The hybridomas thus obtained contain the same nucleotide sequence as the myelomas used in the cell fusion. Selection can be achieved by using a selective medium depending on the selectable marker. For example, HGPRT and T K-deficient cells were grown in HAT medium (containing hypoxanthine, aminopterin, and thymidine). In other words, HAT-sensitive myeloma cells can be selected by culturing them in a medium containing When cells are used for cell fusion, cells that have successfully fused with normal cells are selected in the HAT culture medium. The cells other than the desired hybridoma (non-fused cells) can be selectively grown. The culture is continued in the HAT medium for a suitable period of time. Specifically, the period is generally from several days to several weeks. The desired hybridomas can then be selected by culturing for a period of time. Therefore, screening and single cloning of hybridomas producing the desired antibodies is performed. can be implemented.

[0065] Screening and single cloning of desired antibodies is based on known antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can be used. Such monoclonal antibodies can bind to IL-6R expressed on the cell surface. Clonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a method of analyzing cells that have been contacted with fluorescent antibodies using laser light to identify individual cells. It is possible to measure antibody binding to the cell surface by measuring the fluorescence emitted by the cells. It is a system that

[0066] Screening hybridomas producing the monoclonal antibodies of the present invention by FACS To do this, first prepare cells expressing IL-6R. 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 antibodies to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells can be immobilized on the wells of an ELISA plate. The culture supernatant of the lysoma was brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells were detected. If the monoclonal antibody is of mouse origin, the antibody bound to the cells will be anti-mouse antibody. These screening tests can detect immunoglobulin antibodies. Hybridomas that produce the desired antibodies capable of binding to the antigen can be isolated by limiting dilution or the like. It can be cloned by

[0068] The hybridomas producing the monoclonal antibodies thus produced are cultured in a conventional manner. The hybridomas can be subcultured in liquid nitrogen. It is possible.

[0069] The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody is isolated from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to obtain a human antibody. The former method allows the production of highly purified monoclonal antibodies. It is suitable for obtaining antibodies of the above type.

[0070] Recombinant Antibody The antibody gene is cloned from the antibody-producing cell such as the hybridoma. The cloned antibody gene can be incorporated into an appropriate vector. By incorporating the gene into a host and introducing it into the host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells are described, for example, in For example, it has already been established by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 7 67-775). Methods for producing recombinant antibodies are also known, as described below.

[0071] For example, the variable region ( To do this, the entire R region is first isolated from the hybridoma. As a method for extracting mRNA from cells, for example, the following method is used. It can be used. -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, the mRNA can be purified using the QuickPrep mRNA Purification Kit (GE Healthcare). kits for extracting total mRNA directly from cells, such as those manufactured by Ruscare Biosciences, are available. Using such a kit, mRNA can be obtained from hybridomas. cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. DNA was prepared using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Biochemical Engineering). cDNA can be synthesized by a PCR kit (manufactured by a manufacturer). 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 with vector DNA. In this way, a recombinant vector is created, introduced into E. coli, etc., and colonies are selected. The desired recombinant vector can be prepared from the colonized E. coli. Whether or not the recombinant vector contains the desired cDNA base sequence can be determined by known methods, e.g. For example, it can be confirmed by the dideoxynucleotide chain termination method.

[0074] To obtain the gene encoding the variable region, primers for amplifying the variable region gene are used. It is easy to use the 5'-RACE method using the cDNA is synthesized using RNA as a template, and a 5'-RACE cDNA library is obtained. A library can be synthesized using a commercially available kit such as the SMART RACE cDNA Amplification Kit. do.

[0075] The antibody gene was amplified by PCR using the obtained 5'-RACE cDNA library as a template. Primers for amplifying mouse antibody genes are designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, the subclasses were determined in advance by IsoStrip mouse monoclonal antibody isotypes. Determine this using a commercially available kit such as the ELISA Kit (Roche Diagnostics) It is desirable.

[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 primers used are those that anneal to the region corresponding to the constant region close to the variable region. On the other hand, the 5' primer is the one included in the 5' RACE cDNA library construction kit. 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. Binding antibodies can be screened, for example, as follows: (1) An antibody containing a V region encoded by cDNA obtained from a hybridoma is used to express IL-6R. contacting the cells with the (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. The binding of the antibody to IL-6R-expressing cells can be detected by techniques such as ACS. To evaluate this, fixed specimens of IL-6R-expressing cells can be appropriately used.

[0079] As a method for screening antibodies using binding activity as an index, a phage vector was used. Panning is also suitable. When the subclasses of the light chains were obtained as libraries, the phage vectors were used. A screening method 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 method, phages expressing scFv on their surface can be obtained. After contact, the phages bound to the antigen are collected, and the sc with the desired binding activity is identified. The DNA encoding the Fv can be recovered. This procedure can be repeated as necessary to obtain the desired Fv. scFvs having a binding activity of 100% or more can be enriched.

[0081] After obtaining the cDNA encoding the V region of the desired anti-IL-6R antibody, The cDNA is digested with a restriction enzyme that recognizes the inserted restriction enzyme site. The enzyme recognizes and digests base sequences that appear infrequently in the base sequences that make up antibody genes. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, it is necessary to provide sticky ends. It is preferable to insert a restriction enzyme capable of cleaving the V region of the anti-IL-6R antibody digested as described above. An antibody expression vector can be obtained by inserting the cDNA into an appropriate expression vector. At this time, the gene encoding the antibody constant region (C region) and the gene encoding the V region are If the antibody is fused in-frame, a chimeric antibody is obtained. The origin of the constant region and variable region is different. In addition to chimeric antibodies, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. The V region gene is inserted into an expression vector that already contains a constant region. 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 The restriction enzyme recognition sequences for the restriction enzymes can be appropriately positioned. A chimeric antibody expression vector is constructed by fusing the two sequences in frame.

[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 control of the expression control region. These include, for example, enhancers and promoters. An appropriate signal sequence can be added to the amino terminus, as described in the Examples below. has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) as a signal sequence. Although peptides have been used, other suitable signal sequences may be added. The resulting polypeptide is truncated at the carboxyl terminal portion of the sequence, and the truncated polypeptide The mature polypeptide can then be secreted outside the cell. The host cell is transformed with a recombinant vector that expresses DNA encoding an anti-IL-6R antibody. Recombinant cells can be obtained.

[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, Alternatively, the DNA encoding the heavy and light chains may be expressed in 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] A host for producing antibodies by introducing the isolated antibody genes into a suitable host. Many combinations of cells and expression vectors are known. The method can be applied to isolate specific antigen-binding domains. In this case, animal cells, plant cells, or fungal cells can be used as appropriate. Examples of 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, plant cells such as Nicotiana tabacum may be used. An antibody gene expression system using cells derived from the genus Nicotiana 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 Pichia such as Saccharomyces and Pichia pastoria Genus a -Filamentous fungi: Aspergillus species such as Aspergillus niger pergillus ) genus

[0087] Furthermore, antibody gene expression systems using prokaryotic cells are also known. When the cells are used, bacterial cells such as E. coli and Bacillus subtilis can be appropriately used. An expression vector containing the desired antibody gene is introduced into cells by transformation. The transformed cells are cultured in vitro to produce the desired product. The desired antibody can be obtained.

[0088] In addition to the above 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, antibody genes are genes that encode proteins that are specifically produced in milk. It can be constructed as a fusion gene by inserting it in-frame within a gene. For example, goat β-casein can be used as a protein secreted into the body. The DNA fragment containing the inserted fusion gene is injected into a goat embryo, and the injected embryo The embryo is transferred to a female goat. The transgenic goat (or goat) born from the recipient goat is From the milk produced by the mouse (or its progeny), the desired antibody is synthesized as a fusion protein with a milk protein. In addition, milk containing the desired antibody produced from the transgenic goat can be obtained. Hormones can be administered to transgenic goats to increase the amount of 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 molecules The purpose of this is to reduce heterologous antigenicity to humans as an antigen-binding domain in Therefore, an antigen-binding domain derived from an artificially modified recombinant antibody can be appropriately used. 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] To generate the antigen-binding domain in the antigen-binding molecule described herein The variable region of an antibody used in this study is usually composed of three regions sandwiched between four framework regions (FR). It consists of two complementarity-determining regions (CDRs). The CDRs are essentially the regions that determine the binding specificity of the antibody. On the other hand, the amino acid sequences that make up the FR are highly diverse among antibodies with different binding specificities. However, they often show high identity. Therefore, in general, CDR grafting can be used to 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. For example, humanized antibodies in which the CDRs of a mouse antibody are grafted onto a human antibody are known. General genetic recombination techniques for obtaining antibody clones are also known. As a method for grafting the CDR of a target gene to a human FR, for example, overlap extension PCR has been published. 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. Each primer synthesizes a human FR individually, resulting in the addition of mouse CDRs to each FR. The resulting product contains the DNA encoding the mouse CDR. The human antibody genes are then templated. The overlapping CDR portions of the synthesized products are 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, in which three CDRs and four FRs are finally linked, has annealing sites at its 5' and 3' ends. The full length of the fragment is amplified using primers containing 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. By inserting the fragment into an expression vector as described above, a vector for expressing a human antibody can be prepared. After the integration vector is introduced into a host to establish a recombinant cell, the recombinant cell is cultured. The humanized antibody is produced by cultivating the humanized antibody and expressing the DNA encoding the humanized antibody. The cultured cells produce the α-glucan derivatives (see European Patent Publication EP239400 and International Patent Publication WO1996 / 002576). (see).

[0094] The antigen-binding activity of the humanized antibody prepared as described above is qualitatively or quantitatively measured, By evaluating whether or not the CDRs form a good antigen-binding site when linked via the CDRs, If necessary, the CDRs of the reshaped human antibody can be appropriately selected. The amino acid residues of the FR can be substituted to form a suitable antigen-binding site. For example, The PCR method used to graft mouse CDRs onto human FRs was applied to introduce amino acid sequence mutations into the FRs. Specifically, a partial base sequence mutation can be added to the primer annealing to the FR. The FR synthesized by such a primer contains the base sequence The antigen-binding activity of the mutant antibody with the amino acid substitution is then determined by the above method. By measuring and evaluating the properties, mutant FR sequences having desired properties can be selected (Cancer Re s., (1993) 53, 851-856).

[0095] In addition, transgenic animals with a full repertoire of human antibody genes (International Publication) Open WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1 996 / 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 displayed on a phage as a single-chain antibody (scFv). The scFv that binds to the antigen is expressed on the surface of the phage by the method described above. By analyzing the genes of the selected phages, it is possible to identify human antibodies that bind to the antigen. The DNA sequence encoding the V region of the scFv can be determined. After determining the DNA sequence of the scFv that binds to the antigen, The V region sequence is fused in frame with the C region sequence of a desired human antibody, followed by appropriate expression. An expression vector can be prepared by inserting the vector into an expression vector. The human antibody is then introduced into a suitable expression cell as described above, and the gene encoding the human antibody is expressed. These methods are already known (International Publication WO 2019 92 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 00 1438, see WO1995 / 015388).

[0097] In addition, as a method for obtaining antibody genes, Bernasconi et al. (Science (2002) 298, 2199- 2202) or B cell cloning as described in WO2008 / 081008 (the respective antibody codes Identification and cloning of the sequences, their isolation, and the characterization of the respective antibodies (particularly IgG1, IgG2, The techniques for constructing expression vectors for producing IgG3 or IgG4 are as described above. or may 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 Interactions est (National Institute of Health, Bethesda, Md., 1987 and 1991). In the present specification, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acid sequence of the variable region is The amino acids are numbered according to Kabat numbering, and the amino acids in the constant region are numbered according to Kabat amino acid positions. It is represented according to the EU numbering system.

[0099] FcRn Unlike Fcγ receptors belonging to the immunoglobulin superfamily, human FcRn has a unique structure. Class I polypeptides are structurally similar to those of the major histocompatibility complex (MHC) class I. 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. The α1 and α2 domains of the FcRn in the Fc region of antibodies anchor the protein to the cell surface. It interacts with the binding domain (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. In addition, in the small intestine of neonatal rodents, where FcRn is expressed, FcRn is involved in the transport of ingested FcRn. FcRn is involved in the transport of maternal IgG from milk across the brush border epithelium. It is expressed in a number of other tissues, as well as in various endothelial cell lines. It is also expressed in the 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] A polypeptide containing a signal sequence represented by SEQ ID NO: 4 (FcRn; NP_004098.1) was prepared as follows: The precursor human FcRn binds to the β-microglobulin (SEQ ID NO: 5; NP_004039.1) in vivo. ) Human β2-microglobulin, the polypeptide of which including the signal sequence is described in As will be shown later in the Reference Examples, Soluble human FcRn is produced by conventional recombinant expression techniques. This study was conducted to investigate the effect of soluble human FcRn in a complex with β2-microglobulin. The binding activity of the Fc region of the present invention can be evaluated. FcRn refers to a form capable of binding to the Fc region of the present invention, and examples thereof include human FcRn and human β2- and complexes with microglobulin.

[0102] FcRn-binding domain The antigen-binding molecule of the present invention has an FcRn-binding domain. The molecule is not particularly limited as long as it has binding activity to FcRn in the acidic pH range. It may also be a domain that has binding activity to FcRn directly or indirectly. The main example is an IgG immunoglobulin that has direct binding activity to FcRn. 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 case, a domain having binding activity to FcRn is preferred. Any domain that has binding activity to FcRn in the acidic pH range can be used as is. The domain has no or weak binding activity to FcRn in the acidic pH range. In such cases, the amino acids in the antigen-binding molecule can be modified to confer FcRn-binding activity. In addition, it is possible to develop a domain that already has FcRn-binding activity in the acidic pH range. The amino acids in the FcRn-binding domain may be modified to enhance the FcRn-binding activity. The acid modification was performed by comparing the FcRn-binding activity in the acidic pH range before and after the amino acid modification. By doing so, the desired modification can be found.

[0103] The FcRn-binding domain is preferably a region that directly binds to FcRn. A preferred example of the domain is the Fc region of an antibody. The region capable of binding to polypeptides that have binding activity to FcRn, such as albumin and IgA, is Therefore, the FcRn of the present invention can bind indirectly to FcRn via IgG or the like. As the Rn-binding region, a region that binds to a polypeptide having binding activity to FcRn is preferably used. The Fc region comprises amino acid sequences derived from the constant region of an antibody heavy chain. The hinge region of the papain cleavage site at approximately 216 amino acids, expressed in EU numbering. 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, is As described in the section on sexual activity, it can be measured by methods known to those skilled in the art. Conditions other than pH can be appropriately determined by those skilled in the art. The binding activity and human FcRn binding activity were measured using KD (Dissociation constant), apparent KD ( Apparent dissociation constant, kd (Dissociation rate) tion rate, or apparent dissociation rate 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 binding activity of the FcRn-binding domain to FcRn can be determined appropriately by those skilled in the art. It is possible to select from a wide range of compounds, and there is no particular limitation. For example, the compounds described in WO2009 / 125825 The FcRn-binding domains of the present invention can be measured in MES buffer at 37°C. The main binding activity to FcRn can be measured by methods known to those skilled in the art. For example, it can be measured using Biacore (GE Healthcare) or the like. The binding activity of FcRn can be measured using the FcRn-binding domain or the antigen-binding domain of the present invention. The FcRn or FcRn-binding domain or The antigen-binding molecule of the present invention containing an FcRn-binding domain was evaluated by injecting it as an analyte. It can be valued.

[0106] Conditions under which the FcRn-binding domain contained in the antigen-binding molecule of the present invention has FcRn-binding activity The acidic pH range generally means a pH range of 4.0 to 6.5, preferably a pH range of 5.5 to 6.5. Particularly preferably, it means a pH of 5.8 to 6.0, which is close to the pH in early endosomes in vivo. The temperature used for the conditions was 10°C, which is the binding affinity between the FcRn-binding domain and FcRn. The assay may be performed at any temperature between 100°C and 50°C. Preferably, the assay is performed at a temperature between the FcRn-binding domain and human FcRn. To determine binding affinity, a temperature of 15°C to 40°C is used. More preferably, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C Similarly, any temperature between 20°C and 35°C may be used to measure the binding of the FcRn-binding domain to FcRn. A temperature of 25°C is used to determine affinity in a non-limiting embodiment of the present invention. Here is an example.

[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 human The binding activity to FcRn is 20 μM or stronger, and the binding activity to human FcRn is in the neutral pH range. The binding activity of the antigen-binding molecule to human F under acidic pH conditions is equivalent to that of natural human IgG. An antigen-binding molecule of the present invention having binding activity to cRn can be used. In this study, the antigens with human FcRn-binding activity (KD) of 2.0 μM or stronger under acidic pH conditions were The antigen-binding molecules of the present invention, including the binding molecules, can be used. is an antigen-binding molecule that has a human FcRn-binding activity of 0.5 μM or stronger (KD) under acidic pH conditions. The above KD values ​​can be used in The Journal of Immunology (2009) 182: 7663-7 The method described in 671 (in which antigen-binding molecules were immobilized on a chip and human FcRn was run as an analyte) was used. is determined by

[0108] In the present invention, an Fc region that has binding activity to FcRn under an acidic pH range condition is preferred. The domain already has FcRn-binding activity under acidic pH conditions. The Fc region can be used as it is. If the binding activity is absent or weak, it can be improved by modifying the amino acids in the antigen-binding molecule. An Fc region having the desired binding activity to FcRn can be obtained by modifying the amino acids in the Fc region. The antibody has been modified to have a desired FcRn-binding activity under an acidic pH range, or Enhanced Fc regions can also be suitably obtained. The amino acid modification affects the FcRn-binding activity under acidic pH conditions before and after the amino acid modification. The known amino acid modifications described later in this specification can be found by comparing the amino acid modifications. Those skilled in the art can appropriately modify amino acids using techniques.

[0109] FcRn-binding activity under acidic pH conditions contained in the antigen-binding molecules of the present invention The region can be obtained by any method, but specifically, the starting Fc region can be The amino acid modification of human IgG immunoglobulins increases their ability to bind to FcRn under acidic pH conditions. FcRn-binding domains with or enhanced binding activity can be obtained. Preferred examples of the Fc region of IgG-type immunoglobulin include human IgG (IgG1, IgG2, IgG3, or Examples of the Fc region include IgG4 and its variants. Other amino acid modifications can be made depending on the pH and acidity. It has binding activity to FcRn under acidic conditions or binds to human FcRn under acidic conditions. The amino acid at any position can be modified as long as it can increase the binding activity to the antigen-binding molecule. When the molecule contains the Fc region of human IgG1 as the Fc region, it binds to FcRn under acidic pH conditions. These modifications include those that have the effect of enhancing the binding activity of the Fc region of human IgG1 compared to the binding activity of the starting Fc region of human IgG1. It is preferable that the amino acid be modified in this way. As described in the EU numbering system, the 238th, 252nd, 253rd, 254th, 255th, 2 56th, 265th, 272nd, 286th, 288th, 303rd, 305th, 307th, 309th, 311th, 312th, 317th 340th, 356th, 360th, 362nd, 376th, 378th, 380th, 382nd, 386th, 388th, 400th , 413th, 415th, 424th, 433rd, 434th, 435th, 436th, 439th and / or 447th Ami Similarly, examples of amino acids that can be modified in this way include WO20 251, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 2 5th, 256th, 308th, 309th, 311th, 312th, 385th, 386th, 387th, 389th, 428th, 433rd Suitable examples of such modifications include the amino acids at positions 434 and / or 436. 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, The positions 251, 252, 307, 308, 378, 428, 430, 434 and / or 436 The modification of these amino acids also favorably improves the F of IgG immunoglobulins. The binding of the c region to FcRn is enhanced under conditions in the acidic pH range.

[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 has the effect of improving plasma retention. Any amino acid modification may be introduced as long as it is possible to do so.

[0111] In a non-limiting embodiment of the present invention, the FcRn-binding domain is a polypeptide that binds to FcRn, particularly human FcRn. As described above, an antigen-binding domain having a binding activity similar to that of the present invention can also be used appropriately. A bispecific antibody can also be used as the antigen-binding molecule. One of the epitopes is present on a desired antigen other than FcRn, and the other A preferred example is a bispecific antibody whose epitope is present in FcRn. The structure of a specific antibody consists of a bivalent binding domain with binding valencies for the desired antigen and FcRn, and As long as it contains a carbohydrate receptor-binding domain, it is not limited to a specific structure. For example, Fc Antibody structures such as IgG-type antibodies in which domains are linked may also be used, and "scFv2 (single ch Antibodies such as "antibody Fv2", "diabody", or "F(ab')2" can also be suitably used. When an antibody structure such as The FcRn-binding domain and / or antigen-binding domain may also be included in "scFv2 (single When structures such as "F(ab')2", "diabody", or "F(ab')2" are used The 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, the FcRn-binding domain is an "ion-concentration The binding to FcRn changes depending on the ion concentration conditions, as described later in the section "Conditions of ion concentration." In other words, in a non-limiting embodiment of the present invention, an FcRn-binding domain having an FcRn-binding domain capable of binding to an FcRn-binding domain of ... of an FcRn-binding domain of an FcRn-binding domain of an FcRn-binding domain of an FcRn-binding domain of an FcRn- As will be described later in the section on "Conditions for metal-ionization," the binding of the FcRn-binding domain to FcRn is FcRn-binding domain whose binding activity to FcRn changes depending on ion concentration and pH 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. "Ions" refers to alkali metals and Group I metals such as copper group metals, alkaline earth metals and zinc metals, excluding hydrogen. 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 It refers to the ions of metallic elements such as rhenium. Metal atoms release valence electrons to become cations. Metals with a high ionization tendency have the following chemical properties: It is said to be highly active.

[0114] An example of a metal ion suitable for use in the present invention is calcium ion. Muscle contraction, including skeletal, smooth, and cardiac muscle contraction, Activation of white blood cell movement and phagocytosis, activation of platelet deformation and secretion, activation of lymphocytes Activation of mast cells such as activation of the catecholamine α receptor and acetylcholinesterase inhibitors, and secretion of histamine. Phosphoreceptor-mediated cellular responses, exocytosis, and neurotransmitter transport from neuronal terminals Calcium ions are involved in the release of calcium ions from neurons and the axonal flow of neurons. As an on-receptor, it has multiple calcium ion binding sites and may have a common origin in molecular evolution. Troponin C, calmodulin, parvalbumin, and myosin light chains, which are thought to be derived from Many of the binding motifs are known. For example, cadherin In, EF hand contained in calmodulin, C2 domain contained in protein kinase C, The Gla domain of the blood coagulation protein Factor IX, asialoglycoprotein receptor 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 calcium ion, the calcium ion concentration The conditions for this include low calcium ion concentration and high calcium ion concentration. The fact that the binding activity changes depending on the calcium ion concentration condition means that The binding activity of antigen-binding molecules to antigens varies depending on the conditions of high calcium ion concentration and high calcium ion concentration. For example, the response to an antigen under conditions of low calcium ion concentration is changed. The binding activity of the antigen-binding molecule is higher under high calcium ion concentrations than that of the other antigen-binding molecules. In addition, the binding activity of the antigen-binding molecule is higher than that of the antigen-binding molecule. The binding activity of the antigen-binding molecule to the antigen under conditions of a lower calcium ion concentration is There are also cases where the binding activity of an antigen-binding molecule to an antigen is higher under certain conditions.

[0116] In this specification, the term "high calcium ion concentration" is not limited to a specific numerical value. However, the concentration may be preferably selected from the range of 100 μM to 10 mM. In an embodiment, the concentration may be selected from between 200 μM and 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 400 μM to 1.5 mM. In particular, the concentration of 50, which is close to the calcium ion concentration in plasma (blood) in vivo, can be A preferred concentration is selected from the range of 0 μM to 2.5 mM.

[0117] In this specification, the term "low calcium ion concentration" is not limited to a specific numerical value. However, the concentration may be preferably selected from the range of 0.1 μM to 30 μM. In another embodiment, the concentration may be selected from between 0.2 μM and 20 μM. can be at a concentration selected from between 0.5 μM and 10 μM, and in other embodiments, between 1 μM and 5 μM. The concentration may be selected from the range of 2 μM to 4 μM. In particular, the ionized calcium concentration in the early endosomes in vivo is around 1 μM. A preferred concentration is selected from the range of 0.1 to 5 μM.

[0118] In the present invention, a compound having high antigen-binding activity under a low calcium ion concentration condition is The binding activity to the antigen under the calcium ion concentration condition is lower than that under the calcium ion concentration condition. Antigen binding activity at calcium ion concentrations selected between 0.1 μM and 30 μM However, the binding activity to the antigen at calcium ion concentrations selected between 100 μM and 10 mM was Preferably, the antibody is 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. This means that the binding activity to the antigen is weaker than that at the calcium ion concentration selected. Preferably, the antigen-binding activity is at a calcium ion concentration in early endosomes in vivo. means that the antigen-binding activity is weaker than that at the calcium ion concentration in plasma in vivo, Specifically, the antigen-binding molecule is reacted with the antigen-binding molecule at a calcium ion concentration selected from the range of 1 μM to 5 μM. A calcium ion concentration at which the binding activity to the antigen is 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] The binding activity of the antigen-binding domain of the present invention to an antigen changes depending on the metal ion concentration. Whether or not the binding activity has been increased can be determined by using a known measurement method such as that described above in the section on binding activity. For example, the present invention can be determined by using a method under conditions of low calcium ion concentration. Higher calcium than the antigen-binding activity of the antigen-binding molecule containing the antigen-binding domain of the present invention Binding of an antigen-binding molecule containing the antigen-binding domain to an antigen under ionic concentration conditions To confirm that the combined activity is higher, the calcium ion concentrations were low and high. Antigen-binding molecules containing antigen-binding domains for antigens under calcium ion concentration conditions The binding activities of the antibodies are compared.

[0120] Furthermore, in the present invention, "antigen binding activity under conditions of low calcium ion concentration" The expression "the binding activity to the antigen is lower than that under high calcium ion concentration conditions" The antigen-binding molecule exhibits low antigen-binding activity under high calcium ion concentrations. It can also be expressed as having higher antigen-binding activity than that under calcium ion concentration conditions. In the present invention, the term "binding to an antigen under conditions of low calcium ion concentration" is used. The binding activity to the antigen is lower than that under high calcium ion concentration conditions. The antigen binding ability under calcium ion concentration conditions is higher than that under high calcium ion concentration conditions. In some cases, it is described as "weaker than the binding ability to the antigen" or "low calcium ion The antigen binding activity under high calcium ion concentration conditions was compared with that under high calcium ion concentration conditions. "Decreases antigen binding activity under low calcium ion concentration conditions" was changed to "Enhances antigen binding activity under low calcium ion concentration conditions" When describing "the binding ability to the antigen is weaker than that under calcium ion concentration conditions" There are also.

[0121] The conditions other than calcium ion concentration when measuring the binding activity to an antigen can be determined by those skilled in the art. The incubation conditions can be selected appropriately and are not particularly limited. For example, HEPES buffer, 37°C conditions, For example, Biacore (GE Healthcare) can be used to measure the The binding activity between an antigen-binding molecule containing an antigen-binding domain and an antigen can be measured. When the antigen is a soluble antigen, the measurement is performed by immobilizing an antigen-binding molecule containing the antigen-binding domain. The binding activity of soluble antigens is evaluated by passing the antigen as an analyte through the soluble chip. When the antigen is a membrane-type antigen, the antigen is immobilized on a chip. The binding activity of the antigen-binding molecule containing the binding domain to the membrane antigen is measured by flowing it as an analyte. It is possible to assess the sex.

[0122] The antigen-binding molecules of the present invention bind to antigens under conditions of low calcium ion concentrations. As long as the binding activity is weaker than that for antigens under high calcium ion concentrations The binding activity to antigens under low calcium ion concentrations and high calcium ion concentrations The ratio of binding activity to the antigen under the temperature conditions is not particularly limited, but is preferably KD (Dissociation constant) under low calcium ion concentration conditions The ratio of KD (Ca 3 μM) / KD (Ca 2 mM) under high calcium ion concentration conditions The value of KD (Ca 3 μM) / KD (Ca 2 mM) is preferably 2 or more, and more preferably the value of KD (Ca 3 μM) / KD (Ca 2 mM) is 10 or more. More preferably, the value of KD (Ca 3 μM) / KD (Ca 2 mM) is 40 or more. The upper limit of the value of (μM) / KD (Ca 2 mM) is not particularly limited, and is within the limits that can be prepared by those skilled in the art. It can be any value, such as 400, 1000, or 10000. Also, KD (Ca 3 μM) / KD (Ca 1.2 mM) It can also be specified by the value of KD (3 μM Ca) / KD (1.2 mM Ca) of 2 or more. More preferably, the value of KD (Ca 3 μM) / KD (Ca 1.2 mM) is 10 or more, and even more preferably Preferably, the value of KD (Ca 3 μM) / KD (Ca 1.2 mM) is 40 or more. The upper limit of the value of the calcium phosphate (Ca 1.2 mM) is not particularly limited, and as long as it can be prepared by a person skilled in the art, it is possible to , 1000, 10000, etc., any value is acceptable.

[0123] When the antigen is soluble, the KD (dissociation constant) is used as the value of binding activity to the antigen. However, if the antigen is a membrane antigen, apparent dissociation coefficient (ADC) may be increased. It is possible to use KD (dissociation constant) and apparent dissociation constant. The apparent dissociation constant (KD) of a compound can be measured by a method known to those skilled in the art, for example, Biacore (GE healthcare), Scatchard plot, flow cytometer, etc. It is possible to do so.

[0124] Furthermore, the antigen-binding activity of the antigen-binding molecules of the present invention under conditions of low calcium concentrations Another index showing the ratio of antigen binding activity under conditions of low and high calcium concentrations is, for example, For example, the dissociation rate constant kd (Dissociation rate constant) is also preferred. Instead of KD (dissociation constant), kd (dissociation rate) can be used as an index showing the ratio of binding activity. When using the kd (dissociation rate constant) for the antigen under low calcium concentration conditions, kd (low calcium concentration) is the ratio of kd (dissociation rate constant) under high calcium concentration conditions to kd (dissociation rate constant) under high calcium concentration conditions. The value of kd (high calcium concentration condition) / kd (high calcium concentration condition) is preferably 2 or more, and more preferably The Kd is preferably 5 or more, 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] When the antigen is soluble, kd (dissociation rate constant) is used as the value of antigen binding activity. If the antigen is a membrane antigen, the apparent dissociation rate (Apparent kd) is The apparent dissociation rate constant (kd) and The apparent kd (apparent dissociation rate constant) can be measured by a method known to those skilled in the art. For example, Biacore (GE healthcare), flow cytometer, etc. can be used. In the present invention, the antigen binding molecules at different calcium ion concentrations are When measuring the binding activity to a protein, it is preferable to keep the conditions other than the calcium concentration the same. stomach.

[0126] For example, one embodiment of the present invention is an antibody against low calcium ion concentrations. The binding activity to the antigen under high calcium ion concentration was higher than that under high calcium ion concentration. The antigen-binding domain or antibody having a lower affinity for the antigen-binding domain or antibody can be prepared by a method comprising the steps of: can be obtained by screening antibodies. (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, one embodiment of the present invention is an anti-inflammatory drug under conditions of low calcium ion concentration. The binding activity to the antigen under high calcium ion concentration was higher than that under high calcium ion concentration. The antigen-binding domain or antibody having a lower affinity for the antigen-binding domain or antibody can be prepared by a method comprising the steps of: can be obtained by screening antibodies or libraries thereof. (a) Antigen-binding domains or antibodies or their ligands under high calcium concentration conditions contacting the library with an antigen; (b) detecting the antigen-binding domain or antibody bound to the antigen in step (a) under low calcium concentration conditions; placing down; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0128] Furthermore, one embodiment of the present invention provides an antigen under a condition of low calcium ion concentration. The binding activity to the antigen under the condition of high calcium ion concentration was higher than that under the condition of high calcium ion concentration. The low-density antigen-binding domain or antibody can be prepared by subjecting the antigen-binding domain or antibody to the following steps (a) to (d): They can be obtained by screening antibodies or libraries thereof. (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) detecting the antigen-binding domain or antibody selected in step (b) under high calcium concentration conditions; bonding to the original; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0129] Furthermore, one embodiment of the present invention is an anti-inflammatory drug under conditions of low calcium ion concentration. The binding activity to the antigen under high calcium ion concentration was higher than that under high calcium ion concentration. The antigen-binding domain or antibody having a lower affinity is selected 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) subjecting the antigen-binding domain or antibody bound to the column in step (a) to low calcium concentration conditions; eluting the column under (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0130] Furthermore, one embodiment of the present invention is an anti-inflammatory drug under conditions of low calcium ion concentration. The binding activity to the antigen under high calcium ion concentration was higher than that under high calcium ion concentration. The antigen-binding domain or antibody having a lower affinity is selected by a screening method comprising the following steps (a) to (d): Thus, it can be obtained. (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 was eluted without binding to the column in step (a). The course, (c) subjecting the antigen-binding domain or antibody recovered in step (b) to an antibody reaction under high calcium concentration conditions; bonding to the original; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0131] Furthermore, one embodiment of the present invention is an anti-inflammatory drug under conditions of low calcium ion concentration. The binding activity to the antigen under high calcium ion concentration was higher than that under high calcium ion concentration. The antigen-binding domain or antibody having a lower affinity is selected by a screening method comprising the following steps (a) to (d): Thus, it can be obtained. (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; process, 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 antibody.

[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, a screening method for detecting a low calcium ion concentration The binding activity to the antigen under high calcium ion concentration conditions was The steps (a) to (c) or (a) to (d) are performed to provide an antigen-binding domain or antibody having a lower affinity. The number of repetitions is not particularly limited, but is usually within 10 times.

[0133] In the screening method of the present invention, the antigen-binding domain under low calcium concentration conditions The antigen-binding activity of the antibody or protein was measured at ionized calcium concentrations between 0.1 μM and 30 μM. There is no particular limitation as long as the concentration is active, but the preferred ionized calcium concentration is 0.5 μM to The antigen-binding activity is preferably between 10 μM and 10 μM. The ionized calcium concentration in early endosomes in vivo is one example of this. The antigen-binding activity can be exemplified by the antigen-binding activity at 1 μM to 5 μM. The antigen-binding activity of the antigen-binding domain or antibody at an ionized calcium concentration of 100 μM The antigen-binding activity is not particularly limited as long as it is between 10 mM and 10 mM. The antigen binding activity can be achieved by using a concentration of 200 μM to 5 mM. The ionized calcium concentration in the plasma of the living body can be mentioned as the ionized calcium concentration. Specifically, the antigen-binding activity can be exemplified at 0.5 mM to 2.5 mM.

[0134] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Conditions other than the 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 determined by KD (Dissociation constant : dissociation constant), apparent KD (Apparent dissociation constant), Dissociation rate constant kd (or apparent kd) These can be evaluated as the apparent dissociation rate constant. Measurement can be performed by methods known to commercial manufacturers, for example, Biacore (GE Healthcare), It is possible to use a Catchard plot, FACS, or the like.

[0135] In the present invention, the antigen-binding activity under high calcium concentration conditions is The step of selecting antigen-binding domains or antibodies with higher antigen-binding activity under low calorie conditions is Antigen binding activity under high sodium concentration conditions is higher than that under high calcium concentration conditions. This is synonymous with the process of selecting lower antigen-binding domains or antibodies.

[0136] The antigen-binding activity under high calcium concentration conditions was significantly higher than that under low calcium concentration conditions. As long as the binding activity is higher than that of the antigen, the binding activity under high calcium concentration and low calcium concentration The difference in antigen binding activity under different calcium concentrations is not particularly limited, but is preferably determined under conditions of high calcium concentration. The antigen-binding activity under low calcium concentration conditions is more than twice that under low calcium concentration conditions. It is more preferably 10 times or more, and even more preferably 40 times or more.

[0137] The antigen-binding domain of the present invention screened by the above screening method or The antibody may be any antigen-binding domain or antibody, such as those described above. For example, antibodies can be screened for antigen-binding domains with native sequences. Alternatively, the antigen-binding domains containing the substituted amino acid sequences may be screened. Alternatively, antibodies or antibodies may be screened.

[0138] (2) The binding activity of the antigen-binding domain to the antigen changes depending on the calcium ion concentration. Amino acids that change The antigen-binding domain or The antibody may be prepared in any manner, for example, when the metal ion is calcium ion concentration. In this case, pre-existing antibodies, pre-existing libraries (pharm hybridomas obtained by immunizing animals or B cells from immunized animals antibodies or libraries prepared from these antibodies or libraries, and calcium chelating Mutations of natural amino acids (e.g., aspartic acid and glutamic acid) and unnatural amino acids were introduced. Antibodies or libraries (containing calcium-chelating amino acids (e.g., aspartic acid and glutamic acid) Libraries with high content of glutamic acid or unnatural amino acids, or calcium at specific sites chelatable amino acids (e.g., aspartic acid and glutamic acid) or unnatural amino acids It is possible to use libraries containing mutations, etc.

[0139] As described above, the binding activity of antigen-binding molecules to antigens is changed depending on the ion concentration. For example, when the metal ion is calcium ion, an example of an amino acid that can be used is calcium ion. Any amino acid can be used as long as it forms a calcium-binding motif. Motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2 000) 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. (Struct (2006) 14, 6, 1049-1058). That is, Cs such as ASGPR, CD23, MBR, and DC-SIGN Any known calcium-binding motif, such as that of a lectin-type lectin, may be included in the antigen-binding molecules of the present invention. In addition to the above, preferred examples of such calcium-binding motifs include those represented by SEQ ID NO: 6. The calcium-binding motif contained in the antigen-binding domain may also be mentioned.

[0140] In addition, the binding activity of antigen-binding molecules to antigens changes depending on calcium ion concentration. As an example of an amino acid that chelates, an amino acid that has a metal chelating action can also be suitably used. Examples of amino acids with metal chelating activity include serine (Ser(S)), threonine (Ser(S)), and threonine (Trimethyl)). 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 may be any position. As long as the binding activity of an antigen-binding molecule to an antigen is changed depending on the ion concentration, It can be located anywhere in the heavy chain variable region or light chain variable region that forms the antigen-binding domain. That is, the antigen-binding domain of the present invention exhibits an anti-inflammatory effect depending on the calcium ion concentration. The antigen-binding domain of the heavy chain contains amino acids that change the binding activity of the antigen-binding molecule to the antigen. The antigen-binding molecules can be obtained from a library mainly composed of antigen-binding molecules having different sequences. In another non-limiting aspect, the antigen-binding domain of the present invention is The CDR3 sequences are extracted from a library consisting mainly of antigen-binding molecules with different sequences. In another non-limiting aspect, the antigen-binding domain of the present invention can be obtained by at positions 95, 96, 100a and / or 101 according to the Kabat numbering system in CDR3 of the heavy chain The antigen-binding molecules are obtained from a library mainly composed of antigen-binding molecules having different sequences. obtain.

[0142] In a non-limiting aspect of the present invention, the antigen-binding domain of the present invention binds to calcium ions. The amino acids in the light chain that change the binding activity of the antigen-binding molecule to the antigen depending on the concentration conditions A library consisting mainly of antigen-binding molecules with different sequences contained in the antigen-binding domain. In another aspect, the antigen-binding domain of the present invention can be obtained from the amino acid sequence of the present invention. The light chain CDR1 contains a nucleotide sequence different from each other. In another aspect, the antigen-binding domain of the present invention can be obtained from the amino acid sequence of the The amino acid is present at positions 30, 31, and / or 32 of the CDR1 of the light chain according to the Kabat numbering system. The antigen-binding molecules can be obtained from a library mainly consisting of antigen-binding molecules having sequences different from each other.

[0143] In another non-limiting aspect, the antigen-binding domain of the present invention is an antigen-binding domain in which the amino acid residue is a light chain amino acid residue. A library mainly consisting of antigen-binding molecules with different sequences contained in the CDR2 of In another embodiment, the amino acid residues can be obtained by the Kabat numbering of CDR2 of the light chain. A library consisting mainly of antigen-binding molecules with different sequences contained at positions 50 and 51, represented by the group A library is provided.

[0144] In yet another non-limiting aspect, the antigen-binding domain of the present invention is an antigen-binding domain in which the amino acid residue is a light chain amino acid residue. A library mainly consisting of antigen-binding molecules with different sequences contained in the CDR3 of In another aspect, the antigen-binding domain of the present invention can be obtained by Antigens with different sequences contained at position 92 of the light chain CDR3 expressed by Kabat numbering 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 identical to those of the light chain CD41 described above. Two or three CDRs selected from CDR1, CDR2 and CDR3 have different sequences. In another embodiment of the present invention, the library can be obtained from a library mainly composed of antigen-binding molecules. Furthermore, the antigen-binding domain of the present invention may be a domain in which the amino acid residues are aligned according to the Kabat numbering system of the light chain. 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 The binding activity of antigen-binding molecules to antigens changes depending on calcium ion concentration. These amino acid residues may be contained alone or in combination of two or more of these amino acids. In addition, it may be possible to include a protein that has multiple calcium ion binding sites and is common in molecular evolution. Troponin C, calmodulin, parvalbumin, and myocardium are thought to be derived from the The light chain is known to contain CDR1, CDR2 and / or CDR3. Alternatively, CDR3 can be engineered. For example, a cadherin domain ... EF-hand in lumodulin, C2 domain in protein kinase C, blood coagulation The Gla domain contained in the protein Factor IX, the asialoglycoprotein receptor, 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, "binding of antigen-binding molecules to antigens is controlled by ion concentration conditions" A framework that already contains at least one amino acid residue that changes binding activity The heavy chain variable region was selected as a sequence and the heavy chain variable region was generated as a randomized variable region sequence library. By combining the light chain variable regions of the present invention with the light chain variable regions of the The antigen-binding domains of the present invention can be obtained from libraries containing binding molecules. As a non-limiting example, when the ion concentration is calcium ion concentration, for example, the sequence Heavy chain variable region shown in SEQ ID NO: 7 (6RL#9-IgG1) or SEQ ID NO: 8 (6KC4-1#85-IgG1) The randomized variable region sequence library was then combined with the light chain variable region sequence. A suitable example is a library prepared as a randomized variable region sequence library. Instead of the light chain variable region produced by the method, a light chain variable region having a germline sequence is selected. For example, the sequence SEQ ID NO: 7 (6RL#9-IgG1) or the sequence 6KC4-1#85-IgG1 has the heavy chain variable region sequence and germline sequence described in A suitable example is a library that combines a light chain variable region of the same kind with a light chain variable region of the same kind.

[0148] As used herein, a "library" refers to a library of multiple antigen-binding molecules or a library of multiple antigen-binding molecules. Nucleic acids and polynucleotides encoding the fusion polypeptides or sequences thereof The library contains a plurality of antigen-binding molecules or a plurality of fusion polymers containing antigen-binding molecules. The sequence of the polypeptide is not a single sequence, but rather consists of antigen-binding molecules or antigen-binding molecules with different sequences. The library is a fusion polypeptide containing a recombinant molecule. Methods for displaying fusion polypeptides containing antibody fragments on a surface are known in the art. , for example, WO1992001047 and described herein. No. 6213, WO1993011236 and 1993019172 describe related methods, and those skilled in the art will be able to Other known methods (H.R. Hoogenboom & G. Winter) can be used as appropriate. r (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236) , artificially rearranged variable region genes for various antigens displayed on the phage surface Identification of antibodies by repertoire is shown.

[0149] (3) Hydrogen ion concentration conditions In one embodiment of the present invention, the ion concentration condition is a hydrogen ion concentration condition or a pH In the present invention, the condition for the concentration of protons, i.e., hydrogen atom nuclei, is defined as the hydrogen index The activity of hydrogen ions in an aqueous solution is expressed as aH+, and p H 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 = 1 Since the pH is 0-14, in pure water 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 ion concentration conditions, pH conditions are As conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range, and low hydrogen ion concentration or high The binding activity changes depending on the pH condition. Conditions of hydrogen ion concentration or low pH (acidic pH range) and low hydrogen ion concentration or high pH (neutral pH range) This refers to the change in the binding activity of an antigen-binding molecule to an antigen depending on the conditions. For example, The binding activity of antigen-binding molecules to antigens in the neutral pH range is higher than that in the acidic pH range. In addition, the binding activity of the antigen-binding molecule to the antigen in the neutral pH range is higher. The binding activity of the antigen-binding molecule to the antigen under acidic pH conditions is higher than that under acidic pH conditions. There may also be cases where the binding activity of the antigen-binding molecule to the target antigen is higher.

[0151] In this specification, the neutral pH range is not limited to a specific value, but is preferably Preferably, the pH may be selected from the range of 6.7 to 10.0. In another embodiment, the pH may be selected from the range of 6.7 to 9.5. In a different embodiment, the pH may be selected from the range of 7.0 to 9.0. In one embodiment, the pH can be selected from the range of pH 7.0 to pH 8.0. In particular, the pH in plasma (blood) in vivo A preferred example is a pH of 7.4, which is close to the pH of the aqueous solution.

[0152] In this specification, the acidic pH range is not limited to a specific value, but is preferably In another embodiment, the pH may be selected from the range of pH 4.0 to pH 6.5. In a different embodiment, the pH may be selected from the range of 5.0 to 6.5. In one embodiment, the pH may be selected from the range of 5.5 to 6.5. A preferred example is pH 5.8, which is close to the calcium ion concentration.

[0153] In the present invention, the antigen-binding molecule is cleaved under conditions of high hydrogen ion concentration or low pH (acidic pH range). The binding activity to the antigen in the antibody is low in hydrogen ion concentration or high in pH (neutral pH range). The binding activity of the antigen-binding molecule is lower than that of the antigen-binding molecule at a pH selected from the range of pH 4.0 to pH 6.5. The binding activity to the antigen at a pH selected from the range of pH 6.7 to pH 10.0 is Preferably, the binding activity of the antigen-binding molecule is between pH 4.5 and pH 6.5. The binding activity to the antigen at a selected pH is selected from the range of pH 6.7 to pH 9.5. It means that the binding activity of the antigen-binding molecule is weaker than that of the antigen, and more preferably, the binding activity of the antigen-binding molecule is weaker than that of the antigen-binding molecule at pH 5.0 to pH 6.0. The antigen-binding activity at a pH selected from the range of pH 7.0 to pH 9.0 is It means that the binding activity to the antigen is weaker than that at the pH where the antigen-binding molecule is present. The binding activity to the antigen at a pH selected from the range of pH 5.5 to pH 6.5 is It is particularly preferred that the binding activity to the antigen at a pH selected from the range The antigen-binding activity at the pH in the early endosome in vivo was found to be different from that at the pH in plasma in vivo. Specifically, 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] Does the binding activity of the antigen-binding domain of the present invention to an antigen change depending on pH conditions? Whether or not the binding activity of the antibody can be determined by using a known measurement method such as that described in the above section on binding activity. That is, the binding activity under different pH conditions can be determined by the measurement method. For example, the activity of antigen-binding domains of the present invention under acidic pH conditions is measured. The antigen-binding domain of the present invention exhibits a higher binding activity to the antigen under neutral pH conditions than the antigen-binding activity of the synthetic molecule. To confirm that the binding activity of antigen-binding molecules containing α-glucan is higher than that of the α-glucan-containing ... The binding activity of antigen-binding molecules to antigens under acidic and neutral pH conditions is compared. are compared.

[0155] Furthermore, in the present invention, "under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range" The binding activity to the antigen is low at low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range. The expression "having a binding activity lower than that of an antigen" refers to a low hydrogen ion concentration or The binding activity to antigens at high pH, ​​i.e., in the neutral pH range, is higher than that at high or low hydrogen ion concentrations. It can also be expressed as having higher antigen-binding activity than that under conditions in the acidic pH range. In the present invention, "high hydrogen ion concentration or low pH, i.e., under conditions in the acidic pH range" is used. The binding activity to the antigen at low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, "Lower binding activity to antigens at high hydrogen ion concentrations or low pH, i.e., in the acidic pH range" is defined as " The binding activity to the antigen under the conditions of low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, In some cases, the term "weaker binding ability to antigens than under high hydrogen bond conditions" is used, and in other cases, the term "high hydrogen bond The binding activity to antigens at low concentrations or low pH, i.e., in the acidic pH range, is low. The binding activity to the antigen is lower than that under conditions of high ion concentration or high pH, ​​i.e., neutral pH range. " is used to refer to the antigen-binding activity under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range. The binding ability of the antibody to the antigen under conditions of low hydrogen ion concentration or high pH, ​​i.e., the neutral pH range, is higher than that under conditions of low hydrogen ion concentration or high pH. It may also be written as "weakening the effect."

[0156] Conditions other than hydrogen ion concentration or pH when measuring antigen-binding activity can be determined by those skilled in the art. The buffer may be selected appropriately and is not particularly limited. For example, HEPES buffer, 37°C For example, Biacore (GE Healthcare) can be used. The binding activity of an antigen-binding molecule containing an antigen-binding domain with an antigen can be measured. When the antigen is a soluble antigen, the activity is measured by immobilizing an antigen-binding molecule containing the antigen-binding domain. The binding activity to soluble antigens is evaluated by flowing the antigen as an analyte onto the immobilized chip. If the antigen is a membrane-type antigen, the antigen is immobilized on a chip. By using an antigen-binding molecule containing the original binding domain as an analyte, binding to membrane-type antigens is The activity can be assessed.

[0157] In the antigen-binding molecules of the present invention, high hydrogen ion concentration or low pH, i.e., conditions in the acidic pH range The binding activity to the antigen in the ATP-binding protein is affected by low hydrogen ion concentration or high pH, ​​i.e., neutral pH range. As long as the binding activity to the antigen is weaker than that at high hydrogen ion concentrations or low pH, i.e., pH acidic Antigen binding activity under neutral conditions and low hydrogen ion concentration or high pH, ​​i.e., pH The ratio of binding activity to the antigen under the conditions of the active region is not particularly limited, but is preferably Dissociation (KD) in conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range constant: dissociation constant) and low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range. The ratio of KD (pH 5.8) / KD (pH 7.4) is 2 or more, and more preferably KD (pH 5. 8) / KD (pH7.4) is 10 or more, and more preferably, the value of KD (pH5.8) / KD (pH7.4) is 4 or more. The upper limit of the KD (pH 5.8) / KD (pH 7.4) value is not particularly limited, and it is within the skill of a person skilled in the art to determine the upper limit. Any value such as 400, 1000, 10000, etc. may be used as long as it can be produced.

[0158] When the antigen is soluble, the KD (dissociation constant) is used as the value of binding activity to the antigen. However, if the antigen is a membrane antigen, apparent dissociation coefficient (ADC) may be increased. It is possible to use KD (dissociation constant) and apparent dissociation constant. The apparent dissociation constant (KD) of a compound can be measured by a method known to those skilled in the art, for example, Biacore (GE healthcare), Scatchard plot, flow cytometer, etc. It is possible to do so.

[0159] Furthermore, the antigen-binding molecules of the present invention are capable of binding to conditions of high hydrogen ion concentration or low pH, i.e., in the acidic pH range. The binding activity to antigens at low hydrogen ion concentrations or high pH, ​​i.e., in the neutral pH range, was Another index showing the ratio of binding activity to the antigen in a given antibody is, for example, the dissociation rate constant kd( Dissociation rate constants can also be suitably used. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an index of the ratio, kd (dissociation rate constant) at high hydrogen ion concentration or low pH, i.e., in the acidic pH range and the ratio of kd (dissociation rate constant) at low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range The value of kd (in the acidic pH range) / kd (in the neutral pH range) is preferably is 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably Preferably, it is 30 or more. Kd (in the acidic pH range) / kd (in the 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] When the antigen is soluble, kd (dissociation rate constant) is used as the value of antigen binding activity. If the antigen is a membrane antigen, the apparent dissociation rate (Apparent kd) is The apparent dissociation rate constant (kd) and The apparent kd (apparent dissociation rate constant) can be measured by a method known to those skilled in the art. For example, Biacore (GE healthcare), flow cytometer, etc. can be used. In the present invention, the activity of antigen-binding molecules at different hydrogen ion concentrations, i.e., pH, is When measuring the binding activity to the antigen, conditions other than the hydrogen ion concentration, i.e., pH, should be kept the same. It is preferable that

[0161] For example, in one embodiment of the present invention, a high hydrogen ion concentration or a low pH, i.e., a pH acid The binding activity to the antigen under the conditions of the neutral range is low hydrogen ion concentration or high pH, ​​i.e., pH An antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the serotonin range is The method can be performed by screening for an antigen-binding domain or antibody comprising the steps (a) to (c). do. (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, one embodiment of the present invention provides a method for producing a high hydrogen ion concentration or a low pH, i.e., a pH acid. The binding activity to the antigen under the conditions of the neutral range is low hydrogen ion concentration or high pH, ​​i.e., pH An antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the serotonin range is The screening of antigen-binding domains or antibodies or libraries thereof comprising steps (a) to (c) It can be obtained by scanning. (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 acidic pH conditions; process, and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0163] In addition, one embodiment of the present invention provides a method for producing a compound having a high hydrogen ion concentration or a low pH, i.e., an acidic pH. The binding activity to the antigen under the conditions of low hydrogen ion concentration or high pH, ​​i.e., neutral pH, The antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the following range is Screening of antigen-binding domains or antibodies or libraries thereof, comprising steps (a) to (d) It can be obtained by scanning. (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) binding the antigen-binding domain or antibody selected in step (b) to the antigen under a neutral pH condition; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0164] Furthermore, one embodiment of the present invention provides a method for producing a high hydrogen ion concentration or a low pH, i.e., a pH acid. The binding activity to the antigen under the conditions of the neutral range is low hydrogen ion concentration or high pH, ​​i.e., pH An antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the serotonin range is The antibody can be obtained by a screening method comprising steps (a) to (c) above. (a) Immobilizing a library of antigen-binding domains or antibodies on a column containing immobilized antigens at a neutral pH. contacting the (b) The antigen-binding domain or antibody bound to the column in step (a) is subjected to column chromatography under an acidic pH range. eluting the compound from the emulsion; and (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0165] Furthermore, one embodiment of the present invention provides a method for producing a high hydrogen ion concentration or a low pH, i.e., a pH acid. The binding activity to the antigen under the conditions of the neutral range is low hydrogen ion concentration or high pH, ​​i.e., pH An antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the serotonin range is The antibody can be obtained by a screening method comprising steps (a) to (d) above. (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 was eluted without binding to the column in step (a). The course, (c) binding the antigen-binding domain or antibody recovered in step (b) to an antigen under a neutral pH range. and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0166] Furthermore, one embodiment of the present invention provides a method for producing a high hydrogen ion concentration or a low pH, i.e., a pH acid. The binding activity to the antigen under the conditions of the neutral range is low hydrogen ion concentration or high pH, ​​i.e., pH An antigen-binding domain or antibody having a lower antigen-binding activity under the conditions of the serotonin range is The antibody can be obtained by a screening method comprising steps (a) to (d) above. (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 Call (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 antibody.

[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, a method for screening for an antigen under an acidic pH range is provided. An antigen-binding domain or domains whose binding activity is lower than that in the neutral pH range The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited. However, it is usually less than 10 times.

[0168] In the screening method of the present invention, high hydrogen ion concentration conditions or low pH, i.e., pH acid conditions, The antigen-binding activity of an antigen-binding domain or antibody in the pH range is determined by the antigen-binding activity between pH 4.0 and 6.5. There are no particular limitations as long as the activity is active, but the preferred pH is between 4.5 and 6.6. Other preferred pH values ​​include a pH between 5.0 and 6.5, and the antigen-binding activity is furthermore The antigen binding activity at a pH between 5.5 and 6.5 is particularly preferred. The pH in the early endosomes is one example, and specifically, the antigen binding activity at pH 5.8 is one example. In addition, antigens under low hydrogen ion concentration conditions or high pH, ​​i.e., in the neutral pH range, can be detected. The antigen-binding activity of the binding domain or antibody is particularly high when the pH is between 6.7 and 10. Although not limited thereto, preferred examples of the antigen binding activity include a pH between 6.7 and 9.5. Another preferred pH is between 7.0 and 9.5, and further preferred is between 7.0 and 8.0. As a more preferable pH, the pH of plasma in vivo can be Specifically, the antigen-binding activity at pH 7.4 can be mentioned.

[0169] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Conditions other than the 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 determined by KD (Dissociation constant : dissociation constant), apparent KD (Apparent dissociation constant), Dissociation rate constant kd (dissociation rate constant), or apparent kd (apparent d These can be evaluated by the methods known in the art, such as the apparent dissociation rate constant (apparent dissociation rate constant). Measurement can be performed by methods known to those skilled in the art, for example, Biacore (GE healthcare), Scanner, etc. Chard plots, FACS, etc. can be used.

[0170] In the present invention, antigen binding under conditions of low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, The binding activity is higher than that under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range. The process of selecting high antigen-binding domains or antibodies may be carried out at high hydrogen ion concentration or low pH, i.e. The antigen-binding activity in the acidic pH range was found to be low in hydrogen ion concentration or high in the neutral pH range. the step of selecting an antigen-binding domain or antibody having a lower antigen-binding activity under the conditions is.

[0171] High hydrogen ion binding activity at low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range, As long as the antigen binding activity is higher than that at the on-concentration or low pH, i.e., in the acidic pH range, Antigen binding activity under ionic concentration or high pH, ​​i.e., neutral pH range, and high hydrogen ion concentration The difference in antigen-binding activity under low pH conditions, i.e., in the acidic pH range, is not particularly limited, but is preferably Preferably, the antigen-binding activity is high under conditions of low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range. The antigen-binding activity is at least twice that under conditions of hydrogen ion concentration or low pH, i.e., acidic pH range. It is more preferably 10 times or more, and even more preferably 40 times or more.

[0172] The antigen-binding domain of the present invention screened by the above screening method or The antibody may be any antigen-binding domain or antibody, such as those described above. For example, antibodies can be screened for antigen-binding domains with native sequences. Alternatively, the antigen-binding domains containing the substituted amino acid sequences may be screened. Alternatively, antibodies or antibodies may be screened.

[0173] The antigen-binding domain or The antibody may be prepared in any manner, for example, a pre-existing antibody, libraries that exist for the purpose of antibodies or libraries generated from B cells from immunized animals, The library contains amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and non- Antibodies or libraries into which natural amino acid mutations have been introduced (amino acids with a side chain pKa of 4.0-8.0 ( Libraries enriched with unnatural amino acids (e.g., histidine and glutamic acid) or unnatural amino acids In particular positions, amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) or It is possible to use libraries containing unnatural amino acid mutations, etc.

[0174] Antigens produced from hybridomas obtained by immunizing animals or B cells from immunized animals The binding domain or antibody is then released under conditions of low hydrogen ion concentration or high pH, ​​i.e., in the neutral pH range. The antigen-binding activity in the presence of ATP is higher than that in the presence of ATP under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range. As a method for obtaining an antigen-binding domain or antibody with higher binding activity, for example, WO2009 / 12582 At least one of the amino acids in the antigen-binding domain or antibody as described in 5 is Amino acids with chain pKa values ​​between 4.0 and 8.0 (e.g., histidine and glutamic acid) and unnatural amino acids The antigen-binding domain or antibody has a side chain pKa of 4.0-8.0. Antibodies containing certain amino acids (e.g., histidine and glutamic acid) or unnatural amino acids Preferred examples include natural binding molecules and antibodies.

[0175] Amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids The position where the mutation of the amino acid is introduced is not particularly limited, and the amino acid may be in the acidic pH range compared to before substitution or insertion. The antigen binding activity in the acidic pH range is weaker than that in the neutral pH range (KD(acidic pH range) / KD(p As long as the value of H (neutral range) increases, or the value of kd (acidic pH range) / kd (neutral pH range) increases, For example, when the antigen-binding molecule is an antibody, the variable region or CDR of the antibody may be used. Suitable examples include amino acids with a side chain pKa of 4.0 to 8.0 (e.g., histidine and glutamic acid). The number of amino acids to be substituted with or inserted into the nucleotide sequence (e.g., thiaminic acid, thiaminoacid ... Those skilled in the art can appropriately determine the pKa of one amino acid (e.g., human) whose side chain has a pKa of 4.0 to 8.0. They can be substituted with amino acids (such as stigmine and glutamic acid) or unnatural amino acids, and have side chain pKa values ​​between 4.0 and 8. 0 (e.g., histidine or glutamic acid) or an unnatural amino acid is inserted. It can be composed of two or more amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamine). 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 (eg, histidine and glutamic acid) and unnatural amino acids can be inserted. In addition, amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids Substitution with amino acids or amino acids with side chain pKa of 4.0-8.0 (e.g., histidine and glutamine) In addition to the insertion of amino acids (e.g., amino acids) and unnatural amino acids, other amino acid deletions, additions, insertions and / or substitutions may be made. Amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamine) can be used simultaneously. glutamic acid) or unnatural amino acids or amino acids with a side chain pKa of 4.0-8.0 (e.g. The insertion of amino acids such as histidine and glutamic acid, or unnatural amino acids, can be performed using alanine scanning electron microscopy known to those skilled in the art. Randomization is performed by methods such as histidine scanning, in which alanine in ng is replaced with histidine, etc. The amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamine) can be used. Antigen-binding domains randomly mutated with substitutions or insertions of unnatural amino acids Or, among the antibodies, KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / Antigen-binding molecules with increased kd (neutral pH range) values ​​can be selected.

[0176] As mentioned above, amino acids whose side chains have a pKa of 4.0-8.0 (e.g., histidine and glutamine) The antigen-binding activity in the acidic pH range was increased to that in the neutral pH range. Preferred examples of antigen-binding molecules with lower antigen-binding activity include those with a side chain pKa of 4.0 -8.0 (e.g., histidine or glutamic acid) or mutation to unnatural amino acids The antigen-binding activity in the neutral pH range is determined by the pKa of the side chain of the amino acid (e.g., histidine). The antigen-binding activity in the neutral pH range was comparable to that of the unnatural amino acid (e.g., glutamic acid or guanine) before mutation. In the present invention, preferred examples of antigen-binding molecules include those having a side chain pKa of 4.0 to 8.0. Antigen-binding molecules after mutation of amino acids (e.g., histidine and glutamic acid) or unnatural amino acids , amino acids whose side chains have a pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and non-natural The antigen-binding activity of an antigen-binding molecule is equivalent to that of an antigen-binding molecule before and after the amino acid mutation. .0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids before mutation When the antigen-binding activity of an antigen-binding molecule is taken as 100%, an amino acid sequence having a side chain pKa of 4.0-8.0 is Antigen-binding molecules after mutation of natural amino acids (e.g., histidine and glutamic acid) or unnatural amino acids The original binding activity is at least 10% or more, preferably 50% or more, more preferably 80% or more. More preferably, it means 90% or more of the amino acids whose side chains have a pKa of 4.0-8.0 (e.g., The antigen binding activity at pH 7.4 after mutation of unnatural amino acids (e.g., histidine and glutamic acid) was Amino acids with side chain pKa values ​​between 4.0 and 8.0 (e.g., histidine and glutamic acid) and unnatural amino acids The antigen-binding activity at pH 7.4 may be higher than that before the amino acid mutation. The pKa of the amino acid side chain is 4.0-8. 0 (e.g., histidine or glutamic acid) or unnatural amino acids, or If the antigen-binding activity of the antigen-binding molecule is reduced by the insertion, The antigen-binding activity of the antibody can be improved by substitution, deletion, addition and / or insertion of multiple amino acids. Amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids The antigen binding activity can be made equivalent to that before the substitution or insertion of the amino acid. Amino acids with chain pKa values ​​between 4.0 and 8.0 (e.g., histidine and glutamic acid) and unnatural amino acids Substitution or insertion of one or more amino acids followed by substitution, deletion, addition and / or insertion of 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 containing an antibody constant region, the antigen-binding activity in the acidic pH range is In another preferred embodiment of the antigen-binding molecule, the affinity is lower than the antigen-binding activity in the neutral pH range. Examples of methods include those in which the antibody constant region contained in the binding molecule is modified. Specific examples of the constant region include the constant regions set forth in SEQ ID NO: 9, 10, 11, or 12. A suitable example is a region.

[0178] (4) The binding activity of the antigen-binding domain to the antigen is changed depending on the hydrogen ion concentration. Amino acids The antigen-binding domain or The antibody may be prepared in any manner, for example, under conditions of ion concentration, such as hydrogen ion concentration. If the temperature or pH conditions are such that pre-existing antibodies, pre-existing antibodies Libraries (such as phage libraries), hybridomas obtained by immunizing animals, Antibodies or libraries generated from B cells from immunized animals, and Amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids Antibodies or libraries containing amino acids mutated (amino acids with side chain pKa of 4.0-8.0 (e.g., human) Libraries with high content of unnatural amino acids (such as stigmine and glutamic acid) and specific amino acids Amino acids with side chain pKa values ​​of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids It is possible to use libraries containing mutations of the acid. Examples of amino acids having the formula include natural amino acids such as histidine and glutamic acid, as well as Histidine analogue (US20090035836) or m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr ( unnatural amino acids such as 3,5-I2-Tyr (pKa 7.21) or 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768 are suitable examples. It is known that the hydroxylase activity can be controlled by the hydroxylase activity (Angew. Chem. Int. Ed. (200 5) 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, "antigens against antigens depending on hydrogen ion concentration conditions" is A light chain polymer having at least one amino acid residue introduced therein that alters the binding activity of the original binding molecule. The variable region is combined with a heavy chain variable region prepared as a randomized variable region sequence library. By doing so, it is possible to obtain a library of the present invention containing a plurality of antigen-binding molecules having different sequences. can be produced.

[0180] Non-limiting examples of such amino acid residues include those contained in CDR1 of the light chain. Other non-limiting examples of such amino acid residues include amino acid residues contained in CDR2 of the light chain. Another non-limiting example of such an amino acid residue is an amino acid contained in CDR3 of the light chain. Acid residues are also exemplified.

[0181] As described above, the amino acid residues are non-limiting examples of amino acid residues contained in the light chain CDR1. and the positions 24, 27, 28, 31, and 32 in the CDR1 of the light chain variable region as indicated by Kabat numbering. The amino acid residues at positions 2 and / or 34 of the light chain CD4 Non-limiting examples of amino acid residues contained in R2 include those having Kabat numbering in CDR2 of the light chain variable region. amino acid residues at positions 50, 51, 52, 53, 54, 55 and / or 56, as represented by Furthermore, the amino acid residues included in the CDR3 of the light chain include, but are not limited to, For example, the amino acids at positions 89, 90, 91, and 92 in the CDR3 of the light chain variable region, as represented by the Kabat numbering system, The amino acid residues at positions 91, 92, 93, 94 and / or 95A are also included. The binding activity of the antigen-binding molecule to the antigen changes depending on the hydrogen ion concentration. As long as the amino acid residue is sufficient, it may contain one of these amino acid residues alone or a combination of two or more of these amino acids. may be included together.

[0182] Non-limiting examples of sites substituted with histidine or unnatural amino acids in the present invention include: For example, the following amino acid positions described in WO2009 / 125825 can be mentioned. are shown in Kabat numbering. Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H1 00b, 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 examples when the antigen is an IL-6 receptor (for example, human IL-6 receptor) include: The following points can be cited as new modifications: 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] When multiple sites are substituted with histidine or an unnatural amino acid in combination, preferred combinations are: Specific examples of combinations include H27, H31, and H35, H27, H31, H32, H35, H58, and H6. 2, a combination of H102, a combination of L32 and L53, a combination of L28, L32 and L53, etc. A preferred example of a combination of heavy and light chain substitution sites is a combination of H27, H31, L32, and L53. These positions may include those in which only one position is replaced with histidine or a non-natural amino acid. It may be substituted at multiple positions with histidine or a non-natural amino acid.

[0186] Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, the binding may vary depending on the ion concentration. As another method for changing the binding of an antigen-binding molecule to an antigen, Specific examples of such antibody constant regions include, for example, For example, the antibody constant regions (SEQ ID NO: 13, SEQ ID NO: 14) described in the Examples of WO2009 / 125825 , SEQ ID NO: 15, SEQ ID NO: 16). For example, methods for modifying the constant region include changing the isotype (IgG1, IgG2, IgG3, IgG4) of the constant region. Several studies were carried out and it was found that the antigen binding activity decreased in the acidic pH range (the dissociation rate was faster in the acidic pH range). Furthermore, methods for selecting an isotype that is less likely to produce a wild-type isotype are also proposed. By introducing amino acid substitutions into the amino acid sequence (wild-type IgG1, IgG2, IgG3, and IgG4 amino acid sequences), , decreases antigen-binding activity in the acidic pH range (increases dissociation rate in the acidic pH range) The antibody constant region is differentiated by isotype (IgG1, IgG2, IgG3, IgG4). The amino acid sequence of the hinge region is significantly different, and the difference in the amino acid sequence of the hinge region has a significant effect on antigen binding activity. Therefore, it is important to select an appropriate isotype depending on the type of antigen or epitope. Therefore, the antigen binding activity decreases under ion concentration conditions, for example, in the acidic pH range ( An isotype (which increases the dissociation rate at 1000 kJ / s) can be selected. Since differences in sequence significantly affect antigen-binding activity, we The hinge region is considered to be a desirable location for amino acid substitution in the amino acid sequence.

[0187] The antigen-binding molecules whose antigen-binding activity changes depending on the ion concentration are By using this method, it is possible to substituted or inserted amino acids into antigen-binding molecules that do not have such properties. It is possible to produce such a material by inserting it into the substrate. For example, antigen-binding molecules with such properties can be directly obtained from animals (mouse) , rats, hamsters, rabbits, human immunoglobulin transgenic mice, human immunoglobulin Human immunoglobulin transgenic rats, human immunoglobulin transgenic rabbits The antibodies obtained by immunizing animals (such as llamas and camels) with an antigen were analyzed to determine whether they bind to the antigen in a ion concentration-dependent manner. By screening using the results as an indicator, antibodies with the desired properties can be directly obtained. Furthermore, antibodies against ion concentration-dependent antigens can be obtained from an in vitro displayed antibody library. By screening using the binding of the target antibody as an index, antibodies with the desired properties can be directly obtained. The method is not particularly limited.

[0188] Amino acid modification For the modification of amino acids in the antigen-binding domain, site-directed mutagenesis (Kunkel et al. (Pr oc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR In addition, amino acids other than natural amino acids may be substituted. As a method for modifying amino acids, several known methods can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2 003) 100 (11), 6353-6357). For example, the UAG codon (amber), one of the stop codons, The complementary amber suppressor tRNA of the codon contains a tRNA with an unnatural amino acid attached. A cell-free translation system (Clover Direct (Protein Express)) that can be used is also suitable. .

[0189] Neutralizing activity In a non-limiting embodiment of the present invention, the FcRn-binding domain and the antigen-binding activity thereof are The antigen-binding domain, which changes depending on the conditions, and the binding activity to the sugar chain receptor, An antigen-binding molecule containing one or more carbohydrate receptor-binding domains that change depending on concentration conditions. Generally, the neutralizing activity is the activity of a virus, a toxin, or the like against a cell. It refers to the activity of inhibiting the biological activity of a ligand that has biological activity against a target molecule. A substance with agonistic activity is a substance that binds to the ligand or the receptor to which the ligand binds. It refers to a substance that inhibits the binding of the ligand to the receptor. The receptors whose binding is blocked are unable to exert their biological activity via the receptors. When the antigen-binding molecule is an antibody, the antibody having such neutralizing activity is generally The neutralizing activity of a test substance is determined by the biological activity of the substance in the presence of a ligand. The activity can be measured by comparing the activity 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. The IL-6R, which is a target protein, forms a heteroquatogen with the gp130 receptor, whose dimerization is induced by IL-6. The heterodimer is formed (HEINRICH et al. (Biochem. J. (1998) 334, 297-314)). Tetramer formation activates Jaks associated with the gp130 receptor. The phosphorylation sites of receptors and Jaks are similar to those of Stat3. Stat family molecules with SH2, MAP kinase, PI3 / Akt, and other SH2-containing molecules These act as binding sites for various 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. It can also be involved in the signal cascade as a result of deregulated IL-6 signaling. These genes are observed in the pathology of autoimmune diseases, inflammation, and cancers such as multiple myeloma and prostate cancer. Stat3, which can act as a gene, 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 signal cascades from family members (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

[0191] These intracellular signaling cascades differ depending on the cell type, so it is necessary to select the appropriate signaling cascade for each target cell. The target molecule can be appropriately set for each factor, and is not limited to the factors mentioned above. Neutralizing activity can be evaluated by measuring signal activation. The transcriptional induction effect on target genes downstream of the signal cascade is used as an indicator. It is also possible to detect activation of internal signals. Changes in the transcriptional activity of target genes are reported. Specifically, the transcription factor of the target gene can be detected by the principle of the target assay. Or GFP (Green Fluorescence Protein) or luciferase downstream of the promoter region By placing a reporter gene such as α-glucan and measuring its reporter activity, the transcriptional activity can be determined. The change can be measured as reporter activity. Commercially available reagents can be used as appropriate (e.g., Mercury Pathway Profiling Laboratory). uciferase System (Clontech), etc.).

[0192] Furthermore, EGF receptors, which normally act on signal cascades that promote cell proliferation, As a method for measuring the neutralizing activity of receptor ligands such as the receptor family, The neutralizing activity of a neutralizing antibody can be evaluated by measuring the proliferation activity of the cells. For example, the proliferation of cells whose proliferation is promoted by growth factors of the EGF family, such as HB-EGF, As a method for evaluating or measuring the inhibitory effect of anti-HB-EGF antibodies on proliferation based on the neutralizing activity of the anti-HB-EGF antibody, The following methods are preferably used: One method is to measure the uptake of [3H]-labeled thymidine added to the culture medium by living cells as DNA. A method for measuring trypan blue is used as an index of replication ability. 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 is a method in which living cells are exposed to 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. After a certain period of time has passed, MTT solution is added to the culture medium and left to stand for a certain period of time. As a result, the yellow compound MTT is absorbed into the mitochondria of the cells. This blue product is dissolved and colored. After the incubation, the absorbance is measured and used as an index 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, the same antibody as the anti-HB-EGF antibody can be used as a control antibody. A binding antibody having the same isotype but not having the cell proliferation inhibitory activity is used as the anti-HB-EGF antibody. When used in the same manner, the anti-HB-EGF antibody exhibited stronger cell growth inhibitory activity than the control antibody, demonstrating its activity. Gender can be determined.

[0193] Examples of cells for evaluating activity include cells whose proliferation is promoted by HB-EGF. The RMG-1 cell line, an ovarian cancer cell line, 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 appropriately select cells for evaluating activity. By selecting the appropriate antibody, it can be used for measuring the cell proliferation activity.

[0194] The antigen-binding molecules provided by the present invention can eliminate antigens from plasma, and therefore, It is not necessary for the original binding molecule itself to have neutralizing activity. Receptor-mediated endocytosis transports antigens along with antigen-binding molecules to the Fcγ receptor. It exerts neutralizing activity against antigens until it is taken up into cells expressing the antigen. It is more preferable to block the function of antigens present in plasma.

[0195] Furthermore, the antigen-binding molecules provided by the present invention can be used to bind antigens bound to the antigen-binding molecules extracellularly. This promotes dissociation from antigen-binding molecules within the cell, thereby preventing the release of antigen-binding molecules within the cell. The dissociated antigen is degraded in the lysosome. Therefore, the antigen-binding molecule itself has neutralizing activity. However, it is not necessary for the sugar chain receptor to have an endocytic function. The antigen is taken up into cells that express the sugar chain receptor together with the antigen-binding molecule by endocytosis. While in the blood, it exerts neutralizing activity against antigens, thereby suppressing the function of antigens present in the plasma. It is more preferable to block it.

[0196] Furthermore, the antigen-binding molecules provided by the present invention can reduce the total antigen concentration or free antigen concentration in plasma. Therefore, it is not necessary for the antigen-binding molecule itself to have neutralizing activity. However, the antigen binds to the endocytosis mediated by the sugar chain receptor. The neutralizing activity against the antigen is maintained until the molecule is taken up into cells that express the sugar chain receptor. It is more preferable that the antibody block the function of the antigen present in plasma by exerting its activity. stomach.

[0197] Carbohydrate receptors A glycan is a group of compounds in which various sugars are linked together by glycosidic bonds. Many of the sugar chains in the body exist as complex molecules bound to proteins and lipids, and are called glycoconjugates. Among these, glycoproteins are complex carbohydrates formed by the combination of sugar and protein.

[0198] The sugar chains recognized by the sugar chain receptors to which the sugar chain receptor-binding domains used in the present invention bind are Examples of sugar chains include the sugar chains that make up glycoproteins. Examples of such sugar chains include O-linked sugar chains and N-linked sugar chains. More preferably, the sugar chains of glycoproteins An example is N-linked glycans.

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

[0200] All N-linked glycans of glycoproteins share the same core structure: Man6(Manα1-3)Manβ1-4Glc NAcβ1-4GlcNAc, which is called the trimannosyl core. Based on the structure and location of the added sugar residue, N-linked glycans are classified into three subgroups: .

[0201] Of these, complex-type glycans do not contain any mannose residues other than the trimannosyl core. The reducing end of the amino acid has a GlcNAc residue, and the two α-mannosyl residues of the trimannosyl core It is bonded.

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

[0203] Complex glycans contain one or two α-mannosyl groups, similar to the high mannose type. The Manα1-6 arm of the rimannosyl core binds to the Manα1-3 arm of the core, and the side chain of the complex-type glycan is the same as that of the Manα1-3 arm of the core. The fucose is attached to the C-6 position of GlcNAc at the reducing end of the trimannosyl core. The presence or absence of β-mannosyl residues and the presence or absence of β-GlcNAc at the C-4 position of the β-mannosyl residue (bisecting) The presence or absence of three N-linked N-glycans (called GlcNAc) contributes to the structural diversity of complex glycans. Among the type glycan subgroups, the complex type contains the most diverse structures.

[0204] The sugar chain receptor is a receptor that recognizes and binds to the sugar chains mentioned above. Any molecule can be used as a sugar chain receptor as long as it is expressed on cells. For example, O-linked glycans 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 the type glycans. Furthermore, mannose receptors bind to N-linked glycans whose terminals are mannose. Such receptors are preferably used as sugar chain receptors in the present invention. That is, the antigen-binding molecules of the present invention are those that bind to such receptors. (glycan receptor binding domain). Therefore, the glycan receptor binds to asialoglycoprotein In the case of a receptor, an N-linked sugar chain having a terminal galactose is used in the present invention. The sugar chain receptor-binding domain can be suitably used as a sugar chain receptor-binding domain contained in an antigen-binding molecule in the present invention. When the receptor is a mannose receptor, the N-linked sugar has a terminal mannose. The sugar chain is preferably used as the sugar chain receptor-binding domain contained in the antigen-binding molecule of the present invention. obtain.

[0205] Carbohydrate receptor binding domain The antigen-binding molecules of the present invention bind to one or more glycoproteins that bind to glycoprotein receptors, particularly human glycoprotein receptors. The binding domain for the sugar chain receptor, particularly the human sugar chain receptor, is an antibody. The original binding molecule has binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range. , binding activity to sugar chain receptors in the acidic pH range and binding activity to sugar chain receptors in the neutral pH range The type and number of the sugar chain receptors are not particularly limited as long as they are lower than the sugar chain receptors. In particular, domains having binding activity to human sugar chain receptors can be used. Examples of the enzyme include those that directly bind to sugar chain receptors, particularly human sugar chain receptors. Fc domain of IgG immunoglobulin; carbohydrate receptors, especially human carbohydrate receptors Antibodies against glycoreceptors, especially human glycoreceptors; glycoreceptors, especially human glycoreceptors; In the present invention, a scaffold molecule for a sugar chain receptor can be exemplified. It has binding activity to sugar chain receptors, especially human sugar chain receptors, and exhibits activity in the acidic pH range. The sugar chain receptor binding domain has a lower binding activity to the receptor than to the sugar chain receptor in the neutral pH range. The domain is preferably a saccharide receptor, particularly a human saccharide receptor, in the neutral pH range. It has binding activity to sugar chain receptors in the acidic pH range, but its binding activity with sugar chain receptors in the neutral pH range If the glycan receptor binding domain has lower binding activity to the glycan receptor in A carbohydrate receptor-binding domain having an N-linked glycan with galactose at its terminal. and the asialoglycoprotein receptor, which is a sugar chain receptor that binds to the sugar chain, at an acidic pH. The binding activity in the neutral pH range is preferably lower than the binding activity in the neutral pH range. In addition, the glycoproteins are also shown to be related to glycoreceptor-binding domains with N-linked glycochains whose terminals are mannose. Binding activity to the mannose receptor, a sugar chain receptor that binds to the sugar chain, in the acidic pH range is also a suitable example of a compound having a lower binding activity than that in the neutral pH range.

[0206] Glycan receptor binding domains in the neutral pH range, especially for human glycan receptors If the binding activity is absent or weak, it can be improved by modifying the amino acids in the antigen-binding molecule. Thus, binding activity to sugar chain receptors, particularly human sugar chain receptors, can be acquired. The domains that have binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range are By modifying the amino acids in the glycoprotein, it is possible to bind to glycoreceptors, especially human glycoreceptors. The binding activity can be enhanced. Acid modification is a key step in the analysis of glycoreceptors, especially human glycoreceptors, in the neutral pH range before and after amino acid modification. The desired modification can be found by comparing the binding activity to the

[0207] Binding of the carbohydrate receptor-binding domain to carbohydrate receptors, especially human carbohydrate receptors, in the acidic pH range When the activity is not lower than the binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range. In order to achieve this, we have modified the amino acids in the antigen-binding molecule to inhibit the activity of sugar chain receptors in the acidic pH range. , especially human sugar chain receptors whose binding activity is in the neutral pH range It is possible to obtain a binding activity that is lower than that of the sugar chain receptor, particularly the human sugar chain receptor. The amino acid modification of the receptor-binding domain was performed to determine the activity of the receptor in the acidic pH range before and after the amino acid modification. The binding activity to the sugar chain receptors, especially human sugar chain receptors, and sugar chain receptors in the neutral range, especially By comparing the binding activity to human sugar chain receptors, the desired modification can be found.

[0208] In the present invention, the sugar chain receptor-binding domain is an antigen-binding domain that constitutes an antigen-binding molecule. It can also be introduced into sites other than the FcRn-binding domain. The carbohydrate receptor-binding domain may be incorporated into any part of the structure of the antigen-binding molecule as long as it does not inhibit the binding of the The site may also be introduced into the nucleotide sequence of the target gene, as long as it does not interfere with the binding of the antigen-binding domain to the antigen. It may be introduced into the antigen-binding domain or into other sites. In one embodiment, the carbohydrate receptor binding domain binds to the FcRn binding domain of the antigen-binding molecule and FcRn, particularly human Fc It can be introduced into any site in the structure of the antigen-binding molecule as long as it does not inhibit binding to Rn. For example, the hinge region of IgA antibodies contains the carbohydrate receptor binding domain for binding O-linked glycans. The amino acid sequence can be a candidate for the N-bond Asn-X-Ser / Thr, where X is an amino acid other than Pro. The motif sequence for N-linked glycan addition is a glycan receptor binding domain for N-linked glycan attachment. The amino acid sequences of the antigen-binding molecules containing these amino acid sequences can be candidates for the antigen-binding molecules. The desired sugar chain is bound to the host cell culture medium, which is introduced with the gene to be described later. Antigen-binding molecules of the present invention can be produced that contain a carbohydrate receptor-binding domain.

[0209] In the present invention, the sugar chain receptor binding domain may also be chemically prepared. For example, Galactose derivatives and high mannose-type sugars that mimic N-glycans with galactose terminals The chemical ligands used include mannose derivatives that mimic the chain and derivatives that mimic sialic acid. Such chemical ligands may be covalently conjugated to antigen-binding molecules. Bioorg. Med. Chem. (2011) 19 (8), 2494-2500, Bioorg. Med. Chem. (2009) 17 (20), 7254-7264, Bioorg. Med. Chem. (2008) 16 (9), 5216-5231, J.A. m. Chem. Soc. (2004) 126 (33), 10355-10363, J. Pept. Sci. (2003) 9 (6 ), 375-385, Methods Enzymol. (2010) 478, 343-363, etc. The amino acids of the antigen-binding site to be conjugated include, but are not limited to, Examples of suitable amino acids include, but are not limited to, lysine and cysteine. The method of conjugating to the nucleotide sequence is to replace the amino acid at the conjugation site with cysteine. Alternatively, lysine at the site where conjugation is not desired may be replaced with another amino acid. Methods known to those skilled in the art can be used for conjugation. and the reaction of activated esters with lysine. Methods known to those skilled in the art can be used.

[0210] The conditions other than pH when measuring the binding activity to an antigen or FcRn, particularly human FcRn, can be determined by those skilled in the art. The method is not limited to a specific method and can be appropriately selected depending on the application. For example, Measurement can be performed in MES buffer at 37°C so that the antibody titer of the 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, measurement can be performed 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 measured. The method involves flowing antigen as an analyte onto a chip on which antigen-binding molecules are immobilized. If the antigen is a membrane antigen, the binding activity to the membrane antigen can be evaluated. By flowing antigen-binding molecules as analytes onto the immobilized chip, binding activity to membrane-type antigens can be measured. Measurement of the binding activity of antigen-binding molecules to FcRn, particularly human FcRn. are applied to a chip on which an antigen-binding molecule or FcRn, particularly human FcRn, is immobilized, respectively. It can be evaluated by running FcRn or an antigen-binding molecule as an analyte.

[0211] In the present invention, the binding activity to FcRn, particularly human FcRn, in the acidic pH range is defined as the binding activity to FcRn in the acidic pH range from pH 4.0 to pH 4.0. This means the binding activity of H6.5 to FcRn, particularly human FcRn. The binding activity to FcRn is preferably at any pH between pH 5.5 and pH 6.5, for example, at pH 5.5, 5 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. It is particularly preferable that the binding activity is close to the pH in the early endosome in vivo. Any pH between pH 5.8 and pH 6.0, for example, 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 and 6.00.

[0212] The binding activity was measured at any temperature between 10°C and 50°C. Even if the antigen-binding molecule of the present invention containing an FcRn-binding domain binds to FcRn, particularly human FcRn, Preferably, the antigen-binding molecule of the present invention containing an FcRn-binding domain and FcRn In particular, to measure the binding activity to human FcRn, temperatures ranging from 15°C to 40°C were used. More preferably, the antigen-binding molecule of the present invention containing an FcRn-binding domain and FcRn, particularly human To measure the binding activity to FcRn, for example, , 29, 30, 31, 32, 33, 34, and 35°C, Temperature may also be used.

[0213] In the present invention, not inhibiting the binding of the antigen-binding domain to an antigen means that the antigen-binding domain does not inhibit the binding of the sugar chain receptor to the antigen. The introduction of the receptor-binding domain enhances the antigen-binding activity of the antigen-binding molecule by increasing the activity of the carbohydrate receptor-binding domain. The antigen-binding activity of the antigen-binding molecule before the introduction of the antibody is 20% or more, preferably 50% or more, and This means that the degree of affinity of the sugar chain receptor is preferably maintained at 80% or more, and more preferably at 90% or more. If the antigen-binding activity of the antigen-binding molecule is reduced by the introduction of the binding domain, The glycosylation can be achieved by, for example, substituting, deleting, adding, and / or inserting one or more amino acids in the glycomolecule. The antigen-binding activity can be changed to the same extent as that before the introduction of the receptor-binding domain. Substitution, deletion, or addition of one or more amino acids after introduction of such a carbohydrate receptor-binding domain and / or insertions, thereby achieving equivalent binding activity. Furthermore, not inhibiting the binding of the carbohydrate receptor-binding domain to FcRn, particularly human FcRn, means that The introduction of a carbohydrate receptor-binding domain improves the binding activity of antigen-binding molecules to FcRn, especially human FcRn. The activity of the antigen-binding molecule before the introduction of the sugar chain receptor-binding domain is compared with that of the FcRn, particularly human FcRn, which the antigen-binding molecule has. The binding activity is 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably The introduction of the carbohydrate receptor-binding domain improves antigen binding. When the binding activity of a molecule to FcRn, especially human FcRn, is reduced, one or more of the antigen-binding molecules The carbohydrate receptor binding domain is modified by multiple amino acid substitutions, deletions, additions, and / or insertions. The binding activity to FcRn, particularly human FcRn, can be altered to a level comparable to that before the introduction of the FcRn. In this case, one or more amino acid substitutions or deletions may be made after the introduction of such a carbohydrate receptor-binding domain. The present invention also includes antigen-binding molecules that have equivalent binding activity due to addition and / or insertion. Methods for measuring and determining the binding activity to antigens or FcRn, particularly human FcRn, are described below. can be.

[0214] Glycan Examples of sugar chain receptor-binding domains contained in the antigen-binding molecules of the present invention include those that bind to desired sugar chains. Preferred examples of the desired sugar chain include O-linked sugar chain receptor binding domains. Preferred examples include N-linked glycans and N-linked glycans, but For example, an immortalized mammalian cell line capable of secreting a protein can be used. A cell extract is prepared from cultured cells of an animal cell line, and mRNA encoding an antibody is added to the extract. A method for producing a post-translationally modified antibody in a cell-free protein synthesis system, characterized by adding A series of cell-free enzymatic methods, such as the method (JP 2006-141241), are used to bind the desired sugar chains. Such a method can be adopted for producing a sugar chain receptor binding domain. As one example, a motif sequence for adding a desired sugar chain has been introduced by recombinant gene techniques. The sugar chain receptor-binding domain contained in a natural or artificially produced antigen-binding molecule is encoded. By introducing a gene encoding the desired sugar into a host cell, the desired sugar can be extracted from the culture medium of the host cell. Antigen-binding molecules of the present invention can be produced that contain a carbohydrate chain receptor-binding domain to which a chain is attached.

[0215] When the glycan is an O-linked glycan, the motif sequence for adding the O-linked glycan is based on a publicly known database. By using a glycan such as PEG, it will be possible to design O-linked glycans. The disaccharide portion has an O-linked glycan attached to it, and the previously known O-linked glycans attached to it Gene sequences encoding carbohydrate receptor binding domains are likely candidates for motif sequences. When the glycan is an N-linked glycan, the motif sequence for N-linked glycan addition is Asn-X-Ser / Th. It is known that the motif is a sequence of three consecutive amino acids, r. The motif sequence Asn-X-Ser / Thr, which is used for N-linked glycan addition by genetic techniques, is coded. A gene encoding an antigen-binding molecule containing a carbohydrate receptor-binding domain designed to encode By introducing the above into a host cell as described below, the desired gene can be extracted from the culture medium of the host cell. The antigen-binding molecules of the present invention can be produced by containing a sugar chain receptor-binding domain to which a sugar chain is bound.

[0216] The glycan structure of the antigen-binding molecule produced as described above may be a single one, or multiple glycans. In the present invention, such mixtures may also be produced as a mixture of linked chains. Furthermore, antigen-binding molecules in which specific sugar chains are linked to the sugar chain-binding domains can be suitably used. Children can also be suitably used in the present invention.

[0217] A specific sugar chain is linked to the sugar chain receptor binding domain contained in the antigen-binding molecule of the present invention. As a method for this, several known methods can be adopted. The properties of sugar chains on antigen-binding molecules, whether natural or artificially produced by recombinant gene techniques, etc. One example is a method for purifying antigen-binding molecules with specific sugar chains by utilizing Antibodies with high mannose-type glycans were detected by affinity chromatography using ConA-sepharose. It is known that it can be purified using rafi (Millward (Biologicals (2008) 36, 49-60)). Such a purification method is suitable for the N-linked saccharides of the present invention in which the non-reducing end is mannose. It can be used to create antigen-binding molecules having chains.

[0218] In another embodiment, enzyme treatment is also performed to obtain antigen-binding molecules having specific sugar chains. As will be described later in the Examples, N-type galactose having a non-reducing end may be used. The antigen-binding molecule having a complex glycan is an antigen-binding molecule having a complex glycan in which the non-reducing end is sialic acid. It can be prepared from the original binding molecule by sialidase treatment. The antibody was treated with sialidase and β-galactosidase to remove galactose from its glycan. It is known that the antibody is produced in a form that has been removed from the surface (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, in order to obtain an antigen-binding molecule of the present invention having a specific sugar chain, A recombinant gene encoding a protease-binding molecule was transduced to induce the accumulation of specific glycans ( glycosidase activity, including but not limited to, by genetic or recombinant genetic techniques A method for recovering the high mannose N-linked saccharide from the culture medium of the transformed host cells can also be used appropriately. The antibody having the conjugated glycan is prepared by transfecting a recombinant gene encoding the antibody with an N-acetylglucan. Culture medium of a Lec1 mutant derived from CHO cells lacking glucosaminyltransferase I activity It is known that it is recovered from the steroid hormone (Wright and Morrison (J. Exp. Med. (1994) 180 In the present invention, the non-reducing end of the N-linked sugar chain is mannose. The antigen-binding molecules were cultured in a Lec1 mutant strain transfected with a recombinant gene encoding the molecule. It can be recovered from the liquid.

[0220] In yet another embodiment, the present invention provides a method for obtaining an antigen-binding molecule having a specific sugar chain of the present invention. Inhibition of specific glycosidase reactions during the culture of cells producing the antigen-binding molecule. By adding an agent, antigen-binding molecules having specific sugar chains that have accumulated in the culture medium are recovered. A method of N-linked mannose-type saccharides that do not have fucose at their reducing ends can also be appropriately adopted. Antibodies with glycan-type glycosylation are produced by adding kifunesine to the CHO cells that produce the antibodies. It is known that it can be recovered from the culture medium of the cells by Bioeng. (2008) 99, 652-665). In the present invention, First, an antigen-binding molecule having an N-linked sugar chain in which the non-reducing end is mannose can be used, for example, CHO cells transfected with a recombinant gene encoding the molecule were cultured with kifunesine. Alternatively, the glycosidases mentioned above can be used to recover the glycosidases from the culture medium. By combining the method of adding such inhibitors during the culture of host cells with altered activity, It is also possible to obtain antigen-binding molecules having specific sugar chains of the present invention by the above methods. It is known that antibodies having specific sugar chains can be recovered by such a combination (Kanda et al., 2004). (Glycobiology (2007) 17, 104-118)), antigens having specific sugar chains in the present invention Similar combinations can be suitably employed to obtain binding molecules.

[0221] In addition, by genetically modifying hosts such as Pichia pastoris, it is possible to produce glycoproteins with specific sugar chain structures. Methods for expressing proteins are known (Biochemistry. 2008 Sep 30;47(39):10 294-304., J Biotechnol. 2009 Feb 23;139(4):318-25., Nat Biotechnol. 2006 Feb;24(2):210-5.) By using such methods, it is possible to identify specific glycan structures (e.g., galactose) We created an antigen-binding molecule in which a glycan with a glycan terminus (N-glycosylation chain) was uniformly linked to the N-glycosylation sequence. It is possible.

[0222] The present invention relates to an antigen-binding molecule comprising a sugar chain receptor-binding domain, particularly a sugar chain receptor-binding molecule of human origin. The antigen-binding molecule containing the domain binds to a carbohydrate receptor in a pH-dependent manner, and / or or a sugar chain receptor binding activity, particularly a sugar chain receptor binding activity of human origin, in the neutral pH range. It has the binding activity to the sugar chain receptor in the acidic pH range and the binding activity to the sugar chain receptor in the neutral pH range. If the activity can be lowered below the target value, the uptake of antigens into cells by antigen-binding molecules can be promoted. The administration of the antigen-binding molecule promotes a decrease in the antigen concentration in plasma, and the drug This improves kinetics and increases the number of antigens that can be bound by a single antigen-binding molecule. do.

[0223] Binding activity to carbohydrate receptors In the present invention, the binding to sugar chain receptors in the acidic pH range, particularly sugar chain receptors derived from humans, is The binding activity is the binding activity to glycoreceptors, especially human glycoreceptors, at pH 4.0 to pH 6.5. Preferably, the pH is any pH between 5.5 and 6.5, for example, pH 5.5, 5.6, 5.7, 5.8, Carbohydrate receptors at pHs selected from 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, particularly It means the binding activity to human-derived sugar chain receptors, and particularly preferably to early endogenous sugar chains in vivo. Any pH between pH 5.8 and pH 6.0, close to the pH in the serotonin, e.g., 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. Glycan receptors, particularly human-derived glycans, at pHs selected from 97, 5.98, 5.99, and 6.00 In the present invention, the binding activity to the sugar chain receptor in the neutral pH range is The binding activity to glycoconjugates derived from the human body, especially to glycoconjugate receptors derived from humans, is determined by the glycoconjugate receptors at pH 6.7 to pH 10.0. In particular, it means the binding activity to a sugar chain receptor derived from human. Preferably, it is the binding activity at pH 7.0 to pH 8.0. Any pH, for example, pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8. Binding activity to sugar chain receptors, particularly human sugar chain receptors, at pHs selected from 0 to 100. 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 binding activity to carbohydrate receptors, especially human carbohydrate receptors can be appropriately selected by those skilled in the art, and is not limited to any particular condition. Measurement can be performed in MES buffer at 37°C as described in 2009 / 125825. Measurement of the binding activity of an antigen-binding molecule to a sugar chain receptor, particularly to a human sugar chain receptor, can be carried out by a person skilled in the art. For example, measurement can be performed using Biacore (GE Healthcare) or the like. The binding activity of antigen-binding molecules to carbohydrate receptors, especially human-derived carbohydrate receptors, can be determined. Measurements can be performed, for example, by immobilizing sugar chain receptors, especially human sugar chain receptor molecules, on a chip. By using antigen-binding molecules as analytes, it is possible to detect glycan receptors, especially human-derived glycan receptors. It is possible to evaluate the binding activity of an antigen-binding molecule to a target substance. A solubilized sugar chain receptor, especially a human-derived sugar chain receptor, is applied to a chip on which an antigen-binding molecule is immobilized. By using the body as an analyte, the glycoprotein binds to glycoreceptors, especially those derived from humans. The binding activity can be assessed.

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

[0226] The dissociation constant (KD) is used as a value of binding activity to glycan receptors, especially human glycan receptors. The KD (dissociation constant) can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plot, flow cytometer etc. can be suitably used.

[0227] In addition, the present invention also relates to sugar chain receptors in the acidic pH range, particularly sugar chain receptors derived from humans. Binding activity and binding activity to carbohydrate receptors, especially human carbohydrate receptors, in the neutral pH range Other indicators of the ratio include the dissociation rate constant k d (Dissociation rate constant KD (dissociation constant: nt) can also be suitably used as an index showing the ratio of binding activity. ) instead of k d When using the dissociation rate constant, glycoreceptors, especially human glycoreceptors, k in the acidic pH range d (dissociation rate constant) and k in the neutral pH range d (dissociation rate constant) k d (pH acidic range) / k d The value of (neutral pH range) is preferably 2 or more, more preferably 5 k is preferably 10 or more, more preferably 30 or more. d (pH acidic range) / k d The upper limit of the value of (neutral pH range) is not particularly limited, and can be determined by the common technical knowledge of a person skilled in the art. It can be set to any value possible, such as 50, 100, 200, etc.

[0228] For example, the asialoglycoprotein receptor, which is a type of sugar chain receptor, interacts with galactose. The interaction of the phospholipase C12 with the phospholipase C12 is pH dependent, exhibiting high binding activity in the neutral pH range and low binding activity in the acidic pH range. It is known that the enzymes are soluble in water (J Biol Chem Vol. 274, No. 50, pp. 35400-3540 6, 1999). Similarly, the interaction between mannose and the mannose receptor, which is a sugar chain receptor, The activity is pH-dependent, with high binding activity in the neutral pH range and low binding activity in the acidic pH range. It is known that (J Biol Chem. 1994 Nov 11;269(45):28405-13.) Therefore, in the present invention, the sugar chain / sugar chain acceptor is galactose / asialoglycopeptide. Protein receptors and mannose / mannose receptors can be preferably used.

[0229] Also, the galactose / asialoglycoprotein receptor and mannose / mannose receptor The binding activity of the NO receptor is not only pH-dependent but also calcium ion concentration-dependent. Since most of the sugar chain receptors are C-type lectins, the binding between sugar chain receptors and sugar chains is It has calcium ion concentration dependency. That is, similar to the binding between an antigen-binding molecule and an antigen, The binding of glycan receptors to glycans may be calcium ion concentration dependent, and high calcium ions It is sufficient that the binding at ON concentrations is higher than the binding at low calcium ion concentrations.

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

[0231] antigen binding molecule The present invention relates to a method for producing a nucleic acid molecule comprising: an antigen-binding domain, an FcRn-binding domain, and one or more binding domains to a carbohydrate receptor; An antigen-binding molecule having a binding domain, wherein the number of binding domains for the sugar chain receptor is Increased antigen-binding molecules and antigen-binding activity in the acidic pH range were reduced to that in the neutral pH range. and (c) providing an antigen-binding molecule further comprising an antigen-binding domain having a lower antigen-binding activity than that of the antigen-binding molecule in the The present invention also relates to a method for producing the antigen-binding molecule, and a pharmaceutical composition containing the antigen-binding molecule. Furthermore, the present invention provides a method for producing a glycoprotein comprising the step of culturing the antigen-binding molecule in a cell expressing a sugar chain receptor. the antigen-binding molecule and / or the A method for uptake of an antigen bound by an antigen-binding molecule into a cell, A method for increasing the number of antigens bound to cells, a method for reducing extracellular antigens, and A method for improving the pharmacokinetics of a binding molecule and a method for promoting dissociation of an antigen from the antigen-binding molecule The present invention also provides a method for promoting the production of a glycan-binding domain comprising an antigen-binding domain, an FcRn-binding domain, and a glycan. The sugar chain receptor in an antigen-binding molecule having two or more binding domains to the receptor In vivo or in vitro binding of the antigen, including increasing the number of binding domains for the antigen. To promote the uptake of binding molecules and / or antigens bound by said antigen-binding molecules into cells the number of antigens bound by the antigen-binding molecule per molecule in vivo or in vitro and a method for increasing the antigen-binding molecule's ability to eliminate antigens in vivo or in vitro. a method for improving the pharmacokinetics of the antigen-binding molecule; and a method for improving the pharmacokinetics of the antigen-binding molecule. A method for promoting dissociation of an antigen from a binding molecule is provided.

[0232] Method for producing antigen-binding molecules The present invention relates to an antigen-binding domain, an FcRn-binding domain, particularly a human FcRn-binding domain, and one or more carbohydrate receptors. An antigen-binding molecule having a carbohydrate-binding domain, which binds to a carbohydrate receptor in a neutral pH range. It has binding activity with sugar chain receptors in the acidic pH range, and its binding activity with sugar chain receptors in the neutral pH range is The antigen-binding activity in the acidic pH range is lower than that in the neutral pH range. The present invention also provides a method for producing an antigen-binding molecule that has a lower inflammatory response than that of the human inflammatory process. It has an excellent effect of promoting the reduction of antigen concentration in plasma and has excellent pharmacokinetics. Furthermore, the present invention is particularly useful when used as a pharmaceutical composition. A method for producing an antigen-binding molecule is provided.

[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 step of (b) A candidate motif for the carbohydrate receptor-binding domain in the polypeptide sequence identifying the amino acid sequence; (c) an amino acid sequence that differs from the amino acid sequence identified in (b) by at least one amino acid; a step of designing a motif for a sugar chain receptor binding domain comprising the (d) A polypeptide of an antigen-binding molecule containing the motif of the carbohydrate receptor-binding domain designed in (c). generating a gene encoding the peptide; (e) Extracting antigen-binding molecules from the culture medium of host cells transformed with the gene obtained in (d). The recovery process.

[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 also includes a step of further treating the antigen-binding molecule with an enzyme. It is possible.

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

[0236] Furthermore, the antigen-binding molecules produced by the production method of the present invention are FcRn, particularly The FcRn-binding domain having binding activity to human FcRn is the same as the "FcRn-binding domain" described above. That is, the FcRn-binding domain can be prepared by the method described above. There are no particular limitations as long as the FcRn-binding activity is specific to human FcRn, and the FcRn-binding activity may be directly or indirectly linked to the FcRn. In particular, a domain having binding activity to FcRn, particularly human FcRn, can be used. The domain may be, for example, an IgG-type domain having direct binding activity to FcRn, particularly human FcRn. Fc region of immunoglobulin, albumin, albumin domain 3, anti-human FcRn antibody, anti-human FcR Binding activity to human FcRn, such as peptides, anti-human FcRn scaffold molecules, or indirectly Examples of such molecules include IgG having the above structure and molecules that bind to albumin. A polypeptide having a domain that directly or indirectly binds to FcRn, particularly human FcRn. The sequence may be provided as the polypeptide sequence of the antigen binding domain.

[0237] Sugar chain receptor binding molecules contained in antigen-binding molecules produced by the production method provided by the present invention A preferred example of the domain is a sugar chain receptor binding domain to which a desired sugar chain is bound. The desired sugar chain is preferably an O-linked sugar chain or an N-linked sugar chain. A known method can be used to bind a sugar chain to the carbohydrate-binding domain. One method is to use recombinant gene techniques to create a motif sequence (i.e., a sequence of a desired sugar chain) to which the desired sugar chain is to be added. Natural or artificially produced proteins that contain a carbohydrate receptor-binding domain motif The gene encoding the carbohydrate receptor-binding domain contained in the antigen-binding molecule is introduced into a host cell. By introducing the desired sugar chain from the culture medium of the host cell, the sugar chain receptor binding The antigen-binding molecules of the present invention can be produced comprising the domain.

[0238] In the polypeptide sequences provided above, the motif of the carbohydrate receptor binding domain To identify candidate amino acid sequences for the target protein, the following method is used: The motif sequence for adding an O-linked glycan can be identified by using a publicly known database. Therefore, it can be identified. In addition, when the hinge portion of an antibody is included in the antigen-binding molecule of the present invention, In this way, it is possible to identify which isotype of antibody the hinge portion is derived from. Such a hinge region was identified as a candidate motif for the O-glycan receptor binding domain. In addition, when the sugar chain is an N-linked sugar chain, the motif sequence for adding the N-linked sugar chain (i.e., The motif of the N-glycan receptor binding domain consists of three amino acids, Asn-X-Ser / Thr. It is known that the N motif is a continuous motif. Designed to encode the Asn-X-Ser / Thr motif of the glycan receptor-binding domain In order to design a carbohydrate receptor binding domain, The presence or absence of sequences identical or similar to the Asn-X-Ser / Thr present can be identified.

[0239] An amino acid sequence that differs in at least one amino acid from the amino acid sequence identified above A motif for a carbohydrate receptor binding domain containing a sequence can be designed. For example, By using the above-identified motif sequence for O-linked glycan addition, By substituting amino acids in the candidate polypeptide sequence, O-linked glycan motifs Furthermore, when the hinge region of an antibody is included in the antigen-binding molecule of the present invention, a hinge sequence can be designed. If the hinge portion does not contain a hinge portion derived from an IgA antibody, The O-linked glycan model was obtained by replacing the sequence of the hinge region with the sequence of the hinge region derived from an IgA antibody. On the other hand, when the glycan is an N-linked glycan, the provided polynucleotide sequence can be designed. 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 doing so, the Asn-X-Ser / Thr sequence, which is the motif of the N-linked glycan receptor binding domain, is identified. The analogous sequence may be modified.

[0240] The antigen-binding molecules of the present invention have one or more binding sites to a sugar chain receptor, particularly a human sugar chain receptor. The binding domain for the sugar chain receptor, particularly the human sugar chain receptor, has an antigen-binding domain. The molecule has binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range, and The binding activity with sugar chain receptors in the acidic range is lower than that in the neutral pH range. The type and number of the glycoproteins are not particularly limited as long as they directly or indirectly bind to glycoprotein receptors, particularly glycoprotein receptors. A domain having binding activity to a glycoconjugate receptor can be used. For example, a glycoprotein having direct binding activity to a glycosyltransferase receptor, particularly a glycosyltransferase receptor for human glycosyltransferases, may be used. chain; Fc domain of IgG immunoglobulin; antibody against carbohydrate receptors, especially human carbohydrate receptors ; Anti-glycoprotein receptors, especially human glycoprotein receptor binding peptides; glycoprotein receptors, especially human glycoproteins In the present invention, a sugar molecule in the neutral pH range is used. It has binding activity to sugar chain receptors, especially human sugar chain receptors, and acts as a sugar chain receptor in the acidic pH range. Carbohydrate receptor-binding domain with lower binding activity than the carbohydrate receptor in the neutral pH range Preferably, the domain is previously bound to a sugar chain receptor, particularly a human sugar chain receptor, in a neutral pH range. It has binding activity to sugar chain receptors in the acidic pH range and binding activity to sugar chain receptors in the neutral pH range. Any glycan receptor-binding domain with lower binding activity to the glycan receptor can be used as is. The glycan receptor binding domain has an N-linked glycan with a terminal galactose, and the glycan The asialoglycoprotein receptor, which is a sugar chain receptor that binds to The binding activity is preferably lower than that in the neutral pH range. A glycan receptor binding domain having an N-linked glycan with a mannose terminal and a domain that binds to the glycan The binding activity with the mannose receptor, a sugar chain receptor, in the acidic pH range was also increased in the neutral pH range. This is a suitable example of a compound having a lower binding activity than that of the compound in the

[0241] Glycan receptor binding domains in the neutral pH range, especially for human glycan receptors If the binding activity is absent or weak, it can be improved by modifying the amino acids in the antigen-binding molecule. Thus, binding activity to sugar chain receptors, particularly human sugar chain receptors, can be acquired. The domains that have binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range are By modifying the amino acids in the glycoprotein, it is possible to bind to glycoreceptors, especially human glycoreceptors. The binding activity can be enhanced. Acid modification is a key step in the analysis of glycoreceptors, especially human glycoreceptors, in the neutral pH range before and after amino acid modification. The desired modification can be found by comparing the binding activity to the

[0242] Binding of the carbohydrate receptor-binding domain to carbohydrate receptors, especially human carbohydrate receptors, in the acidic pH range When the activity is not lower than the binding activity to sugar chain receptors, especially human sugar chain receptors, in the neutral pH range. In order to achieve this, we have modified the amino acids in the antigen-binding molecule to inhibit the activity of sugar chain receptors in the acidic pH range. , especially human sugar chain receptors whose binding activity is in the neutral pH range It is possible to obtain a binding activity that is lower than that of the sugar chain receptor, particularly the human sugar chain receptor. The amino acid modification of the receptor-binding domain was performed to determine the activity of the receptor in the acidic pH range before and after the amino acid modification. The binding activity to the sugar chain receptors, especially human sugar chain receptors, and sugar chain receptors in the neutral range, especially By comparing the binding activity to human sugar chain receptors, the desired modification can be found.

[0243] In the present invention, a sugar chain receptor is a glycoprotein that does not inhibit the binding of an antigen-binding domain to an antigen. The binding domain can be introduced into any site in the structure of the antigen-binding molecule. It can also be introduced into the antigen-binding domain as long as it does not inhibit the binding of the binding domain to the antigen. In another embodiment, the sugar chain receptor binding domain is Antigen binding is not possible unless the binding of the FcRn-binding domain of the antigen-binding molecule to FcRn, particularly human FcRn, is inhibited. The antibody can be introduced into any part of the structure of the molecule. For example, the hinge region of an IgA antibody can be introduced into the O-linked It can be a candidate amino acid sequence for the glycan receptor binding domain for glycan binding, and Asn- The motif sequence for N-linked glycan addition, X-Ser / Thr, is a key site for N-linked glycan attachment. These amino acid sequences can be candidates for the carbohydrate receptor binding domain. The desired antigen-binding molecule is extracted from the culture medium of a host cell, as described below, into which a gene encoding the antigen-binding molecule has been introduced. The antigen-binding molecules of the present invention can be produced by using the glycan receptor-binding domain to which the desired glycan is bound. do.

[0244] In the present invention, not inhibiting the binding of the antigen-binding domain to an antigen means that the antigen-binding domain does not inhibit the binding of the sugar chain receptor to the antigen. The introduction of the receptor-binding domain enhances the antigen-binding activity of the antigen-binding molecule by increasing the activity of the carbohydrate receptor-binding domain. The antigen-binding activity of the antigen-binding molecule before the introduction of the antibody is 20% or more, preferably 50% or more, and This means that the degree of affinity of the sugar chain receptor is preferably maintained at 80% or more, and more preferably at 90% or more. If the antigen-binding activity of the antigen-binding molecule is reduced by the introduction of the binding domain, The glycosylation can be achieved by, for example, substituting, deleting, adding, and / or inserting one or more amino acids in the glycomolecule. The antigen-binding activity can be changed to the same extent as that before the introduction of the receptor-binding domain. Substitution, deletion, or addition of one or more amino acids after introduction of such a carbohydrate receptor-binding domain and / or insertions, thereby achieving equivalent binding activity. Furthermore, not inhibiting the binding of the carbohydrate receptor-binding domain to FcRn, particularly human FcRn, means that The introduction of a carbohydrate receptor-binding domain improves the binding activity of antigen-binding molecules to FcRn, especially human FcRn. The activity of the antigen-binding molecule before the introduction of the sugar chain receptor-binding domain is compared with that of the FcRn, particularly human FcRn, which the antigen-binding molecule has. The binding activity is 20% or more, preferably 50% or more, more preferably 80% or more, and even more preferably The introduction of the carbohydrate receptor-binding domain improves antigen binding. When the binding activity of a molecule to FcRn, especially human FcRn, is reduced, one or more of the antigen-binding molecules The carbohydrate receptor binding domain is modified by multiple amino acid substitutions, deletions, additions, and / or insertions. The binding activity to FcRn, particularly human FcRn, can be altered to a level comparable to that before the introduction of the FcRn. In this case, one or more amino acid substitutions or deletions may be made after the introduction of such a carbohydrate receptor-binding domain. The present invention also includes antigen-binding molecules that have equivalent binding activity due to addition and / or insertion. can be.

[0245] In addition, the carbohydrate receptor binding domain is a structural domain that binds to antigens and FcRn. It may also be introduced into sites other than the domain.

[0246] The structure of the antigen-binding molecule of interest in the present invention is not particularly limited, and any antigen-binding molecule may be used. Binding molecules can also be suitably used, and antigen-binding domains, binding domains for FcRn, particularly human FcRn, are preferred. Preferred examples of antigen-binding molecules having two or more carbohydrate receptor domains include: A preferred example of the antibody of the present invention is an IgG antibody. 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. A constant region may be included, and amino acid mutations may be introduced into the constant region. The amino acid mutations include, for example, those that increase or decrease binding to Fcγ receptors (Prγ receptors). oc Natl Acad Sci US A. 1(2006) 103 (11), 4005-10), etc. In addition, an appropriate constant region such as an IgG2 constant region may be selected, but is not limited to these. It is also possible to alter pH-dependent binding by

[0247] When the antigen-binding molecule of the present invention is an antibody, the antibody may be a mouse antibody, a human antibody, a rat antibody, or a Antibodies of any animal origin can be used, such as rabbit antibodies, goat antibodies, and camel antibodies. Furthermore, modified antibodies with amino acid sequence substitutions, such as chimeric antibodies, especially humanized antibodies, are also available. In addition, bispecific antibodies, antibody modifications to which various molecules are bound, and antibodies Polypeptides containing 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] "Humanized antibodies" are antibodies produced by mammals other than humans, also called reshaped human antibodies. The complementarity determining regions (CDRs) of the derived antibody, e.g., mouse antibody, The CDRs are grafted from the CDRs of a human antibody. Methods for identifying CDRs are known. (Kabat et al., Sequence of Proteins of Immunological Interest (198 7), National Institute of Health, Bethesda, Md., Chothia et al., Nature (1989) 342, 877). General genetic recombination techniques are also known (EP125023 , WO1996 / 002576).

[0250] Bispecific antibodies have variable regions that recognize different epitopes within the same antibody molecule. Bispecific antibodies can be antibodies that recognize two or more different antigens, or they can be antibodies that recognize two or more different antigens. It may also be an antibody that recognizes two or more different epitopes on the antigen.

[0251] Furthermore, examples of polypeptides containing antibody fragments include Fab fragments, F(ab')2 fragments, scFv(Na t Biotechnol. (2005) 23 (9), 1126-36)domain antibody (dAb)(WO2004 / 05882 1, WO2003 / 002609), scFv-Fc (WO2005 / 037989), dAb-Fc, Fc fusion protein, etc. Molecules containing an Fc region use the Fc region as a binding domain to FcRn, particularly human FcRn. Furthermore, molecules in which a human FcRn-binding domain is fused to these molecules can also be used. obtain.

[0252] The antigen-binding domain, FcRn-binding domain, and carbohydrate receptor-binding domain designed as above A gene encoding the binding domain can be produced. Methods for producing genes are known, including chemical synthesis. Alternatively, the nucleotides constituting the polynucleotide can be synthesized by PCR or the like. It can also be produced by ligating the DNA by an enzymatic reaction in the presence of a template primer. The antigen-binding domain, FcRn-binding domain, and glycan designed as described above can be used. The receptor-binding domains were each expressed separately in a host cell as described below, and then cultured in a medium The polypeptides recovered from the nucleotide sequence are linked by a chemical reaction in the presence of a cross-linking agent. Synthetic chemical linkers (chemical cross-linking agents) are commonly used for cross-linking peptides. Crosslinkers that are used include N-hydroxysuccinimide (NHS), disuccinimidyl subtilisin (DSB), and dithiobis(succinimidyl) suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl) dithiobis(sulfosuccinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) Dimethylpropional (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), di Succinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST) , bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-B SOCOES), and these crosslinkers are commercially available.

[0253] In addition, the antigen-binding domain, FcRn-binding domain, and sugar chain receptor domain designed as described above can be used. The receptor-binding domains are designed to be linked in-frame by peptide bonds. It can also be obtained by expressing the gene in a host cell as described below and then recovering it from the culture medium, etc. When linked in frame by peptide bonds, each domain is directly linked. The domains can be linked via a linker having a specific peptide sequence. The linker may be any peptide linker that can be introduced by genetic engineering or a synthetic linker. Compound linkers (see, for example, Protein Engineering (1996) 9 (3), 299-305) Although the linkers shown in the drawings can be used, peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but it is usually 3 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. do.

[0255] The gene obtained by the production method of the present invention is usually carried (inserted) into an appropriate vector. The vector is a vector that stably maintains the inserted nucleic acid. For example, if E. coli is used as the host, a cloning vector As a vector, pBluescript vector (Stratagene) is preferred, but various commercially available vectors are also available. Vectors can be used to produce the antigen-binding molecules of the present invention. In this case, an expression vector is particularly useful. There are no particular limitations on the vector, as long as it expresses an antigen-binding molecule in cultured cells or in an individual organism. For example, pBEST vector (Promega) is used for in vitro expression, and pET is used for E. coli. vector (Invitrogen), and for cultured cells, pME18S-FL3 vector (GenBank Accession No. on No. AB009864), and for individual organisms, the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) are preferably used, but are not limited to these. The gene of the present invention can be inserted by a conventional method, for example, by a ligase reaction using a restriction enzyme site. (Current protocols in Molecular Biology ed. Ausubel et al. al. (1987) Publish. John Wiley & Sons.Section 11.4-11.11).

[0256] There are no particular limitations on the host cells, and various host cells can be used depending on the purpose. Examples of cells for expressing the original binding molecule include bacterial cells (e.g., Streptococcus Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., : CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells Examples of methods for introducing a vector into a host cell include calcium phosphate precipitation, electroporation, and the like. Pulse perforation (Current protocols in Molecular Biology ed. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection This can be done by known methods such as the microinjection method or the microinjection method.

[0257] The host cells can be cultured according to known methods. For example, In this case, for example, DMEM, MEM, RPMI1640, or IMDM can be used as the culture medium. The cells were also cultured in a medium supplemented with serum such as fetal calf serum (FCS). The cells can also be cultured in serum-free medium. The pH during culture is about 6 to 8. However, the selection may be made appropriately from among the above, without being limited thereto, for each culture or during the culture period. A different pH can be selected depending on the stage. The 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] The antigen-binding molecule expressed in the host cell is delivered to the lumen of the endoplasmic reticulum, the periplasmic space, or the cytoplasm. For secretion into the external environment, an appropriate secretion signal can be incorporated into the desired polypeptide. These signals may be endogenous to the antigen-binding molecule of interest or may be heterologous. It could also be a signal.

[0259] On the other hand, examples of systems for producing polypeptides in vivo include production systems using animals. These include production systems that use a vector containing the target gene and a plant. The polypeptide encoded by the gene produced in the cells of the animal or plant The "host" in the present invention refers to these animals and plants. Contains.

[0260] In production systems where animals are used as hosts, mammals and insects are used. Examples of animals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SP ECTRUM Biotechnology Applications (1993). In addition, when mammals are used, Alternatively, transgenic animals may be used as appropriate.

[0261] For example, polynucleotides encoding the antigen-binding molecules of the present invention may be used to encode proteins such as goat β-casein. It was prepared as a fusion gene with a gene encoding a polypeptide specifically produced in eel milk. The polynucleotide fragment containing this fusion gene is then injected into a goat embryo, This embryo is then transferred to a female goat. The recipient goat gives birth to a transgenic goat. Alternatively, the antigen-binding molecule of interest can be recovered from milk produced by the offspring. In order to increase the amount of milk containing antigen-binding molecules produced by goats, appropriate hormones are administered. It can be administered to transgenic goats (Ebert et al., Bio / Technology (1994) 1 2, 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, a polynucleotide encoding the antigen-binding molecule of interest is inserted into the silkworm. By infecting silkworms with baculovirus, the target antigen can be extracted from the silkworm's body fluids. The bound molecules can be recovered.

[0263] Furthermore, when plants are used to produce the antigen-binding molecules of the present invention, for example, tobacco is used. When tobacco is used, a polynucleotide encoding the antigen-binding molecule of interest is inserted. The introduced plant expression vector, e.g., pMON 530, is transformed into Agrobacterium tumefaciens. The bacteria are introduced into bacteria such as Agrobacterium tumefaciens. from tobacco leaves infected with, for example, Nicotiana tabacum, The desired antigen-binding molecules can be recovered (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] Examples of sugar chain receptor-binding domains contained in the antigen-binding molecules of the present invention include those that bind to desired sugar chains. When a glycan-bound glycan receptor binding domain is used, the glycan receptor binding domain is The method for linking the chains can be a known method. A cell extract is prepared from cultured cells of a dead mammalian cell line, and the extract is added to a cell line encoding an antibody. Post-translationally modified antibodies in a cell-free protein synthesis system characterized by the addition of mRNA A series of cell-free enzymatic methods such as the method for producing the desired sugar chain (JP 2006-141241 A) The method can be used to produce a sugar chain receptor-binding domain. The production method of the present invention may also include a step of further treating the above with an enzyme.

[0265] The glycan structure of the antigen-binding molecule produced as described above may be a single one, or multiple glycans. In the present invention, such mixtures may also be produced as a mixture of linked chains. Furthermore, antigen-binding molecules in which specific sugar chains are linked to the sugar chain-binding domains can be suitably used. Children can also be suitably used in the present invention.

[0266] A specific sugar chain is linked to the sugar chain receptor binding domain contained in the antigen-binding molecule of the present invention. As a method for this, several known methods can be adopted, and one of such known methods is The sugar chains of natural or artificially produced antigen-binding molecules by recombinant gene techniques, etc. By utilizing the properties of the glycans, an antigen-binding molecule having a specific sugar chain can be purified. Antibodies with high mannose-type glycans are purified by affinity chromatography using ConA-sepharose. It is known that the enzyme can be purified using fluoroscopy (Millward, Biologicals (2008) 36, 49-60)). In the present invention, such a purification method is used to purify N-linked mannose-containing saccharides. It can be used to prepare antigen-binding molecules having sugar chains.

[0267] If necessary, an appropriate protein-modifying enzyme may be applied before or after the purification of the antigen-binding molecule. By this, modifications are optionally made, and modified molecules such as peptides are partially or comprehensively produced. Examples of protein-modifying enzymes include trypsin, chymotrypsin, and lysine. Enzyme endopeptidases, protein kinases, glucosidases, etc. can be used. The production method of the present invention also includes a step of further treating such antigen-binding molecules with an enzyme. It may be included.

[0268] A specific sugar chain is linked to the sugar chain receptor binding domain contained in the antigen-binding molecule of the present invention. Several known methods can be used to obtain antigen-binding molecules having specific sugar chains. As will be described later in the Examples, enzyme treatment can be used to obtain the desired product. An antigen-binding molecule having an N-linked glycan of lactose is synthesized by combining it with a complex glycan having a terminal sialic acid. It can be prepared from an antigen-binding molecule having a high mannose chain by sialidase treatment. Antibodies with galactose-type glycans were treated with sialidase and β-galactosidase to convert them into galactose-type glycans. It is known that the glycoprotein is produced in a form in which its sugar chain is removed (Newkirk (Clin. Exp. Immun.) Nol. (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 Such antigen-binding molecules can be further treated with an enzyme to produce a synthetic molecule. The manufacturing method of the present invention may also include a step of treating using the same.

[0269] By introducing the compound into a host cell as described below, the compound can be used to extract the desired sugar from the culture medium of the host cell. Antigen-binding molecules of the present invention can be produced that contain a carbohydrate chain receptor-binding domain to which a chain is attached.

[0270] In a different aspect, in order to obtain an antigen-binding molecule of the present invention having a specific sugar chain, A recombinant gene encoding a protease-binding molecule was transduced to induce the accumulation of specific glycans ( glycosidase activity, including but not limited to, by genetic or recombinant genetic techniques A method for recovering the high mannose N-linked saccharide from the culture medium of the transformed host cells can also be used appropriately. The antibody having the conjugated glycan is prepared by transfecting a recombinant gene encoding the antibody with an N-acetylglucan. Culture medium of a Lec1 mutant derived from CHO cells lacking glucosaminyltransferase I activity It is known that it is recovered from the steroid hormone (Wright and Morrison (J. Exp. Med. (1994) 180 In the present invention, the non-reducing end of the N-linked sugar chain is mannose. The antigen-binding molecules were cultured in a Lec1 mutant strain transfected with a recombinant gene encoding the molecule. It can be recovered from the liquid.

[0271] In yet another embodiment, the present invention provides a method for obtaining an antigen-binding molecule having a specific sugar chain of the present invention. Inhibition of specific glycosidase reactions during the culture of cells producing the antigen-binding molecule. By adding an agent, antigen-binding molecules having specific sugar chains that have accumulated in the culture medium are recovered. A method of using a high-mannose N-linked sugar chain without fucose can also be used. 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. (2 008) 99, 652-665)). In the present invention, N-type mannose-containing oligosaccharides having no fucose and terminal mannose are An antigen-binding molecule having a conjugated sugar chain can be prepared by, for example, transforming a recombinant gene encoding the molecule. The transfected CHO cells were cultured with the addition of kifunesine and then recovered from the culture medium. In addition, for example, when culturing a host cell in which glycosidase activity has been altered as described above, By combining these methods of adding inhibitors, it is possible to obtain specific sugar chains of the present invention. By using such a combination, it is possible to obtain an antigen-binding molecule having a specific sugar chain. It is known to recover antibodies having the following structure (Kanda et al. (Glycobiology (2007) 17, 104- 118)), and in the present invention, a similar combination was used to obtain antigen-binding molecules having specific sugar chains. 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 multiple glycans. In the present invention, such mixtures may also be produced as a mixture of linked chains. Furthermore, antigen-binding molecules in which specific sugar chains are linked to the sugar chain-binding domains can be suitably used. Children can also be suitably used in the present invention.

[0273] The antigen-binding molecules thus obtained are secreted into host cells or extracellularly (in the culture medium, milk, etc.). The antigen-binding molecule can be isolated from the host and purified as a substantially pure and homogeneous antigen-binding molecule. The methods used for separation and purification are the same as those used for ordinary polypeptide purification. The separation and purification methods are selected appropriately and combined according to the purpose. However, the present invention is not limited to a specific method. For example, a chromatography column, Filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide Mid-gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected and combined to obtain antigen binding. The combined molecules can be separated and purified.

[0274] Examples of chromatography include affinity chromatography and ion exchange chromatography. chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, adsorption Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatography - is obtained by using liquid phase chromatography such as HPLC or FPLC. The column used for affinity chromatography is a protein column. Examples of columns using Protein A include Protein A columns and Protein G columns. Examples of suitable polymers include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0275] The antigen-binding molecule prepared as described above has antigen-binding activity, binding to FcRn, particularly human FcRn. Whether or not the desired properties in terms of the binding activity, sugar chain receptor binding activity, etc. are possessed is determined as described above. The antigen-binding activity, FcRn-binding activity, or carbohydrate receptor-binding activity was evaluated using the assay. As a result of such evaluation, a step of designing a motif for a sugar chain receptor binding domain, and encoding a polypeptide of an antigen-binding molecule containing a motif of a carbohydrate receptor-binding domain. The step of producing a gene having the desired properties is repeated one or more times. An antigen-binding molecule is produced that binds to the target antigen.

[0276] The present invention relates to an antigen-binding molecule comprising a sugar chain receptor-binding domain, particularly a sugar chain receptor-binding molecule of human origin. The antigen-binding molecule containing the domain binds to a carbohydrate receptor in a pH-dependent manner, and / or or a sugar chain receptor binding activity, particularly a sugar chain receptor binding activity of human origin, in the neutral pH range. It has the binding activity to the sugar chain receptor in the acidic pH range and the binding activity to the sugar chain receptor in the neutral pH range. If the activity can be lowered below the target value, the uptake of antigens into cells by antigen-binding molecules can be promoted. The administration of the antigen-binding molecule promotes a decrease in the antigen concentration in plasma, and the drug This improves kinetics and increases the number of antigens that can be bound by a single antigen-binding molecule. do.

[0277] The antigen-binding molecules produced by the production method of the present invention can be administered to reduce the antigen concentration in plasma. Therefore, the production method of the present invention is an antigen-binding molecule that, when administered, promotes a decrease in The method can be used to produce an antigen-binding molecule that promotes a decrease in antigen concentration in plasma.

[0278] Furthermore, the antigen-binding molecules produced by the production method of the present invention have improved pharmacokinetics. Therefore, the production method of the present invention is a method for producing an antigen-binding molecule with improved pharmacokinetics. It can be used as.

[0279] In addition, the antigen-binding molecules produced by the production method can be administered to animals such as humans, mice, and monkeys. When this is done, it is possible to increase the number of antigens that can be bound by a single antigen-binding molecule. Therefore, the production method of the present invention is a method for producing an antigen that can be bound by a single antigen-binding molecule. The method can be used to produce antigen-binding molecules with an increased number of

[0280] Furthermore, the antigen-binding molecules produced by the production method of the present invention can be used in humans, mice, monkeys, etc. When administered to animals, the antigen bound to the antigen-binding molecule outside the cell is released from the antigen-binding molecule inside the cell. Therefore, the production method of the present invention is capable of dissociating the protein from the extracellular matrix. The present invention is used as a method for producing an antigen-binding molecule capable of dissociating a bound antigen within a cell. obtain.

[0281] Furthermore, the antigen-binding molecules produced by the production method of the present invention can be used in humans, mice, monkeys, etc. When administered to animals, the antigen-binding molecules that are taken up into the cells in a bound state with the antigen are called antigen-binding molecules. It is thought that it is possible to release it outside the cell without binding to the antigen. The production method of the present invention allows the antibody to be taken up into cells in a state bound to an antigen, and the antibody to be taken up into cells in a state bound to an antigen. The method can be used to produce antigen-binding molecules that are released extracellularly in a stable state.

[0282] Furthermore, these antigen-binding molecules have a lower plasma concentration upon administration than conventional antigen-binding molecules. It is thought to be particularly excellent as a pharmaceutical product because it has a high effect of reducing the antigen concentration of Therefore, the production method of the present invention is a method for producing an antigen-binding molecule for use as a pharmaceutical composition. It can be used as such.

[0283] Pharmaceutical Composition The present invention also relates to an antigen-binding molecule of the present invention or an antigen produced by the production method of the present invention. The present invention also relates to pharmaceutical compositions containing the antigen-binding molecules of the present invention. The antigen-binding molecules produced by this method have a higher plasma antigen concentration than normal antigen-binding molecules upon administration. The pharmaceutical composition of the present invention is useful as a pharmaceutical composition because it has a high effect of reducing the concentration of steroid hormones. may include a pharmaceutically acceptable carrier.

[0284] In the present invention, the pharmaceutical composition is generally a pharmaceutical composition for treating or preventing a disease, or for testing or diagnosing a disease. This refers to drugs for the purpose of:

[0285] The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. The composition may be a sterile solution or suspension in water or other pharmaceutically acceptable liquid. For example, the pharmaceutical composition of the present invention can be administered parenterally in the form of an injection. Carriers or vehicles used in the preparation of pharmaceuticals, specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, In appropriate combination with surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc. and formulated by blending in unit dosage forms as required by generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is adjusted so that an appropriate dose within the indicated range is obtained. It is set.

[0286] Sterile compositions for injection are prepared according to conventional pharmaceutical practices using a vehicle such as distilled water for injection. The aqueous solution for injection can be, for example, physiological saline, glucose or the like. Other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride) Suitable solubilizing agents include alcohol (ethanol, etc.), Polyalcohol (propylene glycol, polyethylene glycol, etc.), non-ionic surface Active agents (Polysorbate 80(TM), HCO-50, etc.) may also be used in combination.

[0287] The oily liquids include sesame oil and soybean oil, and the solubilizing agent is benzyl benzoate and / or Alternatively, benzyl alcohol may be used in combination. In addition, a buffer (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., Ben The prepared injection solution is usually formulated with an antioxidant (e.g., ethanol and phenol). , and filled into suitable ampoules.

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

[0289] The method of administering the pharmaceutical composition of the present invention is appropriately selected depending on the age and symptoms of the patient. The dosage of a pharmaceutical composition containing an antigen-binding molecule can be, for example, 1 kg of body weight per administration. The dose may be set in the range of 0.0001 mg to 1000 mg per g. For example, the dose may be set in the range of 0.001 mg to 1000 mg per g. The dosage of the pharmaceutical composition of the present invention can be set in the range of 100,000 to 100,000 mg. The dosage and administration method should be determined according to the patient's weight, age, and condition. The dosage and administration method may vary depending on the patient's condition, but those skilled in the art can determine the appropriate dosage and administration method taking these conditions into consideration. Laws can be set.

[0290] It should be noted that the amino acids contained in the amino acid sequences described in the present invention may be modified after translation (e.g., For example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation can be achieved by those skilled in the art. Although amino acids may be post-translationally modified (a well-known modification), The antigen-binding molecules are also antigen-binding molecules specified by the amino acid sequences described in the present invention. is included in the range.

[0291] The method comprises contacting the antigen-binding molecule or the glycoprotein with a cell expressing a glycoprotein receptor in vivo or ex vivo. or a method for promoting the uptake into cells of an antigen that binds to said antigen-binding molecule The present invention further provides a method for detecting an antigen-binding molecule and an antigen that binds to the antigen-binding molecule, comprising: The glycoprotein binds to the glycoprotein receptor-binding domain contained in the conjugated molecule, and is then synthesized in vivo in cells expressing glycoprotein receptors. or contacting the antigen-binding molecule or the antigen-binding molecule in vitro. A method for promoting the uptake of an antigen into a cell is provided.

[0292] In the present invention, "incorporation into cells" refers to the incorporation of an antigen-binding molecule or a compound that binds to the antigen-binding molecule. This means that the antigen is taken up into the cell by endocytosis. In the specification, "promoting uptake into cells" means promoting the uptake of antigen binding molecules that bind to antigens outside the cells. This means that the rate at which the combined molecule is taken up into cells is accelerated. In this method, the uptake of an antigen-binding molecule or an antigen that binds to the antigen-binding molecule into cells is promoted. Whether or not the antigen-binding molecule or the antigen that binds to the antigen-binding molecule is uptaken into the cell is determined by the The rate of antigen uptake into cells can be determined by, for example, whether or not the rate has increased. The antigen-binding molecule and the antigen are added to a culture medium containing cells expressing the chain receptor, and the antigen-binding molecule or the measuring the decrease in the concentration of antigen bound to the antigen-binding molecule in the culture medium over time; or Antigen-binding molecules incorporated into cells expressing sugar chain receptors or antibodies that bind to the antigen-binding molecules The amount of the sugar chain receptor expressed in the cells can be calculated by measuring the amount of the sugar chain receptor expressed in the cells over time. As described above, contact between the cells and the antigen-binding molecules and the antigens that bind to the antigen-binding molecules is carried out in vivo. This can be done ex vivo or in vivo by administering an antigen-binding molecule.

[0293] The present invention provides an antigen-binding molecule and an antigen that binds to the antigen-binding molecule. The glycoprotein binds to the glycoprotein receptor-binding domain contained in the conjugated molecule, and is then synthesized in vivo in cells expressing glycoprotein receptors. or contacting the antigen-binding molecule or the antigen-binding molecule in vitro. Methods for enhancing the rate of antigen uptake into cells, such as (1) antigen-binding molecules and antigen-binding After the plasma containing the antigen that binds to the glycan molecule is extracted from the body, the glycan receptor is expressed. It is then recycled outside the cells after a certain period of time (also called re-secretion or recycling). The plasma containing antigen-binding molecules that do not bind to antigens is returned to the living body, so-called ex vivo or (2) a method of administering an antigen-binding molecule in vivo, thereby In addition, in the method (1), the antibody that binds to the antigen-binding molecule can be effectively eliminated. After the plasma containing the antigen is extracted from the body, the antigen-binding molecules and sugar chain receptors are expressed. Another method that can be used is to contact the plasma with cells that can be cultured for a certain period of time and then return the plasma to the body. This promotes the uptake of the antigen-binding molecule or the antigen that binds to the antigen-binding molecule into cells. Whether or not the antigen-binding molecule has been administered can be determined, for example, by determining the rate at which the antigen present in plasma disappears. Whether the activity is accelerated compared to that without ex vivo methods or antigen-binding molecules By measuring whether the administration of the drug reduces the antigen concentration in the plasma can also be confirmed.

[0294] In addition, whether or not uptake is promoted is confirmed in (1) and (2) above. The rate of antigen elimination in plasma is determined by the activity of human natural IgG, especially human natural IgG1, instead of antigen-binding molecules. The rate of antigen elimination in plasma was confirmed to be faster than that confirmed by the method using This can also be confirmed by checking.

[0295] Furthermore, the present invention also provides a method for producing a compound whose antigen-binding activity changes depending on the ion concentration. Glycans that bind to the glycan receptor-binding domain contained in antigen-binding molecules containing antigen-binding domains The method comprises contacting cells expressing the receptor with the antigen-binding molecule in vivo or ex vivo. Furthermore, the present invention provides a method for promoting the uptake of an antigen into cells. An antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity is changed depending on conditions The glycan receptor binding domain contained in the glycan receptor-binding domain is then bound to cells expressing the glycan receptor in vivo or in vivo. Promoting the uptake of antigens bound to antigen-binding molecules into cells, including contacting them in vitro The present invention further provides a method for detecting a nucleotide sequence in which at least one amino acid in the antigen-binding domain is a nucleotide sequence. The binding activity of the antigen-binding domain to the antigen varies depending on the sodium ion concentration or pH conditions. The amino acid sequence changes depending on the carbohydrate receptor binding domain of the antigen-binding molecule. and contacting the antigen-binding molecule with a cell that expresses a carbohydrate receptor in vivo or ex vivo. The present invention provides a method for promoting the uptake of an antigen that binds to a target molecule into a cell.

[0296] In the present invention, "the binding activity of an antigen-binding domain to an antigen is changed depending on the ion concentration conditions" As a method for "changing the property of an antigen-binding molecule," the method described in the present specification includes the method for producing an antigen-binding molecule. One or a combination of several methods may be used as appropriate.

[0297] In the present invention, a sugar chain receptor that binds to a sugar chain receptor-binding domain contained in an antigen-binding molecule Any cells can be used as long as they express the desired sugar chain receptor. To identify cells expressing the desired glycan receptor, Publicly known databases such as the Protein Atlas (http: / / www.proteinatlas.org / ) are used. Furthermore, by a method for confirming the expression of a gene encoding a desired sugar chain receptor, In addition, the antigen-binding molecule of the present invention can be isolated by immunological techniques using an antibody that binds to a desired sugar chain receptor. It can be determined whether the cells used to contact the child express the receptor. Such a technique is also known. The contact of the antigen with the original binding molecule occurs in vivo as well as in vitro. In this context, contacting an antigen-binding molecule with a cell that expresses a sugar chain receptor means contacting the cell with an antigen-binding molecule that produces the sugar chain receptor. The contact time may be, for example, from 1 minute to several weeks, or from 30 minutes to 1 week. The antigen-binding molecule or the target substance is then incubated for an appropriate time period between 1 hour and 3 days, or between 2 hours and 1 day. The antigen that binds to the antigen-binding molecule is taken up into the cell by endocytosis. The time required for this will be adopted appropriately.

[0298] For example, cells expressing asialoglycoprotein receptors as sugar chain receptors For this purpose, hepatocytes can be used. In addition, cells expressing the mannose receptor as a sugar chain receptor can be used. A wide range of cells can be used, including blood cells.

[0299] The antibody per molecule is then contacted with cells expressing a carbohydrate receptor in vivo or ex vivo. Methods for increasing the number of antigens bound by a natural binding molecule The present invention also provides a method for producing a nucleic acid molecule comprising an antigen-binding domain, an FcRn-binding domain, and one or more sugar chain receptor-binding domains. The antigen-binding molecule having the glycan receptor-binding domain is A method for producing a glycan-binding pro...

Claims

[Claim 1] The invention described in the specification.

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