Antigen-binding molecules that promote the elimination of antigens containing glycosylated receptor-binding domains from plasma.

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 has priority under Japanese Patent Application No. 2011-221400 (filed October 5, 2011). This assertion is incorporated herein by reference.

[0002] Technical field This invention relates to an antigen-binding molecule that promotes the uptake of antigens into cells, and to a reduction in the concentration of antigens in plasma. Antigen-binding molecules that can promote antigen action, antigen-binding molecules that can bind to antigens multiple times, drug action Antigen-binding molecules with improved properties, pharmaceutical compositions containing said antigen-binding molecules, and their manufacturing 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 launched, and many more antibody drugs are currently under development. (Non-patent document 1, Non-patent document 2). On the other hand, as a technology applicable to second-generation antibody drugs Various technologies have been developed to improve effector function, antigen binding ability, pharmacokinetics, and stability. Technologies that increase or reduce the risk of immunogenicity have been reported (non-patent literature). 3) Antibody drugs generally require very high dosages, making it difficult to produce subcutaneous formulations. High manufacturing costs are among the challenges. Methods to reduce the dosage of antibody drugs. As such, methods to improve the pharmacokinetics of antibodies and improve the affinity between antibodies and antigens. The above method can be considered.

[0004] Artificial amino acid substitution in the constant region has been reported as a method to improve the pharmacokinetics of antibodies. is known (Non-Patent Documents 4 and 5). As a technique for enhancing antigen-binding ability and antigen-neutralizing ability, affinity maturation technology (Non-Patent Document 6) has been reported, and it is possible to enhance the binding activity to an antigen by introducing mutations into amino acids such as the CDR region of the variable region. By enhancing the antigen-binding ability, it is possible to improve the in vitro biological activity or reduce the dosage, and it is also possible to improve the drug efficacy in vivo (Non-Patent Document 7). On the other hand, the amount of antigen that can be neutralized per antibody molecule depends on the affinity, and by strengthening the affinity, it is possible to neutralize the antigen with a small amount of antibody, and it is possible to strengthen the affinity of the antibody in various ways (Non-Patent Document 6). Furthermore, if it can be covalently bonded to the antigen to make the affinity infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of divalent) with one molecule of antibody. However, with the methods so far, the stoichiometric neutralization reaction of one molecule of antibody with one molecule of antigen (two antigens in the case of divalent) has a limit,

[0005] and it was impossible to completely neutralize the antigen with an antibody amount less than the antigen amount. That is, there was a limit to the effect of strengthening the affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period, an antibody amount more than the amount of antigen produced in the living body during that period needs to be administered, and there was a limit to reducing the required antibody dosage only by the above-mentioned improvement in the pharmacokinetics of the antibody or affinity maturation technology. Therefore, in order to maintain the neutralizing effect of the antigen for the target period with an antibody amount less than the antigen amount, it is necessary to neutralize multiple antigens with one antibody. As a new method to achieve this, recently, pH-dependent with respect to the antigen bonded, and if the affinity can be made infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of divalent) with one molecule of antibody. However, with the methods so far, the stoichiometric neutralization reaction of one molecule of antibody with one molecule of antigen (two antigens in the case of divalent) has a limit, and it was impossible to completely neutralize the antigen with an antibody amount less than the antigen amount. That is, there was a limit to the effect of strengthening the affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period, an antibody amount more than the amount of antigen produced in the living body during that period needs to be administered, and there was a limit to reducing the required antibody dosage only by the above-mentioned improvement in the pharmacokinetics of the antibody or affinity maturation technology. Therefore, in order to maintain the neutralizing effect of the antigen for the target period with an antibody amount less than the antigen amount, it is necessary to neutralize multiple antigens with one antibody. As a new method to achieve this, recently, pH-dependent with respect to the antigen bonded, and if the affinity can be made infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of divalent) with one molecule of antibody. However, with the methods so far, the stoichiometric neutralization reaction of one molecule of antibody with one molecule of antigen (two antigens in the case of divalent) has a limit, and it was impossible to completely neutralize the antigen with an antibody amount less than the antigen amount. That is, there was a limit to the effect of strengthening the affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period, an antibody amount more than the amount of antigen produced in the living body during that period needs to be administered, and there was a limit to reducing the required antibody dosage only by the above-mentioned improvement in the pharmacokinetics of the antibody or affinity maturation technology. Therefore, in order to maintain the neutralizing effect of the antigen for the target period with an antibody amount less than the antigen amount, it is necessary to neutralize multiple antigens with one antibody. As a new method to achieve this, recently, pH-dependent with respect to the antigen bonded, and if the affinity can be made infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of divalent) with one molecule of antibody. However, with the methods so far, the stoichiometric neutralization reaction of one molecule of antibody with one molecule of antigen (two antigens in the case of divalent) has a limit, and it was impossible to completely neutralize the antigen with an antibody amount less than the antigen amount. That is, there was a limit to the effect of strengthening the affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period, an antibody amount more than the amount of antigen produced in the living body during that period needs to be administered, and there was a limit to reducing the required antibody dosage only by the above-mentioned improvement in the pharmacokinetics of the antibody or affinity maturation technology. Therefore, Antibodies that bind to storage have been reported (Patent Document 1). They bind strongly to antigens under neutral conditions in plasma and dissociate from antigens under acidic conditions within endosomes. pH-dependent antigen-binding antibodies can dissociate from antigens within endosomes. After dissociating from the antigen, the antibody can bind to the antigen again because it can be recycled into the plasma by FcRn. Therefore, it becomes possible to repeatedly bind a single antibody to multiple antigens. Under neutral conditions in plasma, they bind strongly to antigens and dissociate from antigens under acidic conditions within endosomes. pH-dependent antigen-binding antibodies can dissociate from antigens within endosomes. After dissociating from the antigen, the antibody can bind to the antigen again because it can be recycled into the plasma by FcRn. Therefore, it becomes possible to repeatedly bind a single antibody to multiple antigens. After dissociating from the antigen, the antibody can bind to the antigen again because it can be recycled into the plasma by FcRn. Therefore, it becomes possible to repeatedly bind a single antibody to multiple antigens. After dissociating from the antigen, the antibody can bind to the antigen again because it can be recycled into the plasma by FcRn. Therefore, it becomes possible to repeatedly bind a single antibody to multiple antigens.

[0006] In addition, the residence time of antigens in plasma is very short compared to antibodies that bind to FcRn and are recycled. When an antibody with a long residence time in plasma binds to its antigen, the residence time of the antibody-antigen complex in plasma becomes as long as that of the antibody. Therefore, by binding to the antibody, the antigen rather has a longer residence time in plasma, and the plasma antigen concentration increases. In such a case, even if the affinity of the antibody for the antigen is improved, it is not possible to promote the disappearance of the antigen from plasma. The above-mentioned pH-dependent antigen-binding antibody has also been reported to be effective as a method for promoting the disappearance of antigens from plasma (Patent Document 1). In addition, the residence time of antigens in plasma is very short compared to antibodies that bind to FcRn and are recycled. When an antibody with a long residence time in plasma binds to its antigen, the residence time of the antibody-antigen complex in plasma becomes as long as that of the antibody. Therefore, by binding to the antibody, the antigen rather has a longer residence time in plasma, and the plasma antigen concentration increases. In such a case, even if the affinity of the antibody for the antigen is improved, it is not possible to promote the disappearance of the antigen from plasma. The above-mentioned pH-dependent antigen-binding antibody has also been reported to be effective as a method for promoting the disappearance of antigens from plasma (Patent Document 1). The above-mentioned pH-dependent antigen-binding antibody has also been reported to be effective as a method for promoting the disappearance of antigens from plasma (Patent Document 1).

[0007] Thus, pH-dependent antigen-binding antibodies can bind to multiple antigens with a single antibody and promote the disappearance of antigens from plasma compared to normal antibodies, so they have effects that cannot be achieved with normal antibodies. However, to date, no antibody engineering techniques have been reported to further improve the effect of repeatedly binding to antigens and the effect of promoting the disappearance of antigens from plasma of these pH-dependent antigen-binding antibodies. Thus, pH-dependent antigen-binding antibodies can bind to multiple antigens with a single antibody and promote the disappearance of antigens from plasma compared to normal antibodies, so they have effects that cannot be achieved with normal antibodies. However, to date, no antibody engineering techniques have been reported to further improve the effect of repeatedly binding to antigens and the effect of promoting the disappearance of antigens from plasma of these pH-dependent antigen-binding antibodies. However, to date, no antibody engineering techniques have been reported to further improve the effect of repeatedly binding to antigens and the effect of promoting the disappearance of antigens from plasma of these pH-dependent antigen-binding antibodies.

[0008] ​​​On the other hand, glycoproteins present in plasma bind to their sugar chain-specific receptors, and in the plasma... It disappears from (Non-Patent Literature 10). At this time, the glycoprotein is affected by the sugar chain receptors present on the cell surface. It binds to the cell and is taken up, then dissociates from the glycosylation receptor within the cell, and is then processed by lysosomes. Although they are broken down, it is known that glycan receptors are recycled back onto the cell surface. In other words, this glycoprotein and its glycosylation receptor bind strongly under neutral conditions in plasma. It has pH-dependent binding ability that dissociates under acidic conditions within endosomes (non-specific). Reference 11, Non-Patent Document 12). pH-dependent binding of such glycoproteins and glycosylation receptors. This involves sugar chains attached to glycoproteins, and examples of such sugar chains and sugar receptors are given below. As an example, among N-linked glycans, glycans with galacrose at the terminal and asiaglycoprotein Mannose receptor (Non-patent document 11), sugar chain having mannose at the terminal and mannose receptor Examples include (Non-Patent Document 12).

[0009] Due to the properties of these N-linked glycans, functionally unnecessary N-linking is used in biopharmaceuticals, including antibodies. Conferring combined glycans is undesirable from the standpoint of maintaining pharmacological efficacy or plasma retention. It is thought that (Non-Patent Documents 13, 14) that it has the effect of promoting the elimination of antigens from plasma. There have been no instances of this being implemented with the aim of further improvement.

[0010] The references cited herein are as follows: All of the above is incorporated herein by reference. Any of these documents may be found in this specification. This does not constitute prior art to calligraphy. [Prior art documents] [Patent Documents]

[0011]

Patent Document 1

Non-licensed literature

[0012] [Non-licensed document 1] Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology (2005) 23, 1073 - 1078 [Non-licensed document 2] Pavlou AK, Belsey MJ., Eur J Pharm Biopharm. (2005) 59 (3), 389-96 [Non-licensed document 3] Kim SJ, Park Y, Hong HJ., Mol Cells. (2005) 20 (1), 17-29

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0013] The inventors have developed a method to promote the uptake of antigens into cells by antigen-binding molecules having antigen-binding activity. Methods to induce this, methods in which antigen-binding molecules bind to antigens multiple times, and methods in which plasma is produced by administering antigen-binding molecules. Methods to promote the decrease in antigen concentration and methods to improve the retention of antigen-binding molecules in plasma. We conducted intensive research. As a result, the inventors identified an antigen-binding domain, FcRn, particularly human FcRn. It has a binding domain to the glycosylation receptor and a domain that binds to the glycosylation receptor, and it binds to the glycosylation receptor. However, under the conditions of ion concentration within endosomes, the effect is weaker compared to the conditions of ion concentration in plasma. Antigen-binding molecules with binding ability can promote the uptake of antigens into cells, and also in plasma. Compared to antigen binding activity under the ion concentration conditions, early endosome ion concentration An antigen-binding molecule having an antigen-binding domain with weak antigen-binding activity under certain conditions is an antigen-binding molecule. It can further promote the uptake of antigens into cells by synthesizers, and by a single antigen-binding molecule It can increase the number of antigens that can be bound, and the administration of antigen-binding molecules can increase the antigen concentration in plasma. We found that this could accelerate the reduction of antigens and improve the pharmacokinetics of antigen-binding molecules.

[0014] In other words, the present invention provides a method for promoting the uptake of an antigen into cells by an antigen-binding molecule, one minute A method to increase the number of antigens that can be bound by this antigen-binding molecule, and by administering it, Methods to accelerate the decrease in antigen concentration, methods to improve the pharmacokinetics of antigen-binding molecules, and the antigen's finer points Antigen-binding molecules whose internalization into the cell is promoted, antigen-binding molecules whose number of binding antigens has increased, Antigen-binding molecules and drug molecules that can promote a decrease in the concentration of antigens in plasma upon administration. An antigen-binding molecule with improved properties, a pharmaceutical composition containing the antigen-binding molecule, and a method for producing the same. Regarding laws and regulations, more specifically, [1] A method for producing an antigen-binding molecule, comprising the following steps: (a) Provide polypeptide sequences of antigen-binding molecules including antigen-binding domains and FcRn-binding domains. Process to provide (b) Candidate motifs for the glycosylation receptor binding domain in the polypeptide sequence Steps to identify amino acid sequences (c) An amino acid sequence in which at least one amino acid is different from the amino acid sequence identified in (b) The process of designing motifs of glycosylation receptor-binding domains. (d) A polypeptide of an antigen-binding molecule containing the glycosylated receptor-binding domain motif designed in (c) The process of creating the gene that codes for cide (e) Antigen-binding molecules from the culture medium of host cells transformed by the genes obtained in (d) Recovery process [2] The antigen-binding molecule obtained in (e) is further treated with an enzyme, including the step [1 Manufacturing method of ] [3] The binding activity of the antigen-binding domain to the antigen changes depending on the ion concentration conditions. The manufacturing method according to [1] or [2], characterized by: [4] The manufacturing method described in [3], wherein the ion concentration condition is the pH condition. [5] The antigen-binding domain has a binding activity to the antigen in the pH range that is lower than the binding activity in the pH range. The manufacturing method described in [4], which has high binding activity to the antigen under the conditions of the sexual region. [6] At least one amino acid of the antigen-binding domain is a side chain with a pKa of 4.0-8.0 Substituting with a mino acid or at least one side chain with a pKa of 4.0-8.0 in the antigen-binding domain By inserting amino acids, the binding activity to the antigen is improved under pH-acidic conditions. This includes providing an antigen-binding domain that has high binding activity to the antigen under the conditions of the sex zone. The method described in [5], [7] The manufacturing method according to [3], wherein the ion concentration conditions are the calcium ion concentration conditions. law, [8] The antigen-binding domain binds to the antigen under conditions of low calcium ion concentration. The binding activity to the antigen is higher under conditions of high calcium ion concentration than under normal activity conditions. [7] Manufacturing method described above, [9] At least one amino acid of the antigen-binding domain is placed with a calcium-binding motif. By replacing or inserting a calcium-binding motif into the antigen-binding domain, low calcium The binding activity to the antigen under conditions of higher calcium ion concentration is greater than the binding activity under conditions of higher calcium ion concentration. The invention includes providing an antigen-binding domain that has high binding activity to the antigen under the circumstances of [8] The method described in ]

[10] The antigen-binding domain is described in any of [1] to [9], which includes the variable region of the antibody. Method of installation,

[11] The FcRn binding domain is described in any of [1] to

[10] which includes the Fc region of the antibody. Method of installation,

[12] The method according to

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

[13] The IgG antibody is one of IgG1, IgG2, IgG3, or IgG4 as described in

[12] method,

[14] The binding activity of the glycan receptor binding domain to the glycan receptor is subject to the conditions of ion concentration. A method according to any one of [1] to

[12] , characterized by being changed by

[15] The method according to

[14] , wherein the ion concentration condition is the pH condition,

[16] Binding of the glycan receptor binding domain to the glycan receptor under acidic pH conditions. The binding activity to the glycan receptor is higher under conditions in the pH neutral range than the activity [1] to [1 The method described in any of (5)

[17] The person described in

[14] whose ion concentration conditions are the calcium ion concentration conditions law,

[18] Glycan receptor of the glycan receptor binding domain under low calcium ion concentration conditions The binding activity to the glycan receptor under conditions of high calcium ion concentration is greater than the binding activity to the body. The method described in

[17] , which has high binding activity.

[19] The description in any of [1] to

[18] , wherein the glycan receptor binding domain is a glycan. The method,

[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 glycan receptor binding domain allows for the addition of N-linked glycans. The method described in

[21] , which includes designing a motif,

[23] The N-linked sugar chain having a terminal containing galactose as described in

[21] or

[22] method,

[24] The method according to

[23] , wherein the terminal of the N-linked sugar chain comprises three or more galactoses. ,

[25] Since the glycan receptor is an asialoglycoprotein receptor

[21] , The method described in any of

[24]

[26] The method according to

[21] or

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

[27] The method according to

[26] , wherein the glycan receptor is a mannose receptor. Antigen-binding molecules prepared by any of the methods described in

[28] [1] to

[27] ,

[29] The binding activity of the FcRn binding domain to the antigen changes depending on the ion concentration conditions. The binding activity to the antigen-binding domain and glycosylation receptors changes depending on the ion concentration conditions. An antigen-binding molecule containing one or more glycosylated receptor-binding domains,

[30] The binding activity of the antigen-binding domain to the antigen changes depending on the pH conditions [2 9) Antigen-binding molecules described in

[31] The binding activity of the antigen-binding domain to the antigen under conditions of the pH acidity range is greater than pH The antigen-binding molecule described in

[30] has high binding activity to the antigen under neutral conditions.

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

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

[33] The binding activity of the antigen-binding domain to the antigen is determined by the calcium ion concentration. The antigen-binding molecule described in

[29] is characterized by being changed as a result,

[34] The binding of the antigen-binding domain to the antigen under conditions of low calcium ion concentration. The binding activity to the antigen is higher under conditions of high calcium ion concentration than the combined activity [3 3) Antigen-binding molecules described in

[35] At least one amino acid of the antigen-binding domain has a calcium-binding motif The antigen-binding molecules described in

[34] are included.

[36] Any of

[29] to

[35] in which the antigen-binding domain includes the variable region of the antibody The antigen-binding molecules described above,

[37] The FcRn binding domain is one of

[29] to

[36] which includes the Fc region of the antibody. The antigen-binding molecule described,

[38] The antigen-binding molecule described in

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

[39] The IgG antibody is one of IgG1, IgG2, IgG3, or IgG4 as described in

[38] . antigen-binding molecules,

[40] The binding activity of the glycan receptor binding domain to the glycan receptor is subject to the conditions of ion concentration. The antigen-binding component according to any one of

[29] to

[39] is characterized by being changed by child,

[41] The antigen-binding molecule described in

[40] , wherein the ion concentration condition is the pH condition.

[42] Binding of the glycan receptor binding domain to the glycan receptor under acidic pH conditions. The binding activity to the glycan receptor is higher under pH neutral conditions than the activity

[41] . antigen-binding molecule,

[43] The anti- original binding molecule,

[44] Glycan receptor of the glycan receptor binding domain under low calcium ion concentration conditions The binding activity to the glycan receptor under conditions of high calcium ion concentration is greater than the binding activity to the body. Antigen-binding molecules with high binding activity, as described in

[43]

[45] The glycan receptor binding domain is a glycan as described in any of

[29] to

[44] . The antigen-binding molecule listed,

[46] The antigen-binding molecule according to

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

[47] The glycan receptor binding domain contains a motif to which an N-linked glycan is bound [4 Antigen-binding molecules described in [6]

[48] ​​The N-linked sugar chain having a terminal containing galactose as described in

[46] or

[47] antigen-binding molecules,

[49] The N-linked sugar chain comprising three or more terminal galactoses as described in

[48] antigen-binding molecules,

[50] Since the glycan receptor is an asialoglycoprotein receptor

[47] , Antigen-binding molecule as described in any of

[49] ,

[51] The N-linked sugar chain having a terminal containing mannose, as described in

[46] or

[47] . antigen-binding molecules,

[52] The method according to

[46] or

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

[53] The method described in

[28] to

[52] , characterized in that the antigen-binding molecule is an antibody. antigen-binding molecule,

[54] The glycan receptor binding domain is included in the antigen binding domain

[28] to [53 Antigen-binding molecules as described in any of the following:

[55] The glycan receptor binding domain is included in the FcRn binding domain,

[28] to [5 3) Antigen-binding molecule as described in any of the above, A pharmaceutical composition containing an antigen-binding molecule as described in any of

[56] ,

[28] , or

[55] . The antigen-binding molecule described in any of

[57] ,

[28] , to

[55] expresses a glycosylation receptor. This involves bringing an antigen-binding molecule into contact with a cell, either in vivo or in vitro, within the cell. How to import it, The antigen-binding molecule described in any of

[58] ,

[28] to

[55] expresses a glycosylation receptor. This involves contacting cells with an antigen bound to an antigen-binding molecule, either in vivo or in vitro. A method for introducing the substance into the cells of the said cell. The antigen-binding molecule described in any of

[59] ,

[28] to

[55] expresses a glycosylation receptor. This involves contacting cells in vivo or in vivo, where each antigen-binding molecule binds to the cells. Methods to increase the number of antigens, An antigen-binding molecule described in any of

[60] ,

[28] to

[55] expresses a glycosylation receptor. This includes contacting cells in vivo or in vivo to reduce extracellular antigens. How to do it

[61] The method described in

[58] , in which the extracellular space is plasma. The antigen-binding molecule described in any of

[62] ,

[28] to

[55] expresses a glycosylation receptor. A method for improving the pharmacokinetics of antigen-binding molecules, including contact with cells in vivo. The antigen-binding molecule described in any of

[63] ,

[28] to

[55] expresses a glycosylation receptor. This includes contacting cells in vivo or in vivo with antigen-binding molecules outside the cell. A method to promote the dissociation of a combined antigen from its antigen-binding molecule.

[64] Antigen-binding domain, FcRn-binding domain and one or more binding domains to glycosylation receptors Increase the number of binding domains to the glycosylation receptor in the antigen-binding molecule, including the main molecule. A method selected from any of the following, including: (i) Antigen binding into cells expressing the glycan receptor in vivo or in vivo. Methods to promote molecular uptake (ii) Antigen binding into cells expressing the glycan receptor in vivo or in vivo. Methods to promote the uptake of antigens bound to molecules (iii) Increase the number of antigens to which each antigen-binding molecule binds, either in vivo or in vitro. Methods to add (iv) Methods for increasing the antigen-eliminating ability of antigen-binding molecules in vivo or in vitro. (v) Methods for improving the pharmacokinetics of antigen-binding molecules, (vi) Methods for promoting the dissociation of extracellularly bound antigens from antigen-binding molecules

[65] The binding activity of the antigen-binding molecule to the antigen changes depending on the ion concentration conditions. The method described in

[64] ,

[66] The binding activity of the antigen-binding domain to the antigen changes depending on the pH conditions [6 The method described in 4),

[67] The binding activity of the antigen-binding domain to the antigen under conditions of the pH acidity range is greater than pH The method described in

[66] , which has high binding activity to the antigen under neutral conditions,

[68] At least one amino acid of the antigen-binding domain has a side chain pKa of 4.0-8.0 The method described in

[67] , which contains at least one amino acid,

[69] The binding activity of the antigen-binding domain to the antigen is determined by the calcium ion concentration. The method according to

[64] , characterized by the following changes:

[70] The antigen-binding domain's binding to the antigen under low calcium ion concentration conditions. The binding activity to the antigen is higher under conditions of high calcium ion concentration than the combined activity [6] The method described in 9),

[71] At least one amino acid of the antigen-binding domain has a calcium-binding motif The method described in

[70] ,

[72] Any of

[64] to

[71] in which the antigen-binding domain includes the variable region of the antibody Methods used

[73] The FcRn binding domain includes the Fc region of the antibody in any of

[64] to

[72] Method of description,

[74] The method according to

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

[75] The IgG antibody is one of IgG1, IgG2, IgG3, or IgG4 as described in

[74] method,

[76] The binding activity of the glycan receptor binding domain to the glycan receptor is subject to the conditions of ion concentration. A method according to any one of

[64] to

[75] , characterized by being changed by

[77] The method according to

[76] , wherein the ion concentration condition is the pH condition,

[78] Binding of the glycan receptor binding domain to the glycan receptor under acidic pH conditions. The binding activity to the glycan receptor is higher under pH neutral conditions than the activity level

[76] . The method,

[79] The method described in

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

[80] Glycan receptor of the glycan receptor binding domain under low calcium ion concentration conditions The binding activity to the glycan receptor under conditions of high calcium ion concentration is greater than the binding activity to the body. The method described in

[79] , which has high binding activity,

[81] The glycan receptor binding domain is a glycan as described in any of

[64] to

[80] . Method of installation,

[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 motif includes an N-linked glycan that binds to the glycan receptor-binding domain [8 The method described in (3),

[85] The N-linked sugar chain having a terminal containing galactose as described in

[83] or

[84] method,

[86] The N-linked sugar chain comprising three or more terminal galactoses as described in

[85] method,

[87] Since the glycan receptor is an asialoglycoprotein receptor

[84] , The method described in any of

[86]

[88] The N-linked sugar chain having mannose at its terminus, as described in

[83] or

[84] . method,

[89] The method described in

[83] or

[84] , wherein the glycan receptor is a mannose receptor. method,

[90] The method described in

[64] to

[89] , characterized in that the antigen-binding molecule is an antibody. The method,

[91] The glycan receptor binding domain is included in the antigen binding domain

[64] to [90 The method described in any of the following:

[92] The Fc region contains the glycan receptor binding domain, which is included in the FcRn binding domain.

[64] The method described in any of

[90] , To provide. [Brief explanation of the drawing]

[0015] [Figure 1] This schematic diagram illustrates how IgG antibody molecules dissociate from soluble antigens within endosomes, accelerating antigen elimination and allowing them to re-bind to new antigens. [Figure 2] This schematic diagram illustrates how IgG antibody molecules bind to glycosylation receptors in blood vessels, are taken up into cells, and then dissociate from soluble antigens together with lytic receptors in endosomes, accelerating antigen elimination and allowing them to re-bind to new antigens. [Figure 3] This figure shows the detection of heavy and light chains by reduced SDS-PAGE. [Figure 4] This figure shows chromatograms of anion exchange chromatography before and after neuraminidase treatment. [Figure 5] This figure shows a mass chromatogram obtained by RP-LC / ESI-MS analysis of reduced GL-M111, observing the N-linked glycans attached to the light chain. [Figure 6] This figure shows the neurominidase activity observed by anion exchange chromatography. [Figure 7] This figure shows the heavy and light chains detected by reduced SDS-PAGE in a sample whose steady-state region was returned to IgG1. [Figure 8] This figure shows the plasma concentration profile of antibodies in normal mice. [Figure 9] This figure shows the plasma concentration profile of soluble human IL-6 receptor in normal mice. [Figure 10] This figure shows the plasma concentration profile of antibodies in normal mice. [Figure 11] This figure shows the plasma concentration profile of soluble human IL-6 receptor in normal mice. [Figure 12] This figure shows the detection of heavy and light chains by reduced SDS-PAGE. [Figure 13] This figure shows a mass chromatogram obtained by RP-LC / ESI-MS analysis of reduced GL5-G1_kif+, observing the N-linked glycans attached to the light chain. [Figure 14]This figure shows the plasma concentration profile of antibodies in normal mice. [Figure 15] This figure shows the plasma concentration profile of soluble human IL-6 receptor in normal mice. [Figure 16] This figure shows a Biacore sensorgram illustrating the interaction of H54 / L28-IgG1 with soluble human IL-6 receptors at Ca2+ 2mM and Ca2+ 3μM. [Figure 17] This figure shows a Biacore sensorgram illustrating the interaction of FH4-IgG1 with soluble human IL-6 receptors at Ca2+ 2mM and Ca2+ 3μM. [Figure 18] This figure shows a Biacore sensorgram illustrating the interaction of 6RL#9-IgG1 with soluble human IL-6 receptors at Ca2+ 2mM and Ca2+ 3μM. [Figure 19] This figure shows the concentration profiles of antibodies H54 / L28-IgG1, FH4-IgG1, and 6RL#9-IgG1 in normal mouse plasma. [Figure 20] This figure shows the concentration profiles of soluble human IL-6 receptor (hsIL-6R) in normal mouse plasma for H54 / L28-IgG1, FH4-IgG1, and 6RL#9-IgG1. [Figure 21] This figure shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody as determined by X-ray crystallography. [Figure 22] These are ion-exchange chromatograms of an antibody containing the human Vk5-2 sequence and an antibody containing the hVk5-2_L65 sequence, which is a modified version of the human Vk5-2 sequence with a modified glycosylation sequence. The solid line represents the chromatogram of the antibody containing the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 63 and light chain: hVk5-2, a fusion molecule of SEQ ID NO: 6 and SEQ ID NO: 42), and the dashed line represents the chromatogram of the antibody with the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 63), light chain: hVk5-2_L65 (SEQ ID NO: 62)). [Modes for carrying out the invention]

[0016] The following definitions and detailed descriptions facilitate understanding of the invention as described herein. It is provided for this purpose.

[0017] amino acid In this specification, 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 / As represented by I, Val / V, amino acids are coded with one letter, three letters, or both. It is written as follows.

[0018] and / or In this specification, the meanings of the terms "and / or" are as follows: This includes any combination that is appropriate. Specifically, for example, "33rd place, 55th place, and / or The phrase "or the amino acid at position 96 is substituted" includes the following variations of amino acid modification. Born; (a) 33rd, (b) 55th, (c) 96th, (d) 33rd and 55th, (e) 33rd and 96th, (f) 55th 96th, (g) 33rd, 55th, and 96th.

[0019] antigen In this specification, "antigen" means a structure that includes an epitope to which an antigen-binding domain binds. The term "structure" is not limited to a specific structure. In another sense, an antigen can be inorganic or organic. It is possible. An example of an antigen-binding molecule that improves pharmacokinetics by the method of the present invention is, for example, If so, membrane antigens such as receptor proteins (membrane-bound receptors, soluble receptors) and cell surface markers Antigen-binding molecules that recognize antigens, such as cytokines and other soluble antigens, are preferred. Suitable examples include the following molecules as antigens: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8 -Iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, A 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-stimulating 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, Bombeshin 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 (CEA), cancer-associated antigen, cathepsi Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathe 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, complement regulatory factor (decay accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2 EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephaly Nase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-Selecti N, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Cell-activated 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, F 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-Alpha 1, GFR-Alpha 2, GF R-Alpha 3, 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 Glin Alpha 3, Integrin Alpha 4, Integrin Alpha 4 / Beta 1, Integrin Integrin Alpha 4 / Beta 7, Integrin Alpha 5 (Alpha V), Integrin Alpha 5 / Beta 1, Integrin Alpha 5 / Beta 3, Integrin Alpha 6, Integrin Beta 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), Laminin Present 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-cele Kuchin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta Receptors, 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, Neuroturin, Neurotrophin Long 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, P Insulin, 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, polynuclear respiratory 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 receptor (e.g., T cell receptors (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 antigens 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, high-molecular-weight 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 hormones Examples of receptors for ions and growth factors can be cited.

[0020] Epitope, meaning an antigenic determinant present in an antigen, is an anti- This refers to the site on the antigen to which the antigen-binding domain in the proto-binding molecule binds. Therefore, for example, An epitope can be defined by its structure. Furthermore, an antigen that recognizes that epitope may also be defined. The epitope can also be defined by its binding activity to the antigen in the compound molecule. If it is a polypeptide or polypeptide, the epitope is formed by the amino acid residues that make up the epitope. It is also possible to identify the epitope. Furthermore, if the epitope is a sugar chain, a specific sugar can be identified. It is also possible to identify epitopes based on their chain structure.

[0021] A linear epitope is an epitope that contains an epitope whose amino acid primary sequence has been recognized. Linear epitopes typically have at least three, and most commonly at least five. For example, it contains approximately 8 to 10 or 6 to 20 amino acids in a specific sequence.

[0022] In contrast to linear epitopes, three-dimensional epitopes are one of the amino acids containing the epitope. The following sequence is an epitope that is not a single defining component of the recognized epitope (e.g., amino (An epitope whose primary sequence is not necessarily recognized by the antibody that defines the epitope) Yes, there is. Three-dimensional epitopes contain an increased number of amino acids compared to linear epitopes. It may be. Regarding the recognition of three-dimensional structural epitopes, antibodies may recognize peptides or proteins. It recognizes the three-dimensional structure of a substance. For example, it recognizes how protein molecules fold to form a three-dimensional structure. In such cases, a certain amino acid and / or polypeptide that form a three-dimensional epitope is the main component. The chains are arranged in parallel, allowing the antibody to recognize the epitope. Epitope structure Methods for determining this include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spectroscopy. This includes, but is not limited to, pin-labeled and electromagnetic paramagnetic resonance spectroscopy. For example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66 See Morris (ed.).

[0023] Binding activity The following describes the interaction of a test antigen-binding molecule containing an antigen-binding domain with IL-6R to the epitope. Methods for confirming binding are given as examples, but tests include antigen-binding domains for antigens other than IL-6R. The method for confirming the binding of antigen-binding molecules to epitopes may be carried out as appropriate, following the examples below. ru.

[0024] For example, a test antigen-binding molecule containing an antigen-binding domain for IL-6R is present within the IL-6R molecule. Recognizing existing linear epitopes can be confirmed, for example, as follows: For the above purpose, a linear sequence of amino acids constituting the extracellular domain of IL-6R is formed. A peptide is synthesized. This peptide can be synthesized chemically, or from the cDNA of IL-6R. Using the region within it that codes for the amino acid sequence corresponding to the extracellular domain, genetic engineering It is obtained by the following method. Next, a linear peptide consisting of the amino acid sequence that constitutes the extracellular domain is obtained. The binding activity of tide to the test antigen-binding molecule containing the antigen-binding domain for IL-6R was evaluated. For example, by ELISA using an immobilized linear peptide as an antigen, the peptide The binding activity of the antigen-binding molecule to the antigen can be evaluated. Alternatively, the binding activity to IL-6R-expressing cells can be evaluated. Based on the level of inhibition by the linear peptide in the binding of the antigen-binding molecule, The binding activity to peptides can be revealed. These tests can reveal the binding activity to linear peptides. The binding activity of the antigen-binding molecule in question can be revealed.

[0025] Furthermore, the antigen-binding molecule containing the antigen-binding domain for IL-6R is a structural epitope. Recognition can be confirmed as follows: For the above purpose, expressing IL-6R Cells are prepared. A test antigen-binding molecule containing an antigen-binding domain for IL-6R is used to break down IL-6R. While the antigen-binding molecule strongly binds to the cell upon contact, the antigen-binding molecule is immobilized. For a linear peptide consisting of the amino acid sequence that constitutes the extracellular domain of IL-6R, Examples include cases where binding does not occur. Here, "substantially does not bind" refers to human IL-6R-expressing cells. 80% or less of the binding activity to it, usually 50% or less, preferably 30% or less, and especially preferably 15% The following binding activities are used.

[0026] Binding of the test antigen-binding molecule containing the antigen-binding domain for IL-6R to IL-6R-expressing cells. Methods for measuring activity include, for example, 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 acetylcholine spectroscopy) that use IL-6R-expressing cells as antigens. It can be evaluated using the principle of cell sorting.

[0027] In the ELISA format, the test antigen-binding molecule contains the antigen-binding domain for IL-6R. The binding activity of IL-6R to IL-6R-expressing cells is compared by the signal level generated by the enzymatic reaction. This is quantitatively evaluated by immobilizing IL-6R-expressing cells in an ELISA test. The test antigen-binding molecule is added to the test, and the test antigen-binding molecule that binds to the cell is the test antigen-binding molecule. It is detected using an enzyme-labeled antibody that recognizes it. Alternatively, in FACS, the test antigen binds. Create a molecular dilution series and determine the antibody binding titer to IL-6R-expressing cells. This allows for comparison of the binding activity of test antigen-binding molecules to IL-6R-expressing cells.

[0028] The binding of the test antigen-binding molecule to the antigen expressed on the cell surface suspended in a buffer solution is It can be detected by a flow cytometer. For example, the following devices are known. FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are product 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 product names of Beckman Coulter) )

[0029] For example, the binding of a test antigen-binding molecule containing an antigen-binding domain to IL-6R to the antigen. One example of a suitable method for measuring activity is the following: First, the molecules expressing IL-6R are measured. The sample is stained with a FITC-labeled secondary antibody that recognizes the antigen-binding molecule reacted with the cell. By diluting the binding molecule with a suitable buffer, the antigen-binding molecule can be converted to the desired state. It is used after being prepared to the desired concentration. For example, any concentration between 10 μg / ml and 10 ng / ml. It can be used in this way. Next, fluorescence intensity and cell count are measured using FACSCalibur (BD). The amount of antibody binding to the cells will be analyzed using CELL QUEST Software (BD Inc.). This is reflected in the fluorescence intensity obtained, i.e., the Geometric Mean value. By obtaining the eometric mean value, the amount of the test antigen-binding molecule bound to the test antigen can be expressed. The binding activity of antigen-binding molecules can be measured.

[0030] A test antigen-binding molecule containing an antigen-binding domain for IL-6R interacts with a certain antigen-binding molecule and epidermis. Sharing a tope can be confirmed by competition between the two parties for the same epitope. Competition between primal binding molecules can be detected by cross-blocking assays, for example. The ELISA assay is a preferred cross-blocking assay.

[0031] Specifically, in cross-blocking assays, the microtiter plate is used The IL-6R protein coated on the surface reacts in the presence of a candidate competitive antigen-binding molecule, or in the absence of After pre-incubation in the presence of IL, the test antigen-binding molecule is added. IL in the well The amount of antigen-binding molecules bound to the -6R protein competes for binding to the same epitope. It is indirectly correlated with the binding ability of competing antigen-binding molecules that are candidates for binding. In other words, it is identical to the same epidemiological stage. The greater the affinity of the competing antigen-binding molecule to the tope, the greater the IL of the test antigen-binding molecule. Binding activity to wells coated with the -6R protein is reduced.

[0032] The amount of the test antigen-binding molecule bound to the well via the IL-6R protein is the amount of antigen-binding beforehand. By labeling the substance, it can be easily measured. For example, biotin-labeled antibody The original conjugated molecule uses an avidin peroxidase conjugate and an appropriate substrate. This is measured using a cross-blocking assay that utilizes enzyme labeling such as peroxidase. This is specifically called a competitive ELISA assay. Antigen-binding molecules can be detected or measured in other ways. It can be labeled with a labeling substance. Specifically, radioactive labeling or fluorescent labeling are well known.

[0033] Results obtained in a control test conducted in the absence of candidate competing antigen-binding molecules Compared to the combined activity, the competing antigen-binding molecule contains an antigen-binding domain for IL-6R in the test antibody. The binding of the original bonding molecules should be at least 20%, preferably at least 20-50%, and more preferably If it can block at least 50%, then the test antigen-binding molecule is essentially the same as a competing antigen-binding molecule. Antigens that bind to the same epitope, or antigens that compete for binding to the same epitope. It is a compound molecule.

[0034] Structure of the epitope to which the test antigen-binding molecule, containing the antigen-binding domain for IL-6R, binds. If identified, the test antigen-binding molecule and the control antigen-binding molecule share an epitope. This involves introducing amino acid mutations into the peptide that constitutes the epitope. This can be evaluated by comparing the binding activity of the antigen-binding molecules of both parties.

[0035] One method for measuring this binding activity is, for example, using the ELISA format described above. The binding of the test antigen-binding molecule and the control antigen-binding molecule to the linear peptide into which the mutation was introduced. It can be measured by comparing the activity. Other methods besides ELISA involve binding to a column. The binding activity to the mutant peptide was measured in the column, and the binding activity of the test antigen-binding molecule and the control antigen-binding molecule were measured in the column. It can also be measured by quantifying the antigen-binding molecules eluted into the eluate after the molecules have been allowed to flow down. It is possible to adsorb the mutant peptide onto a column as a fusion peptide with, for example, GST. It is public knowledge.

[0036] Furthermore, if the identified epitope is a stereoepitope, the test antigen-binding molecule and the control antibody The sharing of an epitope with the original binding molecule can be evaluated in the following way. First, IL-6R Cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. When these cells are suspended in a cell suspension in an appropriate buffer such as PBS, the target antigen-binding molecule is detected. A irradiant-binding molecule is added. Then, to the cell suspension, which has been washed with buffer as appropriate, A FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added. The fluorescence intensity and cell count of cells stained with labeled antibodies are measured using FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are adjusted using a suitable buffer. It is used after being diluted to the desired concentration. For example, from 10 μg / ml to 10 ng It is used at any concentration between CEL / ml. The amount of labeled antibody bound to the cells is determined by the amount of CEL. The fluorescence intensity obtained by analysis using LQUEST Software (BD Corporation), i.e., Geom This is reflected in the Geometric Mean value. In other words, by obtaining the Geometric Mean value, The binding activity of the test antigen-binding molecule and the control antigen-binding molecule is measured by the amount of labeled antibody that binds to them. It can be determined.

[0037] In this method, for example, "substantially does not bind to mutant IL-6R expressing cells" means the following: This can be determined by the method. First, the cells that express the mutant IL-6R were bound to... The test antigen-binding molecule and the control antigen-binding molecule are stained with the labeled antibody. Then the fluorescence intensity of the cells is determined. When FACSCalibur is used as flow cytometry for fluorescence detection, the results are obtained. The fluorescence intensity can be analyzed using CELL QUEST Software in the presence of an antigen-binding molecule. From the Geometric Mean values ​​in the absence of and , this comparison value (ΔGeo-Mean) is calculated using the following formula By calculating based on this, the percentage increase in fluorescence intensity due to the binding of antigen-binding molecules can be determined. 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 the test antigen-binding molecule obtained from the analysis that binds to mutant IL-6R-expressing cells is reflected. The Geometric Mean comparison value (ΔGeo-Mean value of the mutant IL-6R molecule) is used for the IL-6R antigen-binding molecule of the test antigen. This is compared to a ΔGeo-Mean comparison value that reflects the amount of binding to R-expressing cells. This is used when determining the ΔGeo-Mean comparison value for mutant IL-6R expressing cells and IL-6R expressing cells. It is particularly preferable that the concentrations of the test antigen-binding molecules be the same or substantially the same. It seems so. Antigen-binding molecules that have been previously confirmed to recognize epitopes in IL-6R are, It is used as an irradiant binding molecule.

[0039] The ΔGeo-Mean comparison value of the test antigen-binding molecule against mutant IL-6R-expressing cells is the test antigen-binding component. The ΔGeo-Mean comparison value for the child's IL-6R-expressing cells should be at least 80%, preferably 50%, and further Preferably less than 30%, and especially preferably less than 15%, then "substantially, mutant IL-6R expressing cells are It is assumed that they will not be joined. The formula for calculating the Geo-Mean value (Geometric Mean) is CELL Q This is described in the UEST Software User's Guide (BD Biosciences). If they can be considered substantially equivalent by comparison, then the test antigen-binding molecule and the control molecule The epitopes of the original binding molecules can be considered identical.

[0040] antigen-binding domain In this specification, the "antigen-binding domain" is defined as a domain that binds to the target antigen, in any way. Domains with such structures may also be used. An example of such a domain is, for instance, the heavy chain of an antibody. and the variable region of the light chain, and 35 amicin contained in Avimer, a cell membrane protein present in living organisms. A module called the A domain, which is of the degree of anoacidity (WO2004 / 044011, WO2005 / 040229), cell membrane 10F is a protein-binding domain in fibronectin, a glycoprotein expressed in fibronectin. Adnectin (WO2002 / 032925) containing the n3 domain, Protein A consisting of three helices with 58 amino acids Affibody (WO1995 / 00) uses IgG-binding domains that constitute a bundle of chromosomes as scaffolds. 1937), a turn containing 33 amino acid residues and two antiparallel helices and loops subunits Ankyrin repeats (ARs) are structures in which swabs are repeatedly stacked. DARPins (Designed Ankyrin Repeat proteins) (WO2002) are regions exposed on the surface of the molecule. / 020565), neutrophil gelatinase-associated lipocalin In lipocalin molecules such as lipocalin (NGAL), eight highly conserved antiparallel strands Anticalin, etc., is a four-loop region that supports one side of a barrel structure that is twisted towards the center. WO2003 / 029462), as an acquired immune system of jawless eels such as lampreys and hagfish, Variable lymphocyte receptors that do not possess the structure of noglobulin Leucine-rich repeat (LRR) module of (VLR) The recessed region of the parallel sheet structure inside the horseshoe-shaped structure, which is repeatedly stacked (WO20 08 / 016854) is a preferred example. A preferred example of the antigen-binding domain of the present invention is an antibody Examples include antigen-binding domains that include variable regions of the heavy and light chains. Examples of "in" 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 Examples of suitable examples include / 037989) or Fc fusion proteins. Molecules containing an Fc region are F The c region can be used as a binding domain for FcRn, particularly human FcRn. Molecules to which a human FcRn-binding domain is fused may also be used.

[0041] The antigen-binding domain in the antigen-binding molecule of the present invention binds to the same epitope. This is possible. Here, the same epitope is, for example, sequence number: 1(IL-6R_PP;NP_000556). 1) It can be present in proteins consisting of the amino acid sequence described above. It is present in the protein consisting of amino acids 20 to 365 of the amino acid sequence described in No. 1. They can exist. Alternatively, the antigen-binding domain in the antigen-binding molecule of the present invention can be mutual It can bind to different epitopes. Here, different epitopes are, for example, It can be present in proteins consisting of the amino acid sequence described in column number 1. Also, A protein consisting of amino acids 20 through 365 of the amino acid sequence described in Sequence ID No. 1. It can be present within. For this purpose, the antigen binding contained in bispecific antibodies. Domains can be used as appropriate. Bispecific antibodies have variable regions that recognize different epitopes. An antibody having the same within the antibody molecule. A bispecific antibody can recognize two or more different antigens or can also be an antibody that recognizes two or more different epitopes on the same antigen.

[0042] In addition, as the antigen-binding domain of the present invention, a domain involved in the binding to the target (antigen) in a receptor (receptor protein) that binds to the target can be preferably used. That is, the antigen-binding molecule is a receptor protein that binds to the target (antigen) A protein in which a binding domain for FcRn, particularly FcRn, and a sugar receptor binding domain are fused may be used. Examples of such antigen-binding molecules include, for example, TNFR-Fc fusion protein , IL1R-Fc fusion protein, VEGFR-Fc fusion protein, CTLA4-Fc fusion protein, etc. (Nat Med. (2003) 9 (1), 47-52, BioDrugs. (2006) 20 (3), 151-160). Even if the antigen-binding molecule of the present invention is a fusion protein of these receptor proteins and a binding domain for FcRn, particularly human FcRn, the binding activity to the target molecule changes depending on the ionic concentration conditions, and it has a binding activity to the sugar receptor and a binding activity to FcRn, particularly human FcRn. The antigen-binding molecule can promote the uptake of the antigen into the cell and can promote the decrease in the antigen concentration in the plasma by administration of the antigen-binding molecule. In addition, the pharmacokinetics of such an antigen-binding molecule are improved, and it is possible to increase the number of antigens that one antigen-binding molecule can bind.

[0043] In addition, as the antigen-binding domain of the present invention, a natural or artificial ligand that binds to the target ​​​​​​​​Domains involved in binding to the target in can be preferably used. That is, anti The proto-binding molecule is a natural or artificial molecule that binds to a target and has antagonist activity or neutralizing effect. The protein ligand has a binding domain for FcRn, particularly FcRn, and a glycosylation receptor binding domain. It could also be a fused molecule. Among these antigen-binding domains, an example of an artificial ligand is: For example, artificial ligands such as mutant IL-6 (EMBO J. (1994) 13 (24), 5863-70) can be cited. Even if the antigen-binding molecule of the present invention is one of these artificial ligand fusion molecules, the ion concentration is Depending on the circumstances, the binding to the target molecule changes, affecting the binding activity to the glycosylation receptor and the FcRn characteristic. Antigen-binding molecules that have binding activity to human FcRn promote the uptake of antigens into cells. Furthermore, it is possible to accelerate the decrease in antigen concentration in plasma by administering antigen-binding molecules. Furthermore, the pharmacokinetics of such antigen-binding molecules have been improved, and one antigen-binding molecule can bind to multiple molecules. It is possible to increase the number of active antigens.

[0044] specific "Specific" means that one of the molecules that specifically binds is one or more of the molecules that bind to it. This refers to a state in which no significant binding is observed to molecules other than the specified molecule. The drug is specific to a particular epitope among several epitopes contained in a given antigen. It is also used in cases where the antigen-binding domain binds to multiple different epitopes. If the antigen is contained in the antigen, the antigen-binding molecule having the antigen-binding domain is the epitome It can bind to various antigens, including p.

[0045] antibody In this specification, an antibody is defined as one that is either naturally occurring or partially or completely synthesized. This refers to immunoglobulins that are manufactured from natural materials. Antibodies are naturally occurring substances found in plasma, serum, and other natural materials. It can be isolated from the culture supernatant of hybridoma cells that produce the source or antibodies, or from gene combinations. Antibodies can be partially or completely synthesized by using techniques such as substitution. For example, immunoglobulin isotypes and subclasses of those isotypes are preferably listed. It can be obtained. As human immunoglobulins, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, I Nine classes (isotypes) of gE and IgM are known. The antibodies of the present invention include these The 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. Below, IL An example of a method for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) is given. The matching antibodies can also be appropriately prepared according to the examples below.

[0047] Anti-IL-6R antibodies are produced as polyclonal or monoclonal antibodies using known methods. It can be obtained. Mammalian-derived monoclonal antibodies are preferably used as anti-IL-6R antibodies. It is possible. Mammalian monoclonal antibodies are produced by hybridomas. and host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. This includes substances produced by cells, etc. Furthermore, the monoclonal antibody of the present invention includes "H This includes "T-induced antibodies" and "chimeric antibodies."

[0048] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example. It can be prepared as follows. That is, using the IL-6R protein as a sensitizing antigen, a mammal is immunized according to a conventional immunization method. The obtained immune cells are fused with known parent cells by a conventional cell fusion method. Next, anti-IL-6R antibodies are produced by screening monoclonal antibody-producing cells by a conventional screening method, and a hybridoma can be selected. Specifically, the production of monoclonal antibodies is carried out as shown below. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody acquisition, can be obtained by expressing the IL-6R gene whose nucleotide sequence is disclosed in SEQ ID NO: 2 (IL-6R_PN; NM_000565.3). That is, a suitable host cell is transformed by inserting the gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968), consisting of amino acids 1 to 357, is expressed instead of the IL-6R protein represented by SEQ ID NO: 1. Also, the purified natural IL-6R protein can be used as a sensitizing antigen in the same manner. The purified IL-6R protein can be used as a sensitizing antigen for immunizing mammals.

[0049]

[0050] ​​​​​​​​​​​​​​The IL-6R partial peptide 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 portion of it into an expression vector and expressing it, but the partial peptide and The region and size of the IL-6R peptide used are not limited to any 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 sequence. The number of amino acids that make up the peptide to be used as a sensitizing antigen. It is preferable that it be at least 5, for example, 6 or more, or 7 or more. More specifically, 8~ 50, preferably 10 to 30 residues of peptide, can be used as a sensitizing antigen.

[0051] Furthermore, desired partial polypeptides or peptides of the IL-6R protein can be combined with different polypeptides. The fused proteins can be used as sensitizing antigens. For protein production, for example, antibody Fc fragments or peptide tags are suitably used. The vector expressing the fusion protein can contain two or more desired types of peptides. The genes encoding the fragment are fused in frame, and the fused gene is expressed as described above. It can be produced by insertion into the current vector. The method for producing the fusion protein is Molecula r Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.4 It is described in 7-9.58 (1989) Cold Spring Harbor Lab. press) as a sensitizing antigen. The method for obtaining IL-6R used and the immunization method using it are described in WO2003 / 000883, WO2004 / 022. This is also described in detail in documents such as 754 and WO2006 / 006693.

[0052] The mammals that are immunized by the sensitizing antigen in question are not limited to specific animals. It is preferable to select cells considering their compatibility with the parent cells used for cell fusion. Generally, baldness Rodents, such as mice, rats, hamsters, or rabbits, monkeys, etc., are preferred. It will be used.

[0053] The animals described above are immunized with sensitizing antigens according to known methods. For example, a common method By law, the sensitizing antigen is administered by injection into the abdominal cavity or subcutaneously of a mammal. Immunization is performed. Specifically, with PBS (Phosphate-Buffered Saline) or physiological saline. The sensitizing antigen, diluted to an appropriate dilution ratio, is optionally used with a standard adjuvant, such as Frey. After being mixed with a complete adjuvant and emulsified, the sensitized antigen is exposed to mammals for 4 to 21 days. It is administered several times at a time. Additionally, a suitable carrier may be used during immunization with sensitizing antigens, especially molecular carriers. When small amounts of partial peptides are used as sensitizing antigens, albumin and keyholes are used. Immunizing the sensitizing antigen peptide bound to carrier proteins such as limpet hemocyanin In some cases, this is preferable.

[0054] Furthermore, hybridomas that produce the desired antibody are created using DNA immunization, as follows: DNA immunity can also be produced. DNA immunity is the expression of genes encoding antigen proteins in an immunized animal. In immunized animals to which vector DNA constructed in such a manner can be administered, sensitized antigen This is an immunization method in which immune stimulation is provided by the expression of a substance within the living body of the immunized animal. Compared to common immunization methods in which protein antigens are administered to immunized animals, DNA immunization has the following advantages: Such advantages are expected. - It is possible to maintain the structure of membrane proteins such as IL-6R and deliver immunostimulation. - There is no need to purify immune antigens.

[0055] To obtain the monoclonal antibody of the present invention by DNA immunization, first, the IL-6R protein is The expressed DNA is administered to immunized animals. The DNA encoding IL-6R is processed by known methods such as PCR. Therefore, it can be synthesized. The obtained DNA is inserted into a suitable expression vector and administered to immunized animals. Commercial expression vectors such as pcDNA3.1 are preferably used as expression vectors. It is possible. Commonly used methods for administering vectors to living organisms are employed. For example, gold particles to which an expression vector has been adsorbed can be used to immunize the cells of an animal organism with a gene gun. DNA immunization occurs when it is introduced into the body. Furthermore, the production of antibodies that recognize IL-6R is It can also be prepared using the method described in International Publication WO2003 / 104453.

[0056] In this way, mammals become immunized, and an increase in antibody titers that bind to IL-6R in the serum is confirmed. After approval, immune cells are collected from mammals and subjected to cell fusion. Preferred immune cells Splenocytes, in particular, can be used as cells.

[0057] Mammalian myeloma cells are used as the cells to be fused with the aforementioned immune cells. Roman cells are preferably equipped with appropriate selection markers for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. The selection marker is hypoxanthine-guanine-phosphoribosyltransferase deficiency. Deficiency (hereinafter abbreviated as HGPRT deficiency), or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) Cells lacking HGPRT or TK are known to have hypoxanthine-aminoptase They possess phosphate-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells are HAT selected. They cannot synthesize DNA in selective culture medium and die, but when they fuse with normal cells, they become like normal cells. Because it can continue DNA synthesis using the salvage circuit, it can grow even in HAT selective medium. They will start doing that.

[0058] HGPRT-deficient and TK-deficient cells contain 6-thioguanine and 8-azaguanine, respectively (hereinafter abbreviated as 8AG). These pyri Normal cells that incorporate the pyrimidine analog into their DNA die. On the other hand, these pyrimidine analogs... Cells lacking these enzymes, which are unable to take up logs, can survive in selective media. In addition, a selective marker called G418 resistance is detected by the neomycin resistance gene, specifically 2-deo It confers resistance to xystreptamine antibiotics (gentamicin analogs). Various myeloma cells suitable for this purpose are known.

[0059] Examples of such myeloma cells include P3(P3x63Ag8.653)(J. Immunol.(1979) )123 (4), 1548-1550), P3x63Ag8U.1(Current Topics in Microbiology and I mmunology(1978)81, 1-7), NS-1(C. Eur. 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 others can be preferably used.

[0060] Basically, it is a publicly known method, for example, the method of Köhler and Milstein et al. (Methods Enzyme) Cell fusion between the aforementioned immune cells and myeloma cells was performed in accordance with ol. (1981) 73, 3-46), etc. More specifically, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter, the aforementioned fine Cellular fusion can be performed. Examples of fusion promoters include polyethylene glycol (PEG). Sendai virus (HVJ), etc., is used, and if desired, methyl iontophoresis is added to further enhance fusion efficiency. It is used with the addition of auxiliary agents such as sulfoxides.

[0061] The ratio of immune cells to myeloma cells used can be set arbitrarily. For example, myeloma cells It is preferable to increase the number of immune cells to 1 to 10 times the number of cells. The culture medium used for the cell fusion is For example, RPMI1640 culture medium, MEM culture medium, and the above-mentioned myeloma cell line are suitable for proliferation. In addition, standard culture media used for this type of cell culture are used, and furthermore, fetal bovine serum (FCS) is used. Serum replacement solutions such as ) can be suitably added.

[0062] Cell fusion is performed by thoroughly mixing a predetermined amount of the immune cells and myeloma cells in the culture medium. A PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C is typically used in 30 to 6 It is added at a concentration of 0% (w / v). The mixture is slowly mixed to achieve the desired fusion particles. A cyst (hybridoma) is formed. Then, the appropriate culture medium mentioned above is added sequentially. By repeatedly centrifugating and removing the supernatant, the process becomes more favorable for the growth of hybridomas. Cell fusion agents and the like can be removed.

[0063] Hybridomas obtained in this way can be cultured in a standard selective culture medium, such as HAT culture medium (Hypo Selected by culturing in a culture medium containing xanthine, aminopterin, and thymidine. It is possible. Sufficient time for cells other than the desired hybridoma (non-fused cells) to die. (Typically, the required time is several days to several weeks.) The culture using the above HAT culture medium can be continued. Next, the hybridoma that produces the desired antibody is screened using the standard limiting dilution method. Running and single cloning are performed.

[0064] The hybridoma obtained in this way possesses the properties of the myeloma used in cell fusion. Selection can be achieved by using a selective culture medium corresponding to the selection marker. For example, HGPRT or T Cells lacking K were treated with HAT culture medium (hypoxanthine, aminopterin, and thymidine). They can be selected by culturing in a culture medium containing HAT. That is, HAT-sensitive myeloma cells When cells are used for cell fusion, cells that successfully fused with normal cells are selected in the HAT culture medium. They can proliferate selectively. Sufficient to kill cells other than the desired hybridoma (non-fused cells). The culture using the above HAT culture medium is continued for a certain period of time. Specifically, generally, several days to several weeks. Intermediate culture allows for the selection of the desired hybridoma. Then, using the usual limiting dilution method... Therefore, screening and single cloning of hybridomas that produce the desired antibody. This may be implemented.

[0065] Screening and single cloning of desired antibodies is based on known antigen-antibody reactions. This can be suitably carried out by a screening method. For example, a monoclonal ion bound to IL-6R. The monoclonal antibody can bind to IL-6R expressed on the cell surface. For example, natural antibodies can be detected by FACS (fluorescence-activated cell sorting). FACS can be leaned. FACS analyzes cells that have been contacted with a fluorescent antibody using laser light, and individual By measuring the fluorescence emitted by cells, it is possible to measure the binding of antibodies to the cell surface. This is a system that makes it possible to do so.

[0066] Screening hybridomas producing the monoclonal antibody of the present invention by FACS To do this, first prepare cells that express IL-6R. Preferred for screening The cells are mammalian cells that have been forced to express IL-6R. Transformed cells were used as host cells. By using mammalian cells that have not undergone this process as a control, the response of IL-6R on the cell surface can be determined. Antibody binding activity can be selectively detected. That is, it does not bind to host cells and does not induce IL-6R overactivation. By selecting hybridomas that produce antibodies that bind to present cells, IL-6R monoclonal Hybridomas that produce ronal antibodies can be obtained.

[0067] Alternatively, the binding activity of antibodies against immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. It can be valued. For example, IL-6R expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of lidoma was brought into contact with immobilized cells in the wells, and antibodies that bind to the immobilized cells were detected. It is released. If the monoclonal antibody is derived from a mouse, the antibody bound to the cell is anti-mouse. It can be detected by munoglobulin antibodies. These screenings are selected Hybridomas that produce desired antibodies with the ability to bind to antigens are obtained by methods such as limiting dilution. It can be cloned more easily.

[0068] The hybridomas that produce monoclonal antibodies in this manner can be cultured under normal conditions. It can be subcultured in liquid. Furthermore, the hybridoma can be stored in liquid nitrogen for extended periods. It is possible.

[0069] The hybridoma is cultured according to a standard method, and the desired monoclonal ion is obtained from the culture supernatant. Antibodies can be obtained. Alternatively, hybridomas can be administered to mammals that are compatible with them. By allowing the organism to proliferate, monoclonal antibodies can be obtained from its ascites fluid. The former method yields high purity. It is suitable for obtaining antibodies.

[0070] Recombinant Antibody The antibody gene cloned from the antibody-producing cells such as the hybridoma is used to The cloned antibody gene can also be suitably used. By introducing it into the host, the antibody encoded by that gene is expressed. The isolation of antibody genes, their introduction into vectors, and methods for transforming host cells are examples. For example, it has already been established by Vandamme et al. (Eur.J. Biochem.(1990)192 (3), 7 67-775). As described below, methods for producing recombinant antibodies are also known.

[0071] For example, from hybridoma cells that produce anti-IL-6R antibodies, the variable region of the anti-IL-6R antibody ( The cDNA encoding the V region is obtained. For this purpose, the entire R region is usually first obtained from the hybridoma. NA is extracted. Methods for extracting mRNA from cells include, for example, the following: 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 processed using the mRNA Purification Kit (manufactured by GE Healthcare Biosciences). It can be purified using the following methods: or QuickPrep mRNA Purification Kit (GE Kits for directly extracting total mRNA from cells, such as those made by Ruscare Biosciences. Such kits are also commercially available. Using these kits, mRNA can be obtained from hybridomas. The obtained mRNA can be used to synthesize cDNA encoding the antibody V region using reverse transcriptase. DNA is synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (biochemical engineering). It can be synthesized by (manufacturer's) etc. Furthermore, for cDNA synthesis and amplification, SMART RACE cDNA amplification kit (Clontech) and 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used as appropriate. Furthermore, in the process of synthesizing cDNA, both ends of the cDNA Appropriate restriction enzyme sites, as described later, can be introduced at the edges.

[0073] The desired cDNA fragment is purified from the obtained PCR product and then ligated to the vector DNA. After the recombinant vector is prepared in this way and introduced into E. coli or other bacteria, and colonies are selected... A desired recombinant vector can be prepared from the E. coli that formed the colony. To determine whether a replacement vector has the target cDNA base sequence, please use known methods, examples, etc. For example, it can be confirmed by methods such as dideoxynucleotide chain intermission.

[0074] To obtain the gene encoding the variable region, primers for amplifying the variable region gene are needed. It is convenient to use the 5'-RACE method using [a specific technique]. First, extract from hybridoma cells cDNA is synthesized using RNA as a template, and a 5'-RACE cDNA library is obtained. Commercially available kits, such as the SMART RACE cDNA amplification kit, are used as appropriate for the synthesis of Library A. ru.

[0075] The obtained 5'-RACE cDNA library was used as a template to amplify the antibody gene by PCR. Based on known antibody gene sequences, primers for amplifying mouse antibody genes are designed. These primers have different base sequences for each subclass of immunoglobulin. Therefore, the subclass is pre-defined as an isotype of the Iso Strip mouse monoclonal antibody. Determine the result using a commercially available kit such as a diagnostic kit (Roche Diagnostics). It is desirable.

[0076] Specifically, for example, when the goal is to obtain the gene encoding mouse IgG, Amplification of genes encoding γ1, γ2a, γ2b, and γ3 as the main chains, and κ and λ chains as the light chains is possible. Effective primers can be used. To amplify the variable region gene of IgG, the 3' side is generally used. For the primer, a primer that anneals to the steady-state region, which is close to the variable region, is advantageous. It is used. On the other hand, the 5' side primer is the one included with the 5' RACE cDNA library preparation kit. A primer is used.

[0077] Using the PCR products thus amplified, an immunoglobulin consisting of a combination of heavy and light chains is used. Phosphorus can be reconstituted. The binding activity of the reconstituted immunoglobulin to IL-6R is indicated. As a target, the desired antibody can be screened. For example, obtaining an antibody against IL-6R. When this is the objective, it is even more preferable that the antibody binds to IL-6R specifically. Antibodies that bind can be screened for, for example, as follows: (1) An antibody containing the V region encoded by cDNA obtained from a hybridoma is released into IL-6R The process of bringing the cells into contact with the present cells, (2) A step to detect the binding of IL-6R-expressing cells to an antibody, and (3) A step of selecting an antibody that binds to IL-6R expressing cells.

[0078] Methods for detecting the binding of antibodies to IL-6R-expressing cells are known. Specifically, as mentioned earlier, F The binding of antibodies to IL-6R-expressing cells can be detected using methods such as ACS. Antibody binding activity Fixed specimens of IL-6R-expressing cells may be used as appropriate to evaluate this.

[0079] As a screening method for antibodies that use binding activity as an indicator, phage vectors are used. Panning is also suitably used. Antibody genes are heavy chained from polyclonal antibody-expressing cell populations. And if obtained as a library of light chain subclasses, it utilizes phage vectors. The screening method is advantageous.

[0080] Genes encoding the variable regions of the heavy and light chains can be linked by an appropriate linker sequence. This allows for the formation of a single-chain Fv (scFv) (Nat. Biotechnol. (2005)). 23(9), 1126-1136). Inserting the gene encoding scFv into the phage vector From this, phages expressing scFv on their surface can be obtained. Contact between this phage and the desired antigen. After contact, the phage bound to the antigen is recovered, thereby obtaining sc with the desired binding activity. The DNA encoding Fv can be recovered. By repeating this operation as needed, the desired scFv with binding activity can be enriched.

[0081] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, both ends of the cDNA are modified. The cDNA is digested by restriction enzymes that recognize the inserted restriction enzyme sites. Preferred restriction The enzyme recognizes and digests base sequences that appear infrequently in the base sequences that make up antibody genes. Furthermore, in order to insert one copy of the digested fragment into the vector in the correct orientation, the attachment end is given. Insertion of a restriction enzyme is preferable. The V region of the anti-IL-6R antibody digested as described above is encoded. An antibody expression vector can be obtained by inserting the cDNA into a suitable expression vector. At this time, the gene encoding the antibody constant region (C region) and the gene encoding the V region If the offspring fuses in frame, a chimeric antibody is obtained. Here, a chimeric antibody is... This refers to the fact that the steady-state region and the variable region originate from different sources. Therefore, heterogeneous species such as mouse and human... In addition to chimeric antibodies, human-human allochimeric antibodies are also included in the chimeric antibodies of this invention. By inserting the V region gene into an expression vector that already has a constant region, A melanin antibody expression vector can be constructed. Specifically, for example, the desired antibody constant region (C The V region gene is digested at the 5' end of an expression vector that holds the DNA encoding the region. The restriction enzyme recognition sequences of the restriction enzymes can be appropriately positioned. Both digested with the same combination of restriction enzymes A chimeric antibody expression vector is constructed by fusing the components in-frame.

[0082] To produce anti-IL-6R monoclonal antibodies, the antibody gene is regulated by its expression regulatory region. It is incorporated into the expression vector to be expressed under the following conditions. Expression regulatory region for antibody expression. This includes, for example, enhancers and promoters. Furthermore, expressed antibodies are secreted extracellularly. A suitable signal sequence can be added to the amino terminus. Examples described later. The signal sequence is the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3). Peptides are used, but other suitable signal sequences can also be added. The polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide It can then be secreted extracellularly as a mature polypeptide. Subsequently, this expression vector can be used to process The host cells are transformed, and a set of cells expressing DNA encoding anti-IL-6R antibodies. Replacement cells can be obtained.

[0083] For antibody gene expression, the DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is required. Each is incorporated into a separate expression vector. The vector into which the H chain and L chain are incorporated... By being simultaneously transformed (co-transfected) into the same host cell, it acquires both H chains and L chains. Antibody molecules can be expressed, or DNA encoding the H and L chains can be expressed as a single expression vector. Host cells can be transformed by integration (see International Publication WO 1994 / 011523). (See reference).

[0084] A host for producing antibodies by introducing isolated antibody genes into a suitable host. Many combinations of cells and expression vectors are known. All of these expression systems are based on the original This can be applied to isolate the antigen-binding domain of the molecule. Eukaryotic cells are used as host cells. In such cases, animal cells, plant cells, or fungal cells may be used as appropriate. Specifically, animal 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: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0085] Alternatively, as plant cells, there are species such as Nicotiana tabacum. The expression system for antibody genes using cells derived from the genus Nicotiana is known. Callus-cultured cells can be used as appropriate for cell transformation.

[0086] Furthermore, the following types of fungal cells can be used: - Yeast: Saccharomyces serevisiae and other saccharomyces Pichia, a genus of Saccharomyces, including methanol-utilizing yeasts such as Pichia pastor is. a genus -Filamentous fungi: Aspergillus niger and other Aspergillus species pergillus ) genus

[0087] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, using bacterial cells... If present, bacterial cells such as E. coli and Bacillus subtilis can be used as appropriate. An expression vector containing the target antibody gene is introduced into the cell by transformation. By culturing the transformed cells in vitro, the culture of the transformed cells can be obtained from the resulting The desired antibody may be obtained.

[0088] In addition to the host cells mentioned above, transgenic animals can also be used for the production of recombinant antibodies. In other words, to obtain the antibody from an animal into which the 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 into the gene. Milk As the protein secreted inside, for example, goat β-casein can be used. Antibodies DNA fragments containing fusion genes into which genes have been inserted are injected into goat embryos, and the injected embryos It is introduced into female goats. Transgenic goats are born from the goats that accept the embryo (and From the milk produced by its offspring, the desired antibody is found to be a fusion protein with the milk protein. It can be obtained by doing so. Also, milk containing the desired antibody produced from transgenic goats. To increase the amount, hormones may be administered to transgenic goats (Bio / Technology (1994), 12 (7), 699-702).

[0089] Humanized antibodies, human antibodies When an antigen-binding molecule described herein is administered to a human, the antigen-binding molecule In the antigen-binding domain, the aim is to reduce heterologous antigenicity to humans, etc. An antigen-binding domain derived from a genetically modified recombinant antibody can be appropriately adopted. In addition to the aforementioned chimeric antibodies, genetically modified antibodies include, for example, humanized antibodies. This includes antibodies, etc. These modified antibodies are manufactured as appropriate using known methods.

[0090] To create the antigen-binding domain in the antigen-binding molecule described herein The variable region of the antibody used is typically 3, sandwiched between 4 framework regions (FRs). It is composed of two complementarity-determining regions (CDRs). The CDR is essentially the region that determines the binding specificity of the antibody. The amino acid sequence of the CDR. It is highly diverse. On the other hand, the amino acid sequences that make up FR are different between antibodies with different binding specificities. However, they often show high identity. Therefore, generally, CDR transplantation can lead to the development of certain antibodies. It is believed that the binding specificity of this antibody can be transferred to other antibodies.

[0091] Humanized antibodies are also called reshaped human antibodies. Specifically, they are antibodies derived from non-human animals. Humanized antibodies, such as those obtained by transplanting the CDR of mouse antibodies into human antibodies, are known. Common genetic engineering techniques for obtaining antibodies are also known. Specifically, mouse antibodies Overlap Extension PCR is one method for transplanting a body's CDR into a human FR. It is knowledge. In Overlap Extension PCR, a primer is used to synthesize FR of human antibodies. The primer has a base sequence that codes for the CDR of the mouse antibody to be transplanted added to it. Prepared for each of the four FRs. Generally, in transplantation of mouse CDRs into human FRs Therefore, selecting human FRs that are highly identical to mouse FRs is advantageous in maintaining the function of CDRs. It is believed that there is an amino acid in the FR adjacent to the mouse CDR to be transplanted. It is preferable to use human FR, which has an amino acid sequence that is highly identical to the acid sequence.

[0092] Furthermore, the nucleotide sequences to be linked are designed to connect to each other in-frame. Human FR is synthesized individually by each primer. As a result, mouse CDR is synthesized for each FR. Products with DNA encoding the mouse CDR are obtained. They are designed to overlap each other. Subsequently, the human antibody gene is cast. The overlapping CDR portions of the synthesized product are annealed to each other to form a complementary chain. A reaction takes place. Through this reaction, human FR is linked via the mouse CDR sequence.

[0093] The V region gene, which ultimately consists of three CDRs and four FRs linked together, has an anion at its 5' and 3' ends. The entire length of the enzyme is amplified by a primer to which an appropriate restriction enzyme recognition sequence has been added. The DNA obtained as described above is fused in-frame with the DNA encoding the human antibody C region. By inserting this into an expression vector, a vector for expressing human antibodies can be created. After introducing the embedded vector into a host to establish recombinant cells, the recombinant cells are cultured. By raising the organism and expressing the DNA encoding the humanized antibody, the humanized antibody is Produced in the culture of the cultured cells (see European Patent Publication EP239400, International Publication WO1996 / 002576) see).

[0094] The binding activity of the humanized antibody prepared as described above to the antigen is measured qualitatively or quantitatively. By evaluation, it is determined that when linked via a CDR, the CDR forms a good antigen-binding site. The FR of such a human antibody can be suitably selected. If necessary, the CDR of the reconstituted human antibody is appropriate. It is also possible to substitute amino acid residues of FR to form an antigen-binding site. For example, By applying the PCR method used for transplanting mouse CDRs into human FRs, we introduced amino acid sequence mutations into FRs. This can be done by partially altering the base sequence of the primer that anneals to FR. It is possible to introduce a different type of primer. The FR synthesized by such a primer contains a base sequence. A mutation is introduced. The binding activity of the mutant antibody with the amino acid substitution to the antigen is determined by the method described above. By measuring and evaluating, mutant FR sequences with desired properties can be selected (Cancer Re s., (1993) 53, 851-856).

[0095] Furthermore, transgenic animals possessing the entire repertoire of human antibody genes (international public Open WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1 (See 996 / 033735) can be used as an immunized animal, and the desired human antibodies can be obtained by DNA immunization.

[0096] Furthermore, we also know of a technique to obtain human antibodies by panning using a human antibody library. For example, the V region of a human antibody is displayed as a single-chain antibody (scFv) on a phage display. Phages expressing scFv, which binds to the antigen, are selected. It is possible. By analyzing the genes of selected phages, human antibodies that bind to antigens can be identified. The DNA sequence encoding the V region can be determined. After determining the DNA sequence of scFv that binds to the antigen, After fusing the V region sequence in frame with the sequence of the desired human antibody C region, appropriate development An expression vector can be created by inserting it into the existing vector. The gene encoding the human antibody is introduced into suitable expression cells as described above and expressed. The human antibody is obtained by doing so. These methods are already publicly known (International Publication WO19). 92 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 00 (See 1438, WO1995 / 015388).

[0097] Furthermore, Bernasconi et al. (Science (2002) 298, 2199-) proposed a method for obtaining antibody genes. 2202) or B cell cloning as described in WO2008 / 081008 (code of each antibody) Sequence identification and cloning, isolation thereof, and their respective antibodies (especially IgG1, IgG2, Methods such as the use of expression vectors for producing IgG3 or IgG4 are, in addition to the above. It can be used as appropriate.

[0098] EU numbering and Kabat numbering According to the method used in this invention, the amino acid positions assigned to the CDR and FR of the antibody This is defined according to Kabat (Sequences of Proteins of Immunological Inter). est (National Institute of Health, Bethesda, Md., 1987 and 1991). In the specification, if the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino region of the variable region The acids followed the Kabat numbering, and the amino acids in the constant region corresponded to the amino acid positions in Kabat. It is represented according to EU numbering.

[0099] FcRn Unlike the Fcγ receptor, which belongs to the immunoglobulin superfamily, human FcRn has a structure In terms of structure, it is structurally similar to major histoincompatibility complex (MHC) class I polypeptides. It has 22 to 29% sequence identity with the MHC molecule of 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 embodied transmembrane α or heavy chains, like MHC. The α chain of FcRn consists of three extracellular domains (α1, α2, α3), and a short cytoplasmic domain. The FcRn tethers proteins to the cell surface. The α1 and α2 domains are located within the Fc region of the antibody. 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 it is a precursor to IgG from mother to fetus. It is involved in movement. In addition, in the small intestine of rodent neonates that express FcRn, when FcRn is ingested... It is involved in the migration of maternal IgG from or into milk across the brush border epithelium. FcRn is present in numerous species. It is expressed in numerous other tissues and various endothelial cell lines. It is expressed in human adult blood vessels. It is also expressed in the endothelium, muscular vascular system, and hepatic sinusoidal capillaries. FcRn binds to IgG and By recycling it into serum, it plays a role in maintaining plasma concentrations of IgG. It is thought that the binding of FcRn to IgG molecules is usually strictly pH-dependent, and the optimal binding is... This reaction is observed in the acidic pH range below 7.0.

[0101] A polypeptide containing the signal sequence represented by sequence number 4 (FcRn; NP_004098.1) is used before The human FcRn used as the progenitor is found in vivo (Sequence ID: 5(beta2-microglobulin;NP_004039.1) Human β2-microglobulin (whose polypeptide containing the signal sequence is described in [reference]) It forms a complex with β2-microglobulin, as will be shown later in the reference examples. The soluble human FcRn that forms the body is produced using a standard recombinant expression method. This is the case for soluble human FcRn that forms a complex with β2-microglobulin. The binding activity of the Fc region of the invention can be evaluated. In the present invention, unless otherwise specified, human FcRn refers to a form that can bind to the Fc region of the present invention, for example, human FcRn and human β2- One example is a complex with microglobulin.

[0102] FcRn binding domain The antigen-binding molecule of the present invention has an FcRn-binding domain. The FcRn-binding domain is antigen-binding The molecule is not particularly limited as long as it has binding activity to FcRn in the acidic pH range, and also directly It may be a domain that has binding activity to FcRn indirectly or directly. The main focus is on, for example, IgG-type immunoglobulins that have direct binding activity to FcRn. Fc region, albumin, albumin domain 3, anti-FcRn antibody, anti-FcRn peptide, anti-FcRn foot IgG or albumin molecules that have binding activity to FcRn, such as scaffold molecules, or indirectly to FcRn. Molecules that bind to the compound are preferred examples. In the present invention, in the acidic pH range and the neutral pH range In this case, a domain having binding activity to FcRn is preferred. This domain is pre-configured If the domain has binding activity to FcRn in the acidic pH range, it can be used as is. It may be used. The domain in question has no or weak binding activity to FcRn in the acidic pH range. In some cases, the amino acids in the antigen-binding molecule can be modified to confer binding activity to FcRn. It is possible. In addition, a domerant that has prior binding activity to FcRn in the pH acidic range can be used. The amino acids in the in may be modified to enhance FcRn binding activity. The modification of the acid compares the binding activity to FcRn in the acidic pH range before and after amino acid modification. By doing so, it is possible to find a modification that meets the objective.

[0103] The FcRn binding domain is preferably a region that directly binds to FcRn. A preferred example of this is the Fc region of an antibody. However, albumin The region capable of binding to polypeptides that have binding activity to FcRn, such as IgG, is albumin or I It is possible to indirectly bind to FcRn via gG, etc. Therefore, in the present invention, Fc As the Rn-binding region, a region that binds to a polypeptide having binding activity with FcRn is preferably used. It can be used. The Fc region contains an amino acid sequence derived from the constant region of the antibody heavy chain. The Fc region is Hinge regions of papain cleavage sites in approximately 216 amino acids represented by EU numbering. From the N-terminus of the region, the portion of the antibody's heavy chain constant region that includes the hinge, CH2 and CH3 domains. That is the case.

[0104] The binding activity of the FcRn binding domain in the present invention to FcRn, particularly human FcRn, is the binding activity As stated in the section on properties, it can be measured by methods known to those skilled in the art, Other conditions besides pH can be appropriately determined by those skilled in the art. Antigen binding of antigen-binding molecules Synergistic activity and human FcRn binding activity are related by KD (Dissociation constant) and apparent KD( Apparent dissociation constant (kd), the dissociation rate (Dissociation Dissociation rate (dissociation rate), or apparent kd (apparent dissociation rate) These can be evaluated as follows. These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare, scatchard plots, flow cytometers, etc., may be used.

[0105] Other conditions besides pH when measuring the binding activity of the FcRn-binding domain to FcRn can be determined by those skilled in the art. It is possible to choose, and is not particularly limited. For example, as described in WO2009 / 125825 The MES buffer can be measured under conditions of 37°C. Furthermore, the FcRn-bound dendrogen of the present invention can be measured. The binding activity to the main FcRn can be measured by methods known to those skilled in the art. For example, it can be measured using Biacore (GE Healthcare), etc. FcRn binding domain and FcR The measurement of n binding activity is performed using the FcRn binding domain or the antigen binding of the present invention containing the FcRn binding domain. To the chip immobilized with the composite molecule or FcRn, the FcRn or FcRn-binding domain, respectively, The antigen-binding molecule of the present invention, which contains an FcRn binding domain, is evaluated by flowing it as an analyte. It is worthwhile.

[0106] The conditions under which the FcRn binding domain contained in the antigen-binding molecule of the present invention has binding activity to FcRn The acidic pH range usually refers to a pH of 4.0 to 6.5. Preferably, it refers to a pH of 5.5 to 6.5. Particularly preferred is a pH of 5.8 to 6.0, which is close to the pH in early endosomes in living organisms. The temperature used for the conditions is 10°C, which is the binding affinity between the FcRn binding domain and FcRn. The evaluation may be performed at any temperature of ~50°C. Preferably, the binding of the FcRn binding domain to human FcRn. A temperature range of 15°C to 40°C is used to determine the affinity. More preferably, Any of the following temperatures: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35℃ Similarly, at any temperature between 20°C and 35°C, the binding of the FcRn-binding domain to FcRn occurs. Used to determine affinity. The temperature of 25°C is not limiting to the embodiments of the present invention. This is just one example.

[0107] According to Yeung et al. (J. Immunol. (2009) 182, 7663-7671), the human IgG1 of the natural human type The binding activity to FcRn is KD 1.7 μM in the acidic pH range (pH 6.0), but the activity is lower in the neutral pH range. Almost no detection is possible. Therefore, in a preferred embodiment, human under conditions in the acidic pH range. The binding activity to FcRn is KD 20 μM or stronger, and under neutral pH conditions, it binds to human FcRn. Human F under acidic pH conditions, containing an antigen-binding molecule with binding activity equivalent to that of natural human IgG. The antigen-binding molecule of the present invention having binding activity to cRn may be used. More preferred embodiment In this case, under acidic pH conditions, the human FcRn binding activity is KD 2.0 μM or stronger than that of an antigen. The antigen-binding molecule of the present invention, which includes a binding molecule, may be used. In a more preferred embodiment, This refers to antigen-binding molecules with human FcRn binding activity of KD 0.5 μM or stronger under acidic pH conditions. The above KD values ​​can be used. The above KD values ​​are from The Journal of Immunology (2009) 182: 7663-7 The method described in 671 (immobilizing the antigen-binding molecule on a chip and flowing human FcRn as an analyte) Determined by (su).

[0108] In the present invention, an Fc region having binding activity to FcRn under acidic pH conditions is preferred. The domain in question has prior binding activity to FcRn under acidic pH conditions. If it is in the Fc region, it can be used as is. The domain in question is in the acidic pH range relative to FcRn. If the binding activity is absent or weak, the amino acids in the antigen-binding molecule can be modified. A Fc region having binding activity to the desired FcRn can be obtained, but the amino acids in the Fc region By modifying it, it can have the desired binding activity to FcRn under acidic pH conditions, or Enhanced Fc regions can also be suitably obtained. Amino acid modification alters the binding activity to FcRn under acidic pH conditions before and after amino acid modification. This can be found by comparison. Known, as described later in the section on amino acid modifications in this specification. Those skilled in the art can use the method to modify amino acids as appropriate.

[0109] The antigen-binding molecule of the present invention contains Fc, which has binding activity to FcRn under acidic pH conditions. The region can be obtained by any method, but specifically, it is used as the starting Fc region. By modifying the amino acids of human IgG-type immunoglobulins, the effect on FcRn under acidic pH conditions is achieved. FcRn-binding domains with or enhanced binding activity can be obtained. Examples of the Fc region of IgG-type immunoglobulins include human IgG (IgG1, IgG2, IgG3, and Examples include the Fc region of IgG4 and its variants. Modification to other amino acids is possible with pH acid It has binding activity to FcRn under conditions of the sexual region, or under conditions of the acidic region, it binds to human FcRn. Any amino acid at any position can be modified as long as it enhances the binding activity. If the offspring contains the Fc region of human IgG1 as its Fc region, then under acidic pH conditions, the FcRn is... This modification includes an effect that enhances the binding activity of the starting Fc region of human IgG1. It is preferable that such a modification is possible with amino acids, for example, WO2000 / 042072 As indicated in the EU numbering, 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 No acids are preferred examples. Similarly, examples of amino acids that can be modified in this way include WO20. As noted in 02 / 060919, the EU numbering is 251st, 252nd, 254th, and 25 5th, 256th, 308th, 309th, 311th, 312th, 385th, 386th, 387th, 389th, 428th, 433rd The amino acids at positions 434 and / or 436 are also preferred. Furthermore, such modifications Possible amino acids are represented by EU numbering, as described in WO2004 / 092219. Other amino acids that can be modified include those at positions 250, 314, and 428. As amino acids, they are represented by EU numbering, for example, as described in WO2010 / 045193. The 251st, 252nd, 307th, 308th, 378th, 428th, 430th, 434th and / or 436th A Mino acids are also preferred. By modifying these amino acids, the F of IgG-type immunoglobulins can be obtained. Under acidic pH conditions in the c region, binding to FcRn is enhanced.

[0110] For example, by using these amino acid modifications individually or in combination, Therefore, it is possible to enhance the binding of the Fc region of IgG to FcRn in the acidic pH range, The amino acid modifications introduced are not particularly limited, and the effect is to improve plasma retention. Within limits, any amino acid modification may be introduced.

[0111] Furthermore, in one non-limiting aspect of the present invention, the FcRn binding domain is FcRn, particularly human FcRn. Antigen-binding domains having binding activity may also be used as appropriate. As described above, the present invention Bispecific antibodies may also be used as antigen-binding molecules as appropriate, but when such bispecific antibodies bind One of the epitopes is an epitope present on a desired antigen other than FcRn, and the other is A bispecific antibody whose epitope is an epitope present in FcRn is preferred. The structure of a specific antibody consists of a bivalent binding domain that has a binding titer to the desired antigen and FcRn. As long as it contains a glycosylated receptor-binding domain, it is not limited to a specific structure. For example, Fc Antibody structures such as IgG-type antibodies with linked regions can also be used, as well as "scFv2(single ch "ain Fv 2)", "diabody", or "F(ab')2" can also be suitably used. IgG type antibody When antibody structures like this are used, the glycosylation domain can also be included in the Fc region, It may also be included in the FcRn binding domain and / or antigen binding domain. When structures like "le chain Fv 2)", "diabody", or "F(ab')2" are used The carbohydrate-binding domain may also be included in the FcRn-binding domain and / or antigen-binding domain. ru.

[0112] When such bispecific antibodies are used, the FcRn binding domain is "ion concentration The binding to FcRn changes depending on the ion concentration conditions, as described later in the section on "degree conditions". An FcRn binding domain may be used as appropriate. That is, in one non-limiting aspect of the present invention, " As described later in the section on "Conditions for ON concentration," the binding of the FcRn binding domain to FcRn is metal The FcRn binding domain exhibits changes in binding activity to FcRn depending on ion concentration and pH conditions. It can be used.

[0113] Ion concentration conditions (1) Conditions for metal ion concentration In one non-limiting aspect of the present invention, ion concentration refers to metal ion concentration. "ON" refers to alkali metals excluding hydrogen, Group I metals such as copper, alkaline earth metals, and sub-metals. Group II elements such as lead, Group III elements excluding boron, Group IV elements excluding carbon and silicon, iron elements, and platinum elements. Elements belonging to subgroups A of groups VIII, V, VI, and VII, as well as antimony, bismuth, and polypropylene This refers to ions of metallic elements such as rhonium. Metal atoms release valence electrons to become positive ions. Metals possess a property called ionization tendency. Metals with a high ionization tendency are chemically... It is considered to be highly active.

[0114] Calcium ions are an example of metal ions suitable for use in this invention. The body is involved in regulating many biological phenomena, including the contraction of muscles such as skeletal muscle, smooth muscle, and cardiac muscle. Activation of leukocyte motility and phagocytosis, activation of platelet deformation and secretion, and activation of lymphocytes Activation of mast cells such as sexualization and histamine secretion, catecholamine α receptors and acetylcholine Cellular responses mediated by phosphorus receptors, exocytosis, and neurotransmitters from neuronal terminals Calcium ions are involved in release, axonal flow in neurons, etc. As an ON receptor, it possesses multiple calcium ion binding sites, suggesting a common origin in molecular evolution. Troponin C, calmodulin, parvalbumin, myosin, which are thought to have originated from these substances. Chains and other similar structures are known, and many of their connecting motifs are also known. For example, the cadheridome In, the EF domain contained in calmodulin, the C2 domain contained in Protein kinase C, Gla domain contained in the blood coagulation protein FactorIX, Asialoglycoprotein Rece C-type lectins found in pters and mannose receptors, and A-domains found in LDL receptors. Annexin, thrombospongin type 3 domain, and EGF-like domain are well known. .

[0115] In this invention, when the metal ion is a calcium ion, the calcium ion concentration The conditions for this include low calcium ion concentration and high calcium ion concentration. The binding activity changes depending on the calcium ion concentration conditions, specifically in low calcium ions. The binding activity of antigen-binding molecules to the antigen varies depending on the conditions of concentration and high calcium ion concentration. This refers to a change in sex. For example, the reaction to an antigen under conditions of low calcium ion concentration. The binding activity of the antigen-binding molecule is greater than the binding activity of the antigen under conditions of high calcium ion concentration. One example is when the binding activity of the antigen-binding molecule is higher. Also, in cases of high calcium ion concentration... Conditions where the calcium ion concentration is lower than the binding activity of the antigen-binding molecule to the antigen under the given conditions Another example is when the binding activity of the antigen-binding molecule to the antigen in the given situation is higher.

[0116] In this specification, high calcium ion concentration is not limited to a single, unambiguous value. However, the concentration may preferably be selected from between 100 μM and 10 mM. In some embodiments, the concentration may be selected from between 200 μM and 5 mM. Also, in different embodiments, This can be a concentration selected between 500 μM and 2.5 mM, or in other embodiments, 200 μM. The concentration can be selected from between 2 mM. Furthermore, it can be selected from between 400 μM and 1.5 mM. It can also be at concentrations close to those of calcium ions in plasma (blood) within the body, especially at 50. A concentration selected from between 0 μM and 2.5 mM is preferred.

[0117] In this specification, the term "low calcium ion concentration" is not limited to a single, unambiguous value. However, the concentration may preferably be selected from between 0.1 μM and 30 μM. In some embodiments, the concentration may be selected from between 0.2 μM and 20 μM. Also, in different embodiments... This can be a concentration selected from between 0.5 μM and 10 μM, or in other embodiments from 1 μM to 5 μM. It could also be a concentration selected from between M. Furthermore, it could be a concentration selected from between 2 μM and 4 μM. This is also possible. In particular, a concentration of 1 μM, which is close to the concentration of ionized calcium in early endosomes in living organisms. A concentration selected from between 5 μM is preferred.

[0118] In the present invention, the binding activity to the antigen under low calcium ion concentration conditions is high. Lower than the binding activity to the antigen under the conditions of calcium ion concentration means that the antigen-binding molecule 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 is... This means it is weaker than the sex. Preferably, it is selected from between 0.5 μM and 10 μM of antigen-binding molecule. The binding activity to the antigen at the calcium ion concentration is selected from between 200 μM and 5 mM. This means that the binding activity to the antigen at the selected calcium ion concentration is weaker, especially Furthermore, antigen-binding activity at calcium ion concentrations in early endosomes in living organisms This means that the antigen-binding activity is weaker than that at calcium ion concentrations in plasma within the body. Specifically, at calcium ion concentrations selected from between 1 μM and 5 μM for antigen-binding molecules Calcium ion concentrations selected from between 500 μM and 2.5 mM for antigen binding activity. This means that the binding activity to the antigen is weaker than that of [another device].

[0119] The binding activity of the antigen-binding domain of the present invention to the antigen changes depending on the metal ion concentration conditions. Whether or not it is compounded can be determined using known measurement methods, such as those described in the section on binding activity above. This can be determined by using it. For example, under conditions of low calcium ion concentration Higher calcium content than the antigen-binding activity of the antigen-binding molecule containing the antigen-binding domain of the invention. The binding of an antigen-binding molecule containing the antigen-binding domain to an antigen under certain ion concentration conditions. To confirm that the combined activity changes more significantly, low calcium ion concentration and high calcium Antigen-binding molecules containing an antigen-binding domain for antigens under conditions of high calcium ion concentration. The binding activity of each is compared.

[0120] Furthermore, in the present invention, "binding activity to antigens under low calcium ion concentration conditions" The expression "the binding activity to the antigen is lower than that under conditions of high calcium ion concentration." This is because the antigen-binding activity of the antigen-binding molecule is low under high calcium ion concentration conditions. It can also be described as having higher binding activity to the antigen under specific calcium ion concentration conditions. In this invention, the term "results in antigens under low calcium ion concentration conditions" is used. "The combined activity is lower than the binding activity to the antigen under conditions of high calcium ion concentration" is translated as "low Antigen binding ability under calcium ion concentration conditions is under high calcium ion concentration conditions. It may also be stated that it is "weaker than the binding ability to the antigen," and also that "low calcium ions" Antigen binding activity under concentration conditions vs. antigen binding under high calcium ion concentration conditions "To reduce binding activity" is changed to "To increase antigen binding ability under low calcium ion concentration conditions." When stating "weaker than the binding ability to the antigen under calcium ion concentration conditions" There are also others.

[0121] Other conditions besides calcium ion concentration when measuring the binding activity to the antigen are those that are skilled in the art. It is possible to select as appropriate, and is not particularly limited. For example, HEPES buffer, 37°C It is possible to measure this in the case. For example, using Biacore (GE Healthcare), etc. It is possible to measure the binding activity between an antigen-binding molecule containing an antigen-binding domain and the antigen. If the antigen is a soluble antigen, the measurement involves 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 modified chip. It is possible to do this, and if the antigen is a membrane-type antigen, the antigen is immobilized on a chip. By flowing antigen-binding molecules containing a binding domain as an analyte, binding activity to membrane-bound antigens is activated. It is possible to evaluate sexuality.

[0122] In the antigen-binding molecule of the present invention, under conditions of low calcium ion concentration, As long as the binding activity is weaker than the binding activity to the antigen under high calcium ion concentration conditions , Binding activity to antigens under low calcium ion concentration conditions and high calcium ion concentration The ratio of binding activity to the antigen under these conditions is not particularly limited, but preferably the ratio of binding activity to the antigen The KD (Dissociation constant) under low calcium ion concentration conditions and The ratio of KD under high calcium ion concentration conditions is KD(Ca 3μM) / KD(Ca 2 mM). The value is 2 or greater, and more preferably the KD (Ca 3μM) / KD (Ca 2 mM) value is 10 or greater. Furthermore, it is more preferable that the KD (Ca 3 μM) / KD (Ca 2 mM) value is 40 or higher. There is no particular upper limit to the value of (μM) / KD (Ca 2 mM), and is limited to the value that can be produced by those skilled in the art. Any value is acceptable, such as 400, 1000, 10000, etc. Also, KD (Ca 3 μM) / KD (Ca 1.2 mM) It can also be identified by the value of . That is, the value of KD (Ca 3μM) / KD (Ca 1.2 mM) is 2 or greater. Furthermore, it is preferable that the KD (Ca 3μM) / KD (Ca 1.2 mM) value is 10 or more, and even more preferably In other words, the KD (Ca 3μM) / KD (Ca 1.2 mM) value is 40 or higher. The upper limit of the value of Ca (1.2 mM) is not particularly limited, and should be 400 as long as it can be produced by the art. Any value is acceptable, such as 1000, 10000, etc.

[0123] When the antigen is a soluble antigen, the KD (dissociation constant) is used as the value of the binding activity to the antigen. It is possible, but if the antigen is a membrane-type antigen, apparent dissociation co It is possible to use the apparent dissociation constant (KD). The KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Using Biacore (GE Healthcare), scatchard plots, flow cytometers, etc. It is possible to be there.

[0124] Furthermore, the binding activity of the antigen-binding molecule of the present invention to the antigen under low calcium concentration conditions. And as another indicator showing the ratio of binding activity to the antigen under high calcium concentration conditions, for example, For example, the dissociation rate constant kd is also favorable. It can be used appropriately. Instead of KD (dissociation constant), kd (dissociation rate) can be used as an indicator of the ratio of binding activity. When using the degree constant, kd (dissociation rate constant) under low calcium concentration conditions for the antigen. ) and kd (dissociation rate constant) under high calcium concentration conditions is the ratio of kd (low calcium concentration The value of (condition) / kd (condition of high calcium concentration) is preferably 2 or more, and more preferably It is 5 or more, more preferably 10 or more, and 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. Furthermore, any value such as 50, 100, 200, etc., is acceptable as long as it can be manufactured according to the common technical knowledge of those skilled in the art.

[0125] When the antigen is a soluble antigen, the kd (dissociation rate constant) is used as the value of antigen-binding activity. This is possible, and if the antigen is a membrane-type antigen, the apparent dissociation rate (kD) is calculated. It is possible to use kd (dissociation rate constant), approximately Furthermore, the apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art. Yes, for example, Biacore (GE Healthcare), flow cytometers, etc. can be used. In this invention, the antigen of the antigen-binding molecule at different calcium ion concentrations When measuring the binding activity to [substance], it is preferable to keep all conditions the same except for the calcium concentration. stomach.

[0126] For example, one embodiment of the present invention is the antimicrobial activity under low calcium ion concentration conditions. The binding activity to the source is greater than the binding activity to the antigen under high calcium ion concentration conditions. A low antigen-binding domain or antibody includes the following steps (a) to (c) and This can be obtained by antibody screening. (a) Obtain the antigen-binding activity of the antigen-binding domain or antibody under low calcium concentration conditions. The process, (b) Obtain the antigen-binding activity of the antigen-binding domain or antibody under high calcium concentration conditions. The process, and (c) Antigen binding activity under low calcium concentration conditions is different from that under high calcium concentration conditions. A step of selecting an antigen-binding domain or antibody with lower antigen-binding activity than the specified antigen-binding activity.

[0127] Furthermore, one embodiment of the present invention provides an antimicrobial agent under low calcium ion concentration conditions. The binding activity to the source is greater than the binding activity to the antigen under high calcium ion concentration conditions. A low antigen-binding domain or antibody includes the following steps (a) to (c) and These can be obtained by screening antibodies or libraries thereof. (a) Antigen-binding domain or antibody or their respective under high calcium concentration conditions The process of bringing Ibrali into contact with the antigen, (b) The antigen-binding domain or antibody bound to the antigen in step (a) is subjected to low calcium concentration conditions The process of placing it below, and (c) A step of isolating the antigen-binding domain or antibody dissociated in step (b).

[0128] Furthermore, one embodiment of the present invention is the antigen under low calcium ion concentration conditions. The binding activity to it is greater than the binding activity to the antigen under high calcium ion concentration conditions. A low antigen-binding domain or antibody is an antigen-binding domain or antibody that includes the following steps (a) to (d) It can be obtained by screening antibodies or libraries thereof. (a) Under low calcium concentration conditions, the antigen-binding domain or antibody library is brought into contact with the antigen. The process of (b) A step of selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a), (c) The antigen-binding domain or antibody selected in step (b) is subjected to high calcium concentration conditions. The process of combining with the original, and (d) A step of isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0129] Furthermore, one embodiment of the present invention provides an antimicrobial agent under low calcium ion concentration conditions. The binding activity to the source is greater than the binding activity to the antigen under high calcium ion concentration conditions. Antigen-binding domains or antibodies with low levels of binding are screened using a screening method that includes the following steps (a) to (c). Therefore, it may be acquired. (a) A column on which an antigen has been immobilized, with an antigen-binding domain or antibody under high calcium concentration conditions. The process of bringing Ibrali into contact, (b) The antigen-binding domain or antibody bound to the column in step (a) is subjected to low calcium concentration conditions The process of eluting from the column below, and (c) A step of isolating the antigen-binding domain or antibody eluted in step (b).

[0130] Furthermore, one embodiment of the present invention provides an antimicrobial agent under low calcium ion concentration conditions. The binding activity to the source is greater than the binding activity to the antigen under high calcium ion concentration conditions. Antigen-binding domains or antibodies with low levels of binding are screened using a screening method that includes the following steps (a) to (d). Therefore, it may be acquired. (a) An antigen-binding domain or antibody is attached to a column with the antigen immobilized under low calcium concentration conditions. The process of passing through Ibrali (b) A process to recover the antigen-binding domain or antibody that was eluted in step (a) without binding to the column. To the extent, (c) The antigen-binding domain or antibody recovered in step (b) is subjected to high calcium concentration conditions. The process of combining with the original, and (d) A step of isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0131] Furthermore, one embodiment of the present invention provides an antimicrobial agent under low calcium ion concentration conditions. The binding activity to the source is greater than the binding activity to the antigen under high calcium ion concentration conditions. Antigen-binding domains or antibodies with low levels of binding are screened using a screening method that includes the following steps (a) to (d). Therefore, it may be acquired. (a) Under high calcium concentration conditions, the antigen-binding domain or antibody library is brought into contact with the antigen. The process of (b) A step of obtaining an antigen-binding domain or antibody bound to an antigen in step (a) above, (c) Place the antigen-binding domain or antibody obtained in step (b) under low calcium concentration conditions. Process, and (d) The antigen-binding activity in step (c) is weaker than the criteria selected in step (b) A step to isolate the yeast or antibody.

[0132] The above steps may be repeated two or more times. Therefore, according to the present invention, the above-mentioned S In the cleaning method, the process of repeating steps (a) to (c) or (a) to (d) two or more times is Furthermore, under conditions of low calcium ion concentration obtained by a screening method including The binding activity to the antigen is the binding activity to the antigen under conditions of high calcium ion concentration. A lower antigen-binding domain or antibody is provided. Steps (a) to (c) or (a) to (d) There is no particular limit to the number of repetitions, but it is usually no more than 10 times.

[0133] In the screening method of the present invention, antigen-binding dormancy under low calcium concentration conditions The antigen-binding activity of the ionized calcium or antibody is measured at an ionized calcium concentration between 0.1 μM and 30 μM. While not particularly limited as long as it is compatible, a preferred ionized calcium concentration is 0.5 μM~ Antigen binding activity between 10 μM can be observed. A more preferred ionized calcium concentration. One example is the concentration of ionized calcium in early endosomes in living organisms, specifically Antigen binding activity at concentrations of 1 μM to 5 μM can be cited. Furthermore, under high calcium concentration conditions... The antigen-binding domain or antigen-binding activity of the antibody in this case is measured at an ionized calcium concentration of 100 μM. While there are no particular limitations as long as the antigen-binding activity is between ~10 mM, ionized calcium is preferred. Antigen binding activity can be found in concentrations between 200 μM and 5 mM. A more preferred io As an example of calcium ionide concentration, the concentration of ionized calcium in plasma within a living organism can be cited. Specifically, we can cite the antigen-binding activity in the range of 0.5 mM to 2.5 mM.

[0134] The antigen-binding domain or the antigen-binding activity of an antibody can be measured by methods known to those skilled in the art. It is possible, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art. It is possible. The antigen-binding domain or the antigen-binding activity of the antibody is determined by KD (Dissociation constant). : Dissociation constant), Apparent dissociation constant (KD), The dissociation rate kd (dissociation rate constant), or the apparent kd (apparent kd). It is possible to evaluate these as dissociation (apparent dissociation rate constant), etc. It can be measured using methods known to the vendor, such as Biacore (GE Healthcare) and Ski. Catchered plots, FACS, etc., can be used.

[0135] In the present invention, the antigen-binding activity under high calcium concentration conditions is compared to the low calcium concentration conditions. The step of selecting an antigen-binding domain or antibody with higher antigen-binding activity in the following case is low calorie Antigen binding activity under high calcium concentration conditions vs. Antigen binding activity under high calcium concentration conditions This is equivalent to the process of selecting a lower antigen-binding domain or antibody.

[0136] Antigen binding activity under high calcium concentration conditions is different from antigen binding activity under low calcium concentration conditions. As long as the binding activity is higher, antigen binding activity under high calcium concentration conditions and low calcium concentration The difference in antigen-binding activity under 3°C conditions is not particularly limited, but preferably high calcium concentration Antigen binding activity under these conditions is more than twice as high as antigen binding activity under low calcium concentration conditions. It is more preferably 10 times or more, and 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, for example, the antigen-binding domain or It is possible to screen for antibodies. For example, antigen-binding antibodies with natural sequences. You may screen for an antigen-binding domain with a substituted amino acid sequence. You may screen for antibodies or other substances.

[0138] (2) The binding activity of the antigen-binding domain to the antigen depends on the calcium ion concentration. Changing amino acids The antigen-binding domain of the present invention screened by the aforementioned screening method or The antibody may be prepared in any way, for example, if the metal ions are at the calcium ion concentration In such cases, pre-existing antibodies and pre-existing libraries (fur) are used. (e.g., dilibraries), hybridomas obtained from animal immunization, or B cells from immunized animals Antibodies or libraries produced from these antibodies or libraries, and calcium chelates these antibodies or libraries. We introduced natural amino acids (such as aspartic acid and glutamic acid) and unnatural amino acid mutations. Antibodies or libraries (amino acids capable of chelating calcium (e.g., aspartic acid or glycerides) Libraries with a high content of lutamic acid or non-natural amino acids, or calcium in specific locations. Chelateable amino acids (e.g., aspartic acid or glutamic acid) or unnatural amino acids It is possible to use libraries with introduced mutations, etc.

[0139] As described above, the binding activity of antigen-binding molecules to the antigen is changed depending on the ion concentration conditions. As an example of an amino acid, for instance, if the metal ion is a calcium ion, then Any amino acid that forms a calcium bond motif is acceptable, regardless of its type. The motif is well known to those skilled in the art and has 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 ure. (2006) 14, 6, 1049-1058). That is, C such as ASGPR, CD23, MBR, DC-SIGN. Any known calcium-binding motif, such as type lectin, is included in the antigen-binding molecule of the present invention. Obtain. A suitable example of such a calcium-binding motif is, in addition to the above, SEQ ID NO: The calcium-binding motif included in the antigen-binding domain described in 6 can also be cited.

[0140] Furthermore, the binding activity of antigen-binding molecules to the antigen changes depending on the calcium ion concentration. As an example of amino acids that undergo metal chelating, amino acids that have metal chelating activity can also be suitably used. Examples of amino acids that have metal chelating activity include, for example, serine (Ser(S)) and threonine. Thr(T)), Asparagine (Asn(N)), Glutamine (Gln(Q)), Aspartic acid (Asp(D)) and glutamic acid (Glu(E)) are preferred examples.

[0141] The position of the antigen-binding domain containing the aforementioned amino acids is not limited to a specific position, and calcium As long as the binding activity of antigen-binding molecules to the antigen is changed by the um ion concentration conditions, It can be located at any position within the heavy chain variable region or light chain variable region that forms the antigen-binding domain. In other words, the antigen-binding domain of the present invention can be obtained by depending on the calcium ion concentration. The heavy chain's antigen-binding domain contains amino acids that alter the binding activity of antigen-binding molecules to the source. It can be obtained from a library mainly consisting of antigen-binding molecules with different sequences. In another non-limiting embodiment, the antigen-binding domain of the present invention is such that the amino acid is part of the heavy chain. The CDR3 library is primarily composed of antigen-binding molecules with different sequences. It may be obtained. In other non-limiting embodiments, the antigen-binding domain of the present invention is the amino acid These are represented by the Kabat numbering of the heavy chain CDR3 at positions 95, 96, 100a and / or 101. It is obtained from a library mainly consisting of antigen-binding molecules with different sequences. obtain.

[0142] Furthermore, in one non-limiting aspect of the present invention, the antigen-binding domain of the present invention is a calcium ion The amino acids in the light chain change the binding activity of antigen-binding molecules to the antigen depending on the concentration conditions. It mainly consists of antigen-binding molecules with different sequences contained within the antigen-binding domain. It can be obtained from Blar. In another embodiment, the antigen-binding domain of the present invention is the ami The rhyncholytic acid is mainly composed of antigen-binding molecules with different sequences that are contained in the CDR1 of the light chain. It can be obtained from Brari. In other embodiments, the antigen-binding domain of the present invention is the ami The no acids are located at positions 30, 31, and / or 32 in the Kabat numbering of CDR1 in the light chain. It can be obtained from a library mainly consisting of antigen-binding molecules with different sequences.

[0143] In another non-limiting embodiment, the antigen-binding domain of the present invention is such that the amino acid residue is a light chain From a library mainly consisting of antigen-binding molecules with different sequences contained in CDR2 It can be obtained. In other embodiments, the amino acid residue is the Kabat number of CDR2 of the light chain. The ly is mainly composed of antigen-binding molecules with different sequences that are located at position 50, represented by the 'g'. A burari will be provided.

[0144] In yet another non-limiting embodiment, the antigen-binding domain of the present invention is such that the amino acid residue is a light chain From a library mainly consisting of antigen-binding molecules with different sequences contained in CDR3 It can be obtained. In other embodiments, the antigen-binding domain of the present invention is such that the amino acid residue Antigens with different sequences located at position 92 of the CDR3 in the light chain, as represented by Kabat numbering. It can be obtained from a library consisting mainly of binding molecules.

[0145] Furthermore, the antigen-binding domain of the present invention has an amino acid residue which is the CD of the light chain described above. Two or three CDRs selected from R1, CDR2, and CDR3 contain different sequences from each other. In a different aspect of this invention, antigen-binding molecules can be obtained from a library mainly consisting of such molecules. Furthermore, the antigen-binding domain of the present invention is such that the amino acid residue is Kabat numbered in the light chain. Included in one or more of the 30th, 31st, 32nd, 50th and / or 92nd positions represented by It can be obtained from a library mainly consisting of antigen-binding molecules with different sequences.

[0146] Furthermore, these amino acid residues form calcium-binding motifs, and / or The binding activity of antigen-binding molecules to the antigen changes depending on the calcium ion concentration conditions. These amino acid residues may be present individually, or two or more of these amino acids may be present in combination. It may be included as such. Furthermore, it has multiple calcium ion binding sites and is common in molecular evolution. Troponin C, calmodulin, parvalbumin, myo, which are thought to have originated from this source Syn light chains are known, and light chains CDR1, CDR2 and / Alternatively, CDR3 can be designed. For example, for the above purpose, the cadherin domain, Lumodulin contains EF domain, Protein kinase C contains C2 domain, blood coagulation The Gla domain contained in the protein FactorIX, the Asialoglycoprotein receptor, and C-type lectins contained in mannose receptors, A-domains contained in LDL receptors, and anesthesia Syn, thrombospongin type 3 domain, and EGF-like domain may be used as appropriate.

[0147] In one aspect of the present invention, "the binding of antigen-binding molecules to an antigen is determined by the ion concentration conditions." A framework that pre-includes "at least one amino acid residue that alters the synergistic activity." The heavy chain variable region selected as the sequence and the randomized variable region sequence library were created. By combining the light chain variable region, the present invention provides a plurality of antigens with different sequences. The antigen-binding domain of the present invention can be obtained from a library containing binding molecules. As a non-limiting example, if the ion concentration is calcium ion concentration, for example, the arrangement Heavy chain variable region described in number: 7 (6RL#9-IgG1) or sequence number: 8 (6KC4-1#85-IgG1) Combining a region sequence with a light chain variable region created as a randomized variable region sequence library. The following libraries are preferred. Also, as a randomized variable region sequence library, Instead of the generated light chain variable region, select from among the light chain variable regions having germline sequences. It can be produced by selecting as appropriate. For example, sequence number: 7 (6RL#9-IgG1) or sequence It possesses the heavy chain variable region sequence and germline sequence described in number: 8 (6KC4-1#85-IgG1). A library combining a variable light chain region is a preferred example.

[0148] In this specification, "library" means multiple antigen-binding molecules or multiple antigen-binding molecules containing multiple antigen-binding molecules. A number of fused polypeptides, or nucleic acids, polynucleotides that encode these sequences. U. Multiple antigen-binding molecules or multiple fusion molecules containing antigen-binding molecules contained in the library. The lipeptide sequence is not a single sequence, but rather a series of antigen-binding molecules or antigen-binding molecules with different sequences. It is a fusion polypeptide containing compound molecules. Bacteriophages are an example of such a library. Methods for presenting fusion polypeptides containing antibody fragments on a surface are known in the art. For example, WO1992001047 and described herein. Also, WO1992020791, WO199300 Related methods are described in 6213, WO1993011236 and 1993019172, and those skilled in the art will understand The methods can be used as appropriate. Other publicly available literature (HR Hoogenboom & G. Winter r (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236) artificially rearranged variable region genes in response to various antigens presented on the phage surface. The identification of antibodies by repertoire is shown.

[0149] (3) Conditions for hydrogen ion concentration Furthermore, in one aspect of the present invention, the ion concentration condition is the hydrogen ion concentration condition or pH This refers to the condition. In this invention, the condition for the concentration of protons, i.e., the nuclei of hydrogen atoms, is the hydrogen index. This is treated as synonymous with the (pH) condition. If the activity of hydrogen ions in an aqueous solution is represented by aH+, then p H is defined as -log10aH+. The ionic strength in aqueous solution is (for example, 10- 3 If it is lower, aH+ This 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 0-14, in pure water aH+=aOH=10-7. In this case, pH=7 is neutral, and pH 7 is A solution with a pH lower than 7 is acidic, and a solution with a pH greater than 7 is alkaline.

[0150] In this invention, when pH conditions are used as ion concentration conditions, the pH conditions As conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range and low hydrogen ion concentration or high One example is a pH, or rather, a pH in the neutral range. The fact that binding activity changes depending on the pH condition means that high Conditions of low 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 antigen-binding molecules to an antigen due to differences in the number of antigens. For example, The binding activity of antigen-binding molecules to antigens is higher under neutral pH conditions than under acidic pH conditions. One example is when the binding activity of the antigen-binding molecule to the antigen is higher. Also, in the pH neutral range. The binding activity of the antigen-binding molecule to the antigen under certain conditions is greater than the binding activity of the antigen under conditions in the pH acidic range. Another example is when the binding activity of the antigen-binding molecule is higher than that of the antigen-binding molecule.

[0151] In this specification, the pH neutral range is not limited to a single, unambiguous value, but In some cases, the pH can be selected from between 6.7 and 10.0. In another embodiment, the pH can be selected from between 6.7 and 9.5. It can be selected from between. Also, in a different embodiment, it can be selected from between pH 7.0 and pH 9.0, and In that embodiment, a pH can be selected from between pH 7.0 and pH 8.0. In particular, pH in plasma (blood) in living organisms A pH of 7.4, close to the specified value, is preferred.

[0152] In this specification, the pH acidic range is not limited to a single, unambiguous value, but is preferable The pH can be selected from between pH 4.0 and pH 6.5. In another embodiment, the pH can be selected from between pH 4.5 and pH 6.5. It can be selected from among. In other embodiments, it can be selected from between pH 5.0 and pH 6.5, and other Depending on the embodiment, a pH between 5.5 and 6.5 may be selected. In particular, in early endosomes in living organisms A pH of 5.8, close to the concentration of calcium oncide, is preferred.

[0153] In the present invention, under conditions of high hydrogen ion concentration or low pH (pH acidic range) of antigen-binding molecules The binding activity to the antigen is low under conditions of low hydrogen ion concentration or high pH (pH neutral range). Lower than the binding activity to the antigen means that the pH is selected from between pH 4.0 and pH 6.5 of the antigen-binding molecule. The binding activity to the antigen is selected from pH 6.7 to pH 10.0. This means that the binding activity is weaker than the binding activity. Preferably, the pH of the antigen-binding molecule is between 4.5 and 6.5. The binding activity to the antigen at the selected pH is the same as the binding activity to the antigen at the selected pH between pH 6.7 and pH 9.5. This means that the binding activity to the antigen is weaker than that of the antigen, and more preferably, the pH of the antigen-binding molecule is from 5.0 to p The binding activity to the antigen at a pH selected from between H6.5 is selected from between pH7.0 and pH9.0. This means that the binding activity to the antigen at the specified pH is weaker. Furthermore, preferably the antigen-binding component The binding activity to the antigen at a pH selected between pH 5.5 and pH 6.5 is between pH 7.0 and pH 8.0. This means that the binding activity to the antigen is weaker than that at a pH selected from among the options. Particularly preferred This means that the antigen-binding activity at pH in early endosomes in vivo is affected by the pH in plasma in vivo. This means that the antigen-binding activity is weaker than that of the antigen-binding molecule at pH 5.8. This means that the binding activity 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 the antigen change depending on the pH conditions? Whether or not this is determined by using known measurement methods, such as those described in the section on binding activity above. Therefore, it can be determined. That is, the binding activity under different pH conditions in the measurement method is It is measured. For example, the antigen binding domain of the present invention is measured under conditions in the pH acid range. The antigen-binding domain of the present invention under pH neutral conditions is more important than the binding activity of the combined molecule to the antigen. To confirm that the binding activity of antigen-binding molecules containing changes to a higher level for the antigen, The binding activity of antigen-binding molecules to antigens under conditions of acidic and neutral pH ranges is relatively low. They are compared.

[0155] Furthermore, in the present invention, "under conditions of high hydrogen ion concentration or low pH, i.e., pH in the acidic range" The binding activity to the antigen is under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. The expression "lower than the binding activity to the antigen" means that the hydrogen ion concentration of the antigen-binding molecule is low or The binding activity to the antigen under high pH conditions, i.e., in the pH neutral range, is high hydrogen ion concentration or low It can also be expressed as having higher binding activity to the antigen than under pH, or acidic conditions. In this invention, "high hydrogen ion concentration or low pH, i.e., pH acidic conditions" The binding activity to the antigen is low under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. Lower than the binding activity to the antigen" is defined as "high hydrogen ion concentration or low pH, i.e., acidic pH range". The binding activity to the antigen under these conditions is low hydrogen ion concentration or high pH, ​​i.e., in the pH neutral range. It may also be stated that "its binding ability to the antigen is weaker than under certain conditions," and also that "high hydrogen The binding activity to the antigen at low ion concentrations or low pH, i.e., in the pH acidic range, is low hydrogen ion. To reduce the binding activity to the antigen under conditions of high concentration or high pH, ​​i.e., pH neutral range. " to "binding activity to antigens under conditions of high hydrogen ion concentration or low pH, i.e., pH acidic range" The ability to bind to antigens under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutrality. It may also be written as "to weaken the effect."

[0156] Conditions other than hydrogen ion concentration or pH when measuring binding activity to an antigen are, as can be determined by those skilled in the art. It is possible to select as appropriate, and is not particularly limited. For example, HEPES buffer, 37°C Measurement is possible under certain conditions. For example, using Biacore (GE Healthcare), etc. It is possible to measure the binding activity of an antigen-binding molecule containing an antigen-binding domain to the antigen. Sex measurement involves, if the antigen is a soluble antigen, fixing the antigen-binding molecule containing the antigen-binding domain. The binding activity to soluble antigens is evaluated by flowing the antigen as an analyte through a standardized chip. It is possible to evaluate the antigen, and if the antigen is a membrane-type antigen, the antigen is immobilized on a chip and then the antibody By flowing an antigen-binding molecule containing the protobinding domain as an analyte, binding to membrane-bound antigens occurs. It is possible to evaluate the activity.

[0157] In the antigen-binding molecule of the present invention, under conditions of high hydrogen ion concentration or low pH, i.e., pH in the acidic range. The binding activity to the antigen in this case is under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral 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 acidity Binding activity to antigens under sexual conditions and low hydrogen ion concentration or high pH, ​​i.e., pH The ratio of binding activity to the antigen under sexual conditions is not particularly limited, but preferably the antigen KD (Dissociation) under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range Constant (dissociation constant) and under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. The KD ratio, KD(pH5.8) / KD(pH7.4), is 2 or greater, and more preferably KD(pH5. 8) The value of KD (pH7.4) is 10 or more, and more preferably the value of KD (pH5.8) / KD (pH7.4) is 4 It is greater than or equal to 0. There is no particular upper limit to the KD (pH5.8) / KD (pH7.4) value, and it is subject to the skills of those skilled in the art. Any value is acceptable, such as 400, 1000, 10000, etc., as long as it can be manufactured.

[0158] When the antigen is a soluble antigen, the KD (dissociation constant) is used as the value of the binding activity to the antigen. It is possible, but if the antigen is a membrane-type antigen, apparent dissociation co It is possible to use the apparent dissociation constant (KD). The KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Using Biacore (GE Healthcare), scatchard plots, flow cytometers, etc. It is possible to be there.

[0159] Furthermore, under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range, of the antigen-binding molecule of the present invention The binding activity to the antigen and the conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. Other indicators that show the ratio of binding activity to an antigen include, for example, the dissociation rate constant kd( The dissociation rate constant can also be suitably used. When using kd (dissociation rate constant) instead of KD (dissociation constant) as an index to show the ratio, the antigen In contrast, kd (dissociation rate constant) under conditions of high hydrogen ion concentration or low pH, i.e., in the pH acidic range. and the ratio of kd (dissociation rate constant) under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. The value of kd(under conditions in the acidic pH range) / kd(under conditions in the neutral pH range) is preferably is 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably The value is 30 or higher. Kd (under acidic pH conditions) / kd (under neutral pH conditions) There is no particular upper limit to the value, and as long as it can be manufactured using the common technical knowledge of those skilled in the art, the values ​​may be 50, 100, 200. Any value is acceptable.

[0160] When the antigen is a soluble antigen, the kd (dissociation rate constant) is used as the value of antigen-binding activity. This is possible, and if the antigen is a membrane-type antigen, the apparent dissociation rate (kD) is calculated. It is possible to use kd (dissociation rate constant), approximately Furthermore, the apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art. Yes, for example, Biacore (GE Healthcare), flow cytometers, etc. can be used. In this invention, antigen-binding molecules at different hydrogen ion concentrations, i.e., pH levels, When measuring the binding activity to the antigen, all conditions except for the hydrogen ion concentration, i.e., pH, should be kept the same. It is preferable to do so.

[0161] For example, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the sexual range is low hydrogen ion concentration or high pH, ​​i.e., in pH. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the sex range are as follows: It can be obtained by screening an antigen-binding domain or antibody, including steps (a) to (c). ru. (a) A step to obtain the antigen-binding activity of an antigen-binding domain or antibody under conditions in the pH range. , (b) A step to obtain the antigen-binding activity of an antigen-binding domain or antibody under conditions in the pH neutral range. , and (c) Antigen binding activity under acidic pH conditions is equal to antigen binding activity under neutral pH conditions. A step of selecting a lower antigen-binding domain or antibody.

[0162] Furthermore, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the sexual range is low hydrogen ion concentration or high pH, ​​i.e., in pH. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the sex range are as follows: The script of the antigen-binding domain or antibody or library thereof, including steps (a) to (c) It can be obtained through training. (a) Antigen-binding domain or antibody or their library under pH neutral conditions The process of bringing the antigen into contact with the substance. (b) The antigen-binding domain or antibody bound to the antigen in step (a) above is placed under conditions in the pH range of acidic range. Process, and (c) A step of isolating the antigen-binding domain or antibody dissociated in step (b).

[0163] Furthermore, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the region is low hydrogen ion concentration or high pH, ​​i.e., pH neutral. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the region are as follows: Screen of antigen-binding domains or antibodies or libraries thereof including steps (a) to (d) It can be obtained through learning. (a) A process in which an antigen-binding domain or antibody library is brought into contact with an antigen under conditions in the pH range. To the extent, (b) A step of selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a), (c) The antigen-binding domain or antibody selected in step (b) is bound to the antigen under conditions of a neutral pH. The process of causing, (d) A step of isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0164] Furthermore, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the sexual range is low hydrogen ion concentration or high pH, ​​i.e., in pH. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the sex range are as follows: This can be obtained by a screening method that includes steps (a) to (c). (a) A library of antigen-binding domains or antibodies on a column immobilized with the antigen under pH neutral conditions. The process of bringing them into contact, (b) The antigen-binding domain or antibody bound to the column in step (a) above is heated under conditions in the pH range. The process of eluting from the mu, and (c) A step of isolating the antigen-binding domain or antibody eluted in step (b).

[0165] Furthermore, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the sexual range is low hydrogen ion concentration or high pH, ​​i.e., in pH. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the sex range are as follows: This can be obtained by a screening method that includes steps (a) to (d). (a) A library of antigen-binding domains or antibodies on a column immobilized with the antigen under conditions of an acidic pH range. The process of passing through (b) A process to recover the antigen-binding domain or antibody that was eluted in step (a) without binding to the column. To the extent, (c) The antigen-binding domain or antibody recovered in step (b) is bound to the antigen under conditions of a neutral pH. The process of causing, (d) A step of isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0166] Furthermore, one embodiment of the present invention is a high hydrogen ion concentration or low pH, i.e., pH acidity. The binding activity to the antigen under the conditions of the sexual range is low hydrogen ion concentration or high pH, ​​i.e., in pH. Antigen-binding domains or antibodies with lower binding activity to the antigen under the conditions of the sex range are as follows: This can be obtained by a screening method that includes steps (a) to (d). (a) A process in which an antigen-binding domain or antibody library is brought into contact with an antigen under conditions of pH neutrality. To the extent, (b) A step of obtaining an antigen-binding domain or antibody bound to an antigen in step (a) above, (c) A step of placing the antigen-binding domain or antibody obtained in step (b) under conditions in the pH range of acidity, call (d) The antigen-binding activity in step (c) is weaker than the criteria selected in step (b) A step to isolate the yeast or antibody.

[0167] The above steps may be repeated two or more times. Therefore, according to the present invention, the above-mentioned S In the cleaning method, the process of repeating steps (a) to (c) or (a) to (d) two or more times is Furthermore, the screening method obtained for the antigen under conditions in the pH acidic range was also included. The antigen-binding domain has lower binding activity than the antigen-binding activity under neutral pH conditions. The antibody is provided. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited. It's not always possible, but it's usually within 10 times.

[0168] In the screening method of the present invention, high hydrogen ion concentration conditions or low pH conditions, i.e., pH acid The antigen-binding domain or antigen-binding activity of the antibody in the sexual region is determined by antigen binding at a pH between 4.0 and 6.5. While not particularly limited as long as it is active, a preferred pH is an antigen-binding activity between 4.5 and 6.6. One example is the antigen-binding activity between pH 5.0 and 6.5. Antigen-binding activity can be cited as occurring at a pH between 5.5 and 6.5. A more preferable pH is... The pH within early endosomes is cited, specifically the antigen-binding activity at pH 5.8. It is possible to also test antigens under low hydrogen ion concentration conditions or high pH, ​​i.e., in the pH neutral range. The antigen-binding activity of the binding domain or antibody is particularly important if the antigen-binding activity is between pH 6.7 and 10. While not limited to this, a preferred pH range for antigen-binding activity is between 6.7 and 9.5. Another preferred pH range is between 7.0 and 9.5 for antigen binding activity, and between 7.0 and 8.0. The antigen-binding activity can be cited. A more preferred pH is the pH in plasma in vivo. One example that can be cited is the antigen-binding activity at a pH of 7.4.

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

[0170] In the present invention, antigen formation under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range. The combined activity is greater than the antigen-binding activity under conditions of high hydrogen ion concentration or low pH, i.e., acidic pH range. The step of selecting a highly antigen-binding domain or antibody involves a high hydrogen ion concentration or low pH, i.e. Antigen binding activity under acidic pH conditions is low hydrogen ion concentration or high pH, ​​i.e., in the pH neutral range. This has the same meaning as the step of selecting an antigen-binding domain or antibody with lower antigen-binding activity than the given conditions. That is the case.

[0171] Antigen binding activity under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range, is high hydrogen ion As long as the ion concentration or the antigen-binding activity under low pH conditions, i.e., in the pH acidic range, is higher, low hydrogen Antigen binding activity and high hydrogen ion concentration under conditions of high ion concentration or high pH, ​​i.e., pH neutral range. Alternatively, the difference in antigen-binding activity under low pH, i.e., acidic pH conditions, is not particularly limited, but Or, the antigen-binding activity is high under conditions of low hydrogen ion concentration or high pH, ​​i.e., in the pH neutral range. The antigen-binding activity is more than twice the hydrogen ion concentration or the low pH, i.e., the acidic pH range. More preferably, it is 10 times or more, and 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, for example, the antigen-binding domain or It is possible to screen for antibodies. For example, antigen-binding antibodies with natural sequences. You may screen for an antigen-binding domain with a substituted amino acid sequence. You may screen for antibodies or other substances.

[0173] The antigen-binding domain of the present invention screened by the aforementioned screening method or Antibodies can be prepared in any way, for example, by using pre-existing antibodies. Existing libraries (phage libraries, etc.), hives obtained from animal immunization Antibodies or libraries produced from B cells from ridomas or immunized animals, these antibodies and Ibrali contains amino acids with a side chain pKa 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) For example, libraries with a high content of histidine or glutamic acid or non-natural amino acids, At a specific site, an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or It is possible to use libraries containing unnatural amino acid mutations, etc.

[0174] Antigens produced from hybridomas obtained from animal immunization or from B cells from immunized animals. From the binding domain or antibody, under conditions of low hydrogen ion concentration or high pH, ​​i.e., pH neutral range... Antigen-binding activity is affected by high hydrogen ion concentration or low pH, i.e., acidic pH conditions. For example, WO2009 / 12582 is a method for obtaining antigen-binding domains or antibodies with higher synergistic activity. At least one of the antigen-binding domains or amino acids in the antibody, as described in 5, Amino acids with a chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids. The mutation is substituted or the side chain pKa is 4.0-8.0 in the antigen-binding domain or antibody. Antioxidants that have certain amino acids (such as histidine or glutamic acid) or non-natural amino acids inserted into them Suitable examples include protoconjugated molecules or antibodies.

[0175] Amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids. The location where the mutation of the no acid is introduced is not particularly limited, and the pH becomes more acidic compared to before the substitution or insertion. The antigen-binding activity at this pH range is weaker than the antigen-binding activity at 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 range) / kd (neutral range) increases, Any part is acceptable. For example, if the antigen-binding molecule is an antibody, the variable region or CDR of the antibody can be used. These are some examples of suitable amino acids. Amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and gluten) The number of amino acids that are substituted with tamic acid or non-natural amino acids, or the number of amino acids that are inserted. A person skilled in the art can appropriately determine one amino acid (e.g., hi) whose side chain pKa is 4.0-8.0. It can be substituted with stidine or glutamic acid or non-natural amino acids, and the side chain pKa is 4.0-8 A single amino acid with a value of 0 (e.g., histidine or glutamic acid) or a non-natural amino acid is inserted. It is possible to combine two or more amino acids with side chain pKas of 4.0-8.0 (for example, histidine or glycerin). It can be substituted with glutamic acid or non-natural amino acids, and the side chain pKa is 4.0-8.0. The above amino acids (e.g., histidine and glutamic acid) and non-natural amino acids can be inserted. Furthermore, amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids Amino acids that are substituted with amino acids or have a side chain pKa of 4.0-8.0 (e.g., histidine and glutamine) In addition to the insertion of acids and non-natural amino acids, deletion, addition, insertion and / or placement of other amino acids are also possible. Substitutions and other processes can occur simultaneously. Amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glycerin) (e.g., substitution with lutamic acid or non-natural amino acids, or amino acids with a side chain pKa of 4.0-8.0) Insertion of histidine or glutamic acid or non-natural amino acids is known to those skilled in the art as alanine scanni By replacing alanine in ng with histidine, etc., and using methods such as histidine scanning, This can be done with amino acids whose side chain pKa is 4.0-8.0 (for example, histidine and glutamine). Antigen-binding domains into which mutations such as substitutions or insertions of acids or non-natural amino acids are randomly introduced. Alternatively, from among the antibodies, KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / Antigen-binding molecules with a high kd (pH neutral range) value may be selected.

[0176] As mentioned above, amino acids whose side chain pKa is 4.0-8.0 (for example, histidine and glutamine) Mutations occur to acids or non-natural amino acids, and antigen-binding activity in the acidic pH range is reduced in the neutral pH range. A preferred example of an antigen-binding molecule with lower antigen-binding activity is one whose side chain pKa is 4.0. Amino acids with a -8.0 value (e.g., histidine and glutamic acid) or mutations in non-natural amino acids Antigen-binding activity in the pH neutral range is determined by amino acids whose side chain pKa is 4.0-8.0 (e.g., histidine). The antigen-binding activity in the pH neutral range is equivalent to that of the amino acids (such as glutamic acid) or non-natural amino acids before mutation. Suitable antigen-binding molecules include those whose side chain pKa is 4.0-8.0. The antigen-binding molecules after mutation of amino acids (e.g., histidine and glutamic acid) and non-natural amino acids , amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids. Having antigen-binding activity equivalent to the antigen-binding molecule before the amino acid mutation means that the pKa of its side chain is 4 Amino acids with a molecular weight of 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 set to 100%, the pKa of its side chain is 4.0-8.0. Anti-antigen binding molecules of mutated non-amino acids (e.g., histidine and glutamic acid) and non-natural amino acids The primordial binding activity is at least 10%, preferably 50%, and more preferably 80% or more. Preferably, it means 90% or more. The side chain pKa of the amino acid is 4.0-8.0 (for example) The antigen-binding activity at pH 7.4 after mutation of histidine, glutamic acid, and non-natural amino acids is Amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids The antigen-binding activity at pH 7.4 may be higher than that of the pre-mutation mino acid. Its side chain pKa should be 4.0-8. Substitution of an amino acid that is 0 (e.g., histidine or glutamic acid) or a non-natural amino acid or If the antigen-binding activity of the antigen-binding molecule decreases due to insertion, then one or The antigen-binding activity is altered by the substitution, deletion, addition, and / or insertion of multiple amino acids. Amino acids with a side chain pKa 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 acid. In the present invention, such side Amino acids with a chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids. Perform substitution, deletion, addition, and / or insertion of one or more amino acids after substitution or insertion. This also includes antigen-binding molecules whose binding activity has become equivalent as a result.

[0177] Furthermore, if the antigen-binding molecule is a substance that includes an antibody constant region, antigen-binding activity in the acidic pH range will be reduced. Another preferred embodiment of an antigen-binding molecule whose activity is lower than that of an antigen-binding molecule in the pH neutral range is an antigen One example is a method in which the constant region of the antibody contained in the binding molecule is modified. (Modified antibody) A concrete example of the steady-state region is the steady state described in sequence numbers 9, 10, 11, or 12. The following are preferred examples.

[0178] (4) The binding activity of the antigen-binding domain to the antigen is changed depending on the hydrogen ion concentration conditions. amino acids The antigen-binding domain of the present invention screened by the aforementioned screening method or The antibody may be prepared in any way, for example, if the ion concentration conditions are such that the hydrogen ion concentration conditions If the conditions are for one case or pH, the antibodies that are already present, Libraries (phage libraries, etc.), hybridomas obtained from animal immunization, and Antibodies or libraries produced from B cells of immunized animals, and these antibodies or libraries Amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids Antibodies or libraries into which acid mutations have been introduced (amino acids with a side chain pKa of 4.0-8.0 (e.g., hi Libraries and specific locations with a high content of stidine and glutamic acid, and non-natural amino acids. Amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) and unnatural amino acids It is possible to use libraries (such as those with acid mutations introduced). Amino acids that have this include natural amino acids such as histidine or glutamic acid, Histidine analog (US20090035836) or m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr ( Non-natural amino acids such as 3,5-I2-Tyr (pKa 7.21) or 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Che m. (2003) 11 (17), 3761-2768 is a preferred example. Non-natural amino acids are artificially given pKa It is known that it can be controlled (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] One non-limiting aspect of the present invention is "Anti-antigens depending on the hydrogen ion concentration conditions." A light chain containing "at least one amino acid residue that alters the binding activity of the original binding molecule" has been introduced. Combining variable regions with heavy chain variable regions created as a randomized variable region sequence library. By doing so, the present invention provides a library containing multiple antigen-binding molecules with different sequences. It can be manufactured.

[0180] An example of a non-limiting amino acid residue in question is the amino acid residue contained in CDR1 of the light chain. In addition, as a non-specific example of the amino acid residue in question, the amino acid residue contained in CDR2 of the light chain is As an example, the amino acid residue in question is found in CDR3 of the light chain. anoacid residues are also given as examples.

[0181] As described above, the amino acid residue in question is an unrestricted example of an amino acid residue included in the CDR1 of the light chain. Then, the 24th, 27th, 28th, 31st, and 3rd positions in the Kabat numbering within the light chain variable region CDR1 are The amino acid residues at positions 2 and / or 34 are also included. As an example of an unrestricted amino acid residue included in R2, the Kabat number in CDR2 of the light chain variable region The amino acid residues at positions 50, 51, 52, 53, 54, 55 and / or 56, represented by . This includes the following. Furthermore, the amino acid residue is included in the CDR3 of the light chain, and the amino acid residue is not limited. For example, positions 89, 90, 91, and 92 in the Kabat numbering within the light chain variable region of CDR3. Examples include the amino acid residues at positions 93, 94, and / or 95A. The acid residues change the binding activity of antigen-binding molecules to the antigen depending on the hydrogen ion concentration. As long as these amino acid residues are present individually, or in combination of two or more amino acids They may be included together.

[0182] In the present invention, non-limiting examples of sites substituted with histidine or unnatural amino acids include: For example, the section described in WO2009 / 125825 shown below could be cited. Note that the amino acid position This is indicated by Kabat numbering. Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H1 00b, H102 Light chains: L24, L27, L28, L32, L53, L54, L56, L90, L92, L94

[0183] Of these modified sections, H32, H61, L53, L90, and L94 are considered to be highly universal modifications. However, it is not limited to this and can be designed appropriately depending on the purpose.

[0184] Furthermore, although not particularly limited, the preference is for the antigen to be an IL-6 receptor (e.g., human IL-6 receptor). The following are examples of areas that have been significantly altered. Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, H102 Light chains: L24, L27, L28, L32, L53, L56, L90, L92, L94

[0185] Preferred combinations when substituting histidine or non-natural amino acids in combination of multiple sites. Specific examples of combinations include, for instance, the combinations H27, H31, H35, H27, H31, H32, H35, H58, and H6 2. Possible combinations include H102, L32 and L53, and L28, L32, and L53. A preferred combination of heavy and light chain substitution sites is the combination of H27, H31, L32, and L53. These could be cited as examples. These locations should be filled with only one histidine or non-natural amino acid. It can be replaced, and multiple sites can be substituted with histidine or non-natural amino acids.

[0186] Furthermore, if the antigen-binding molecule is a substance containing the antibody constant region, depending on the ion concentration conditions... Another method for altering the binding of an antigen-binding molecule to an antigen is to use the constant region of the antibody. Methods involving the modification of amino acids can also be mentioned. Specific examples of such antibody constant regions include: For example, the constant region of the antibody described in the example of WO2009 / 125825 (SEQ ID NO: 13, SEQ ID NO: 14 One possible method is to substitute with (Sequence ID: 15, Sequence ID: 16). Also, the antibody constant region One method for modifying the region is, for example, the isotypes of the steady-state region (IgG1, IgG2, IgG3, IgG4). Multiple studies revealed that antigen-binding activity decreases in the acidic pH range (the dissociation rate in the acidic pH range is fast). One method is to select an isotype that is less likely to be wild-type. By introducing amino acid substitutions into the acid sequence (wild-type IgG1, IgG2, IgG3, IgG4 amino acid sequence) It reduces antigen binding activity in the acidic pH range (it increases the dissociation rate in the acidic pH range). Methods include: Depending on the isotype (IgG1, IgG2, IgG3, IgG4), the constant region of the antibody The sequence of the hinge region differs significantly, and the difference in the amino acid sequence of the hinge region greatly affects antigen-binding activity. Because it has a significant impact, it is important to select the appropriate isotype depending on the type of antigen or epitope. Therefore, the ion concentration conditions, for example, the antigen binding activity decreases in the acidic pH range (in the acidic pH range) An isotype that increases the dissociation rate may be selected. Also, the amino acids in the hinge region Since differences in the sequence significantly affect antigen-binding activity, wild-type isotype amino For amino acid substitution sites in acid sequences, hinge regions are considered desirable.

[0187] Antigen-binding molecules whose binding activity to antigens changes depending on the ion concentration conditions are as described above. By using this method, amino acid substitutions or insertions can be made into antigen-binding molecules that do not possess these properties. It is possible to produce it by implementing the input process, but other methods include this One method is to directly obtain antigen-binding molecules that have the properties of a mouse. For example, animals (mouse) , rats, hamsters, rabbits, human immunoglobulin transgenic mice, human immunoglobulin Epidemic globulin transgenic rats, human immunoglobulin transgenic rabbits Antibodies obtained by immunizing animals (such as llamas and camels) with an antigen are used to determine the ion concentration-dependent antibody response to the antigen. By screening using the combination as an indicator, antibodies with the desired properties can be directly obtained. Furthermore, antibody libraries presented in vitro can be used to identify ion concentration-dependent antigens. By screening using binding as an indicator, antibodies with the desired properties can be directly obtained. The method is not particularly limited.

[0188] Amino acid modification For modifying the amino acids of the antigen-binding domain, site-directed mutagenesis (Kunkel et al. (Pr oc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR Other known methods may be used as appropriate. In addition, substitution with amino acids other than natural amino acids may be used. Several known methods can also be employed as methods for modifying amino acids (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), which is one of the stop codons. The tRNA contains a non-natural amino acid attached to the complementary amber suppressor tRNA of the codon. Cell-free translation systems such as Clover Direct (Protein Express) are also suitably used. .

[0189] Neutralizing activity In one non-limiting aspect of the present invention, the FcRn binding domain, the binding activity to the antigen, and the ion concentration The antigen-binding domain and its binding activity to the glycosylation receptor change depending on the conditions. An antigen-binding molecule containing one or more glycosylated receptor-binding domains that changes depending on the concentration conditions. A pharmaceutical composition containing this is provided. Generally, neutralizing activity refers to the ability to neutralize viruses, toxins, and other substances that affect cells. This refers to the activity of inhibiting the biological activity of a ligand that has biological activity. In other words, A substance that has binding activity is one that binds to the ligand or the receptor to which the ligand binds. This refers to a substance that inhibits the binding of the ligand to the receptor. Its neutralizing activity inhibits the binding of the ligand to the receptor. Receptors whose binding is blocked can no longer exert biological activity through those receptors. It becomes impossible. When the antigen-binding molecule is an antibody, antibodies with such neutralizing activity are generally not found. These are called neutralizing antibodies. The neutralizing activity of a test substance is determined by its biological activity in the presence of a ligand. Activity can be measured by comparing conditions under which the test substance is present or absent.

[0190] For example, the primary ligand for IL-6R is represented by sequence number 17. IL-6 is a preferred example. Its amino terminus forms an extracellular domain. The substance IL-6R, along with the gp130 receptor whose dimerization is induced by IL-6, forms a heterotetragonal. It forms a mer (HEINRICH et al. (Biochem. J. (1998) 334, 297-314)). The formation of the tetramer activates Jak, which is associated with the gp130 receptor. It performs phosphorylation of the receptor and the receptor. The phosphorylation sites of the receptor and Jak are similar to those of Stat3. Molecules belonging to the Stat family that possess SH2, as well as MAP kinases, PI3 / Akt, and other molecules that possess SH2 It acts as a binding site for proteins and adapters. Next, the gp130 receptor Stat bound to Jak is phosphorylated by Jak. The phosphorylated Stat forms a dimer and enters the nucleus. It transitions to and regulates the transcription of target genes. Jak or Stat acts via other classes of receptors. It can also participate in the signal cascade. Decontrolled IL-6 signal cascade This is observed in the pathology and inflammation of autoimmune diseases, as well as in cancers such as multiple myeloma and prostate cancer. Stat3, which can act as a gene, is constitutively activated in many cancers. Prostate cancer and In multiple myeloma, the signaling cascade from IL-6R and the epidermal growth factor receptor (EGFR) There is crosstalk between the signal cascade from family members (Ishikawa et al.) (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

[0191] These intracellular signaling cascades differ from cell type to cell type, therefore, each target cell type Target molecules can be set as appropriate, and are not limited to the factors mentioned above. Neutralizing activity can be evaluated by measuring the activation of Gunar. Using the transcriptional induction effect on target genes located downstream of the signal cascade as an indicator, It is also possible to detect the activation of signals within the body. Changes in the transcriptional activity of target genes are reported. It can be detected by the principle of the TER assay. Specifically, the transcription factor of the target gene. Alternatively, GFP (Green Fluorescence Protein) or luciferase may be present downstream of the promoter region. By inserting reporter genes such as these and measuring their reporter activity, transcriptional activity can be measured. Changes can be measured as reporter activity. Measurement kit for the activation of biological signals. You can use commercially available products as appropriate (for example, Mercury Pathway Profiling L uciferase System (Clontech), etc.).

[0192] Furthermore, EGF receptors act on signal cascades that normally work to promote cell proliferation. As a method for measuring the neutralizing activity of receptor ligands such as the receptor family, the target is fine The neutralizing activity of a neutralizing antibody can be evaluated by measuring the proliferation activity of cells. Example For example, the proliferation of cells whose growth is promoted by growth factors of the EGF family, such as HB-EGF. As a method for evaluating or measuring the inhibitory effect on proliferation based on the neutralizing activity of anti-HB-EGF antibodies, The following method is preferably used: Evaluate or measure the cell proliferation inhibitory activity in a test tube. One method involves observing the uptake of [3H]-labeled thymidine added to the culture medium by living cells. A method is used to measure replication ability. A simpler method is tripamping. Dye exclusion methods, such as the MTT method, are used to measure the ability to remove dyes such as these from cells under a microscope. The latter is a living cell that has a tetrazolium salt called MTT(3-(4,5-dimethylthiazol-2-yl)-2 It has the ability to convert ,5-diphenyl tetrazolium bromide into a blue formazan product. This method utilizes the following: More specifically, the test antibody is added to the culture medium of the test cells along with the ligand. After a certain period of time has elapsed, the MTT solution is added to the culture medium and left to stand for a certain period of time, thereby producing MTT The substance is taken up by the cells. As a result, the yellow compound MTT is absorbed into the mitochondria within the cells. It is converted into a blue compound by succinate dehydrogenase. This blue product is dissolved and colored The absorbance after this is measured to serve as an indicator of the number of viable cells. Reagents such as MTS, XTT, WST-1, and WST-8 are also commercially available (e.g., nacalai tesque) and are suitable. It can be used. When measuring activity, the same antibody as the anti-HB-EGF antibody should be used as the control antibody. A conjugated antibody having an isotype that does not exhibit the cell proliferation inhibitory activity is the same as an anti-HB-EGF antibody. By using it in this way, the anti-HB-EGF antibody exhibits stronger cell proliferation inhibitory activity than the control antibody, thus activating It can determine sex.

[0193] Cells used to evaluate activity include, for example, cells whose proliferation is promoted by HB-EGF. This includes the RMG-1 cell line, which is an ovarian cancer cell line, and the extracellular domain of human EGFR and mouse GCSF receptors. hEGFR / mG-CSFR is a fusion protein in which intracellular domains of the body are fused in-frame. Mouse Ba / F3 cells transformed with a vector bound to express the gene that controls the expression of the mouse Ba / F3 cell. Cells and the like can also be suitably used. Thus, those skilled in the art can appropriately select cells for evaluating activity. By selecting this, it can be used to measure the aforementioned cell proliferation activity.

[0194] The antigen-binding molecule provided by the present invention can eliminate antigens from plasma, thus providing an anti- It is not necessarily required that the original binding molecule itself possesses neutralizing activity. However, Fcγ Endocytosis via the receptor allows the antigen to be transferred to the Fcγ receptor along with the antigen-binding molecule. Until it is taken up into cells expressing the antigen, it exerts neutralizing activity against the antigen. Furthermore, it is preferable to block the function of antigens present in the plasma.

[0195] Furthermore, the antigen-binding molecule provided by the present invention is an antigen that has been bound to the antigen-binding molecule outside the cell. Because it can promote dissociation from antigen-binding molecules within the cell, The dissociated antigen is degraded in lysosomes. Therefore, the antigen-binding molecule itself has neutralizing activity. Having it is not necessarily required. However, endosites via glycosylation receptors Through cis, the antigen, along with the antigen-binding molecule, is taken up into cells that express glycosylation receptors. During this time, by exhibiting neutralizing activity against the antigen, the function of the antigen present in the plasma is affected. Blocking it is even more preferable.

[0196] Furthermore, the antigen-binding molecule provided by the present invention can reduce the total antigen concentration or free antigen concentration in plasma. Since it can be reduced, it is not necessarily required that the antigen-binding molecule itself possesses neutralizing activity. No. However, antigen binding occurs via endocytosis mediated by glycosylated receptors. Until it is taken up into cells expressing glycosylation receptors along with the molecule, it is neutralized against the antigen. By exhibiting properties, it is even more preferable to block the function of antigens present in the plasma. stomach.

[0197] Glycan receptors Glycans are a group of compounds in which various sugars are linked together by glycosidic bonds. Many of these sugar chains exist as complex molecules bound to proteins and lipids, and are known as complex carbohydrates. These are collectively referred to as such. Among them, complex carbohydrates in which sugar and protein are bound together are called glycoproteins.

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

[0199] O-linked sugar chains in glycoproteins are formed by the hydroxyl group of a serine or threonine residue in the protein and the O-linked sugar chain. It forms a lycosidic bond. Sugars that directly bind to serine or threonine residues are N-acetylgalactosate. It is often methyl (GalNAc), and its core structure is (1) N-acetylgalactosamine (Ga (1) Galactose (Gal) linked to (1) GalNAc via β1-3 linkage, (2) GalNAc linked to β1-3 linkage Gal bound by β1-6 linkage and N-acetylglucosamine (GlcNAc), (3) (4) GlcNAc bound to GalNAc via β1-3 linkage, (5) GlcN bound to GalNAc via β1-3 linkage (5) GlcNAc bound to Ac and β1-6 bonds, (6) GlcNAc bound to GalNAc and β1-6 bonds (6) GalNAc bound to GalNAc via β1-6 linkage, (7) GalNAc bound to GalNAc via β1-3 linkage It consists of eight Gals.

[0200] All N-linked sugar chains in glycoproteins share a common nuclear structure: Man 6(Manα1-3)Manβ1-4Glc It contains NAcβ1-4GlcNAc, which is called a trimannosylcore. Based on the structure and location of the added sugar residues, N-linked glycans are classified into three subgroups. .

[0201] Of these, the complex glycans do not contain mannose residues except for the trimannosyl core. Side chain portion The reducing end of the molecule contains a GlcNAc residue, and the two α-mannosyl residues of the trimannosyl core are They are joined together.

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

[0203] Complex glycans have one or two α-mannosyl groups, similar to the high-mannose type. The Manα1-6 arms of the limannosyl core are bound to the Manα1-3 arms of the core, and the same side chains as the complex glycan are attached to the Manα1-3 arms of the core. It is attached to the arm. Fucoir to the C-6 position of GlcNAc located at the reducing end of the trimannosylcore. The presence or absence of binding, and the binding of β-GlcNAc to the C-4 position of the β-mannosyl residue (bisecting). The presence or absence of what is called GlcNAc contributes to the structural diversity of complex glycans. Three N-bonds Among the subgroups of glycan types, the complex type contains the most diverse structures.

[0204] A glycan receptor is a receptor that recognizes and binds to the glycans mentioned above. Any molecule can be used as a glycosylation receptor as long as it is expressed on the cell. It is a receptor that does this. For example, the O-linked glycans on CD99 are PILR (paired Ig-like type 2 receptor). It has been shown to play an important role in binding by (The Journal of Immunology) gy (2008) vol.180 (3), 1686-1693). Also, the N bond whose terminus is galactose. It is known that asiaglycoprotein receptors bind to glycans of this type. Furthermore, mannose receptors bind to N-linked sugar chains whose terminal ends are mannose. It is known that they combine. Such receptors are suitably utilized in the present invention as glycosylation receptors. It is used. That is, the antigen-binding molecule in the present invention has a domain that binds to such receptors. It has a (glycan receptor binding domain). Therefore, the glycan receptor is asialoglycoprotein If it is a receptor, the N-linked sugar chain whose terminal is galactose is used in the present invention. It can be suitably used as a glycosylated receptor binding domain contained in an antigen-binding molecule. If the receptor is a mannose receptor, then an N-linked sugar whose terminus is mannose. The chain is suitably used as the glycosylated receptor binding domain contained in the antigen-binding molecule in the present invention. obtain.

[0205] Glycan receptor binding domain The antigen-binding molecule of the present invention binds to one or more glycosylation receptors, particularly human glycosylation receptors. It has a binding domain. The binding domain to glycan receptors, especially human glycan receptors, is anti The primordial binding molecule has binding activity to glycan receptors, particularly human glycan receptors, in the pH neutral range. , the binding activity to glycan receptors in the acidic pH range is compared to the binding activity to glycan receptors in the neutral pH range. If the level is lower, the type and number are not particularly limited. Also, directly or indirectly, glycan receptors In particular, domains that have binding activity to human glycosylation receptors may be used. Examples include those that directly have binding activity to glycan receptors, particularly human glycan receptors. Glycans; Fc domain of IgG-type immunoglobulins; glycan receptors, especially human glycan receptors Antibodies; anti-glycan receptors, especially binding peptides to human glycan receptors; glycan receptors, especially human Examples of Scaffold molecules for glycosylation receptors include the Scaffold molecule. In the present invention, in the pH neutral range... It has binding activity to glycan receptors, particularly human glycan receptors, and glycan receptors in the acidic pH range. The binding activity to the glycan receptor is lower than the binding activity to the glycan receptor in the pH neutral range. The main domain is preferred. The domain is pre-treated in a pH neutral range to target glycosylation receptors, particularly human ones. It has binding activity to glycan receptors, and its binding activity to glycan receptors in the pH acidic range is in the pH neutral range. If the glycosylation receptor binding domain has lower binding activity than the one in the above, it can be used as is. It is possible. A glycan receptor binding domain having an N-linked glycan whose terminus is galactose. The pH of the asialoglycoprotein receptor, which is a glycan receptor that binds to the glycan, is acidic. Examples of binding activity in the pH range being lower than binding activity in the pH neutral range are suitable. Furthermore, the glycan receptor binding domain has an N-linked glycan whose terminus is mannose. Binding activity in the acidic pH range with mannose receptors, which are glycosylation receptors that bind to the glycans. This is also a suitable example of a binding activity lower than that in the pH neutral range.

[0206] The glycan receptor binding domain to glycan receptors, particularly human glycan receptors, in the pH neutral range. If the binding activity is absent or weak, the amino acids in the antigen-binding molecule can be modified. Therefore, binding activity to glycan receptors, particularly human glycan receptors, can be acquired. Also, In the pH neutral range, it has binding activity to glycan receptors, especially human glycan receptors. By modifying the amino acids in the enzyme, it is possible to target glycosylation receptors, particularly human glycosylation receptors. Synergistic activity may be enhanced. The amino of the binding domain to glycan receptors, particularly human glycan receptors. Acid modification affects glycan receptors, particularly human glycan receptors, in the pH neutral range before and after amino acid modification. The desired modification can be identified by comparing the binding activity to the target.

[0207] Binding of the glycosylation receptor binding domain to glycosylation receptors, particularly human glycosylation receptors, in the acidic pH range. If the activity is not lower than the binding activity to glycan receptors, especially human glycan receptors, in the pH neutral range. This involves modifying the amino acids in antigen-binding molecules to enable glycosylation receptors in the pH acidic range. In particular, the binding activity to human glycan receptors in the pH neutral range, especially human glycan receptors A binding activity lower than that of the receptor can be acquired. Glycan receptors, especially human glycan receptors. Amino acid modification of the binding domain to the substance is performed in the acidic pH range before and after amino acid modification. Binding activity to glycan receptors, especially human glycan receptors, and glycan receptors in the neutral range, especially The desired modifications can be identified by comparing the binding activity to human glycosylation receptors.

[0208] In the present invention, the glycosylated receptor-binding domain is the antigen-binding domain that constitutes the antigen-binding molecule. It can also be introduced into sites other than the FcRn binding domain. Furthermore, the antigen-binding domain can be introduced into the antigen. Unless the binding is inhibited, the glycosylated receptor binding domain can be attached to any part of the structure of the antigen-binding molecule. It can also be introduced into this. Unless the site inhibits the binding of the antigen by the antigen-binding domain It can be introduced into the antigen-binding domain, or into other sites. In this embodiment, the glycosylation receptor binding domain is the FcRn binding domain of the antigen binding molecule and FcRn, particularly human Fc Unless binding to Rn is inhibited, it can be introduced into any part of the antigen-binding molecule's structure. Example For example, the hinge portion of an IgA antibody is a glycan receptor binding domain for binding O-linked glycans. It could be a candidate for the amino acid sequence, and it is an N-bond as Asn-X-Ser / Thr where X is an amino acid other than Pro. The motif sequence for attaching type glycans is a glycan receptor binding domain for attaching N-linked glycans. These amino acid sequences could be candidates for the antigen-binding molecule. The gene is introduced, and the desired sugar chain is bound to the culture medium of the host cell, as described later. The antigen-binding molecule of the present invention, which includes a glycosylation receptor-binding domain, can be produced.

[0209] Furthermore, in the present invention, the glycosylation receptor binding domain may be chemically synthesized. For example, Galactose derivatives and high-mannose type sugars that mimic N-linked sugar chains with galactose termini Chemical ligands such as mannose derivatives that mimic chains and derivatives that mimic sialic acid are used together. It may also be conjugated to an antigen-binding molecule in a bound manner. As such a chemical ligand, 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. Examples include, but are not limited to, the amino acids of the conjugated antigen-binding site and Examples include lysine and cysteine, but are not limited to these. Specific sites of antigen-binding molecules One method of conjugation involves replacing the amino acid at the conjugation site with cysteine. Alternatively, one could replace the lysine in the area where conjugation is not desired with another amino acid. Methods known to those skilled in the art can be used. One method of conjugation is maleimide. Methods such as reacting cysteine ​​thiols with lysine, or reacting activated esters with lysine. Methods known to those skilled in the art can be used.

[0210] Other conditions besides pH when measuring the binding activity to antigens and FcRns, especially human FcRns, are known to those skilled in the art. It is not limited to a specific method, as it can be selected as appropriate. For example, as described in WO2009 / 125825 It can be measured using MES buffer at 37°C. Furthermore, the antigen-binding molecule's resistance The measurement of primordial binding activity and binding activity to FcRn, particularly human FcRn, is known to those skilled in the art. This can be done by law. For example, by measuring using Biacore (GE Healthcare), etc. This is possible. If the antigen is a soluble antigen, the binding activity of the antigen-binding molecule and the antigen can be measured. This process involves dissolving the antigen as an analyte by passing it through a chip on which an antigen-binding molecule has been immobilized. It is possible to evaluate the binding activity to type antigens, and if the antigen is a membrane-type antigen, the antigen can be evaluated. By flowing antigen-binding molecules as analytes onto an immobilized chip, the binding activity to membrane-type antigens is determined. It is possible to evaluate this. Measurement of the binding activity of antigen-binding molecules and FcRn, particularly human FcRn. This involves immobilizing an antigen-binding molecule or FcRn, particularly human FcRn, onto a chip, and then applying FcRn, particularly human FcRn, to each chip. This can be evaluated by flushing FcRn or antigen-binding molecules as analytes.

[0211] In this invention, the binding activity to FcRn, particularly human FcRn, in the acidic pH range is defined as pH 4.0 to p This refers to the binding activity of FcRn, particularly human FcRn, at H6.5. The binding activity to FcRn is preferably any pH between pH 5.5 and pH 6.5, for example, pH 5.5, 5 Antimicrobial activity at pH values ​​selected from 0.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5 This refers to binding activity to the gen, and is particularly preferably close to the pH within early endosomes 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, o This refers to antigen-binding activity at a pH selected from 6.00.

[0212] When measuring binding activity, any temperature between 10°C and 50°C is used. Even if there is an FcRn binding domain, the antigen-binding molecule of the present invention and the binding to FcRn, particularly human FcRn. Activity can be evaluated. Preferably, the antigen-binding molecule of the present invention containing an FcRn binding domain and FcRn In particular, temperatures ranging from 15°C to 40°C were used to measure binding activity to human FcRn. It is possible. More preferably, the antigen-binding molecule of the present invention containing an FcRn binding domain and FcRn in particular To measure the binding activity to FcRn, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28 , any of the temperatures between 20°C and 35°C, such as 29, 30, 31, 32, 33, 34, and 35°C Temperature can also be used.

[0213] In the present invention, not inhibiting the binding of the antigen to the antigen by the antigen-binding domain means that the glycosylation receptor The introduction of a receptor-binding domain enhances the antigen-binding activity of antigen-binding molecules, and the glycosylation receptor-binding domain The antigen-binding activity of the antigen-binding molecule before the introduction of the agent is increased by 20% or more, preferably 50% or more, and further... This refers to maintaining a concentration of preferably 80% or more, and more preferably 90% or more. Glycan receptor If the antigen-binding activity of the antigen-binding molecule is reduced by the introduction of a binding domain, the antigen binding activity of the antigen-binding molecule will decrease. By substitution, deletion, addition and / or insertion of one or more amino acids in the compound molecule, the sugar chain The antigen-binding activity can be altered to be equivalent to that before the introduction of the receptor-binding domain. In this invention, Following the introduction of such a glycosylation receptor-binding domain, one or more amino acids may be substituted, deleted, or added. This also includes antigen-binding molecules whose binding activity is equivalent through and / or insertion. Furthermore, the fact that it does not inhibit the binding of the glycosylation receptor domain to FcRn, particularly human FcRn, means that The introduction of a glycosylation receptor-binding domain enhances the binding activity of antigen-binding molecules to FcRn, particularly human FcRn. The sex of the antigen-binding molecule before the introduction of the glycosylation receptor-binding domain is particularly related to FcRn, especially human FcRn. 20% or more of the binding activity, preferably 50% or more, more preferably 80% or more, even more preferably This means that it maintains a level of 90% or higher. Antigen binding occurs through the introduction of a glycosylation receptor binding domain. When the binding activity of a molecule to FcRn, particularly human FcRn, decreases, one of the antigen-binding molecules This involves the substitution, deletion, addition, and / or insertion of multiple amino acids, thereby creating glycosylation receptor-binding domains. The binding activity of FcRn, particularly human FcRn, can be altered to be equivalent to that of FcRn before introduction. Therefore, after the introduction of such a glycosylation receptor-binding domain, one or more amino acid substitutions or deletions occur. This also includes antigen-binding molecules whose binding activity is equivalent through addition and / or insertion. The methods for measuring and determining the binding activity to antigens or FcRns, particularly human FcRns, will be described later. It can be done.

[0214] Glycans As an example of a glycosylation receptor binding domain included in the antigen-binding molecule in the present invention, a desired sugar A preferred example is a glycan receptor-binding domain to which a chain is attached. A desired glycan is an O-linked glycan. Preferred examples include type glycans or N-linked glycans, but the glycan is bound to the glycan receptor binding domain. Known methods may be employed for this purpose. For example, immortalized calf with protein-secreting ability. 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 post-translationally modified antibodies in a cell-free protein synthesis system, characterized by the addition of [a specific component]. A series of cell-free enzyme methods, such as the method (Japanese Patent Publication No. 2006-141241), are used to attach the desired sugar chain. It can be used in a method for producing glycosylation receptor-binding domains. For example, motif sequences that attach desired sugar chains have been introduced using recombinant gene techniques. The glycosylated receptor-binding domain contained in naturally or artificially produced antigen-binding molecules By introducing a gene into a host cell, the desired sugar can be extracted from the culture medium of the host cell. The antigen-binding molecule of the present invention, which includes a glycosylation receptor-binding domain to which the chain is bound, can be produced.

[0215] If the sugar chain is an O-linked sugar chain, the motif sequence for attaching the O-linked sugar chain is a known database. By using such as, it will be possible to design O-linked glycans. IgA antibody O-linked glycans are attached to the di portion, and these already known O-linked glycans are attached Gene sequences encoding glycosylation receptor-binding domains would likely be candidates for motif sequences. If the glycan is an N-linked glycan, the motif sequence for attaching the N-linked glycan is Asn-X-Ser / Th It is known that it is a motif of three consecutive amino acids called r. Therefore, recombinant The motif sequence Asn-X-Ser / Thr, which adds N-linked glycans using genetic techniques, is used to create A gene encoding an antigen-binding molecule that contains a glycosylated receptor-binding domain designed to do so. By introducing it into host cells as described later, the desired substance can be obtained from the culture medium of the host cells. The antigen-binding molecule of the present invention, which includes a glycosylation receptor-binding domain to which a sugar chain is bound, can be produced.

[0216] As described above, the sugar chain structure of the antigen-binding molecule produced may be single, but it may also consist of multiple sugars. It can also be produced as a mixture of linked chains. In this invention, such mixtures are also It can be suitably used. Also, antigen-binding components in which a specific sugar chain is linked to its sugar chain-binding domain. Children can also be suitably used in the present invention.

[0217] In the present invention, a specific glycan is linked to the glycan receptor binding domain contained in the antigen-binding molecule. Several known methods can be employed as a way to do this. One such known method is to use the The properties of the sugar chains of antigen-binding molecules, whether naturally occurring or artificially produced by recombinant gene techniques, etc. One method involves utilizing quality to purify antigen-binding molecules that possess specific sugar chains. Antibodies with high mannose-type glycans were found using affinity chromatography with ConA-sepharose. It is known to be purified using roughing (Millward (Biologicals (2008) 36, 49-60)). This purification method is used for N-linked sugars in which the non-reducing end is mannose in the present invention. It can be used to create antigen-binding molecules that have chains.

[0218] In another embodiment, enzymatic treatment is also performed for the purpose of obtaining antigen-binding molecules having a specific sugar chain. It may be adopted as appropriate. As will be described later in the examples, N is a galactose at the non-reducing end. Antigen-binding molecules having linked glycans have complex glycans with sialic acid at the non-reducing end. It can be produced from the original linked molecule by sialidase treatment. It also possesses a high mannose-type glycan. When antibodies are treated with sialidase and β-galactosidase, galactose is converted to its sugar chain. It is known that it is produced in a form in which these are removed (Newkirk(Clin.Exp.Immunol. (1996)). 106, 259-264)). Preparation including sialidase and β-galactosidase treatment. The method involves an antigen-binding molecule having an N-linked sugar chain whose non-reducing end is mannose. It can be used to manufacture.

[0219] In a different embodiment, for the purpose of obtaining an antigen-binding molecule having a specific sugar chain of the present invention, the antibody A recombinant gene encoding the original binding molecule was introduced, causing a specific sugar chain to accumulate. Its glycosidase activity (including but not limited to genetic or recombinant gene techniques) Methods for recovery from the culture medium of altered host cells can also be employed as appropriate. High-mannose type N-tubules An antibody having a combined glycan is transduced into N-acetylcholine, which has a recombinant gene encoding the antibody. Culture medium of a Lec1 mutant cell derived from CHO cells lacking luglucosaminyltransferase I activity. It is known to be recovered more often (Wright and Morrison (J. Exp. Med. (1994) 180) , 1087-1096)). The present invention has an N-linked glycan whose non-reducing end is mannose. The antigen-binding molecule is cultured in a Lec1 mutant strain into which the recombinant gene encoding the molecule has been transduced. It can be recovered from the liquid.

[0220] In yet another embodiment, with the aim of obtaining an antigen-binding molecule having a specific sugar chain of the present invention, Inhibition of a specific glycosidase reaction during the culture of cells that produce the antigen-binding molecule. By adding the agent, antigen-binding molecules with specific sugar chains accumulated in the culture medium can be recovered. Other methods may be adopted as appropriate. A high-mannose type N bond that does not have fucose at its reducing end. When adding kifunesine to the culture of CHO cells that produce antibodies containing type glycans... It is known that the cells can be recovered from the culture medium by (Zhou(Biotechnol . Bioeng. (2008) 99, 652-665)). In the present invention, fucose is provided at its reducing end. Furthermore, antigen-binding molecules having an N-linked sugar chain with a non-reducing end being mannose are, for example, the CHO cells, into which recombinant genes encoding molecules have been transduced, are cultured with the addition of kifunesine. It can be recovered from the culture medium by doing so. Also, for example, the glycosidase mentioned above Combining methods such as adding these inhibitors during the culture of host cells with altered activity is possible. By this method, it is also possible to obtain antigen-binding molecules having specific sugar chains according to the present invention. It is known that antibodies with specific sugar chains can be recovered by such combinations (Kanda et al. (Glycobiology (2007) 17, 104-118)) describe antigens having specific sugar chains in the present invention. Similar combinations may be used as appropriate for the purpose of obtaining bound molecules.

[0221] Furthermore, by genetically modifying the host, such as Pichia pastoris, it is possible to obtain a specific sugar chain structure. 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 this method, a specific sugar chain structure (e.g., galac An antigen-binding molecule was created by uniformly attaching N-linked glycans (with tose ends) to an N-linked glycan attachment sequence. It is possible.

[0222] Antigen-binding molecule containing a glycosylation receptor-binding domain of the present invention, particularly human-derived glycosylation receptor-binding molecule. Antigen-binding molecules containing domains bind to glycosylation receptors in a pH-dependent manner, and, , or, in the pH neutral range, glycan receptor binding activity, particularly human-derived glycan receptor binding activity It possesses binding activity to glycan receptors in the acidic pH range and binding activity to glycan receptors in the neutral pH range. If the activity level can be reduced to a lower level, it will promote the uptake of antigens into cells by antigen-binding molecules. Therefore, the administration of antigen-binding molecules promotes a decrease in the antigen concentration in the plasma, and the drug of antigen-binding molecules It is possible to improve dynamics and increase the number of antigens that a single antigen-binding molecule can bind to. ru.

[0223] Binding activity to glycosylation receptors In this invention, binding to glycan receptors in the pH acidic range, particularly to human-derived glycan receptors, is achieved. Synchronous activity refers to the binding activity to glycan receptors, particularly human-derived glycan receptors, at pH 4.0 to pH 6.5. This means any pH between pH 5.5 and pH 6.5, for example, pH 5.5, 5.6, 5.7, 5.8. Glycan receptors at pH values ​​selected from 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, particularly This refers to binding activity to human-derived glycosylation receptors, and is particularly preferred for early endogenous activity in vivo. Any pH between pH 5.8 and pH 6.0, close to the pH inside the somole, 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. Glycan receptors, particularly human-derived glycans, at pH values ​​selected from 97, 5.98, 5.99, and 6.00. This refers to the binding activity to the receptor. Furthermore, in this invention, the glycan receptor in the pH neutral range. Binding activity to glycan receptors, particularly those of human origin, refers to the binding activity to glycan receptors at pH 6.7 to pH 10.0. This specifically refers to binding activity to human-derived glycan receptors. Preferably, 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. The binding activity to glycan receptors, particularly human-derived glycan receptors, at pH levels selected from 0. This means, and especially preferably, a glycan receptor at pH 7.4, which is close to the pH of plasma in the body, particularly human-derived This refers to the binding activity to the glycosylation receptor.

[0224] Other conditions besides pH when measuring binding activity to glycan receptors, especially human-derived glycan receptors Since these can be appropriately selected by those skilled in the art, they are not limited to any particular conditions. For example, WO As described in 2009 / 125825, it can be measured under the conditions of MES buffer and 37°C. Also, The measurement of the binding activity of antigen-binding molecules to glycosylation receptors, particularly human-derived glycosylation receptors, is performed by those skilled in the art. This can be carried out by publicly known methods. For example, by using Biacore (GE Healthcare), etc. It can be determined that the binding activity of antigen-binding molecules to glycosylation receptors, particularly human-derived glycosylation receptors, is... The measurement is performed, for example, on a chip immobilized with glycan receptors, particularly human-derived glycan receptor molecules. By flowing antigen-binding molecules as analytes, glycosylation receptors, especially human-derived glycosylation receptors, can be activated. It is possible to evaluate the binding activity of antigen-binding molecules to the substance. Conversely, A chip immobilized with antigen-binding molecules is used to solubilize glycan receptors, particularly human-derived glycan receptors. By flushing the body as an analyte, it targets glycan receptors, especially human-derived glycan receptors. The binding activity can be evaluated.

[0225] In this invention, binding to glycan receptors in the pH acidic range, particularly to human-derived glycan receptors, is achieved. As long as the combined activity is weaker than the antigen-binding activity in the pH neutral range, the glycosylation receptor in the pH acidic range, In particular, binding activity to human-derived glycan receptors and glycan receptors in the pH neutral range, especially human-derived The ratio of binding activity to the original glycan receptor is not particularly limited. Preferably, the glycan receptor, in particular KD (Dissociation constant) at pH 5.8 for human-derived glycan receptors The ratio of KD (pH 5.8) to KD (pH 7.4), which is the ratio of KD at pH 7.4, is 2 or greater, and furthermore, More preferably, the KD (pH5.8) / KD (pH7.4) value is 10 or higher, and more preferably KD (pH5.8 The value of ) / KD (pH7.4) is 40 or higher. There is no particular upper limit to the value of KD (pH5.8) / KD (pH7.4). Not applicable, and any value such as 400, 1000, 10000, etc., is acceptable as long as it can be manufactured using the art of a person skilled in the art. stomach.

[0226] The KD (dissociation constant) is a value representing the binding activity to glycan receptors, particularly human-derived glycan receptors. The following can be used. The KD (dissociation constant) can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), scatchard plots, flow cytometers. These can be suitably used.

[0227] In addition, as another index indicating the ratio of the binding activity to a sugar chain receptor in the pH acidic range, particularly a human-derived sugar chain receptor, and the binding activity to a sugar chain receptor in the pH neutral range, particularly a human-derived sugar chain receptor, for example, k , which is the dissociation rate constant, can also be preferably used. When using k (Dissociation rate consta d (Dissociation rate constant) instead of KD (dissociation constant) as an index indicating the ratio of the binding activity, the ratio of k (Dissociation rate constant) in the pH acidic range to k (Dissociation rate constant) in the pH neutral range for a sugar chain receptor, particularly a human-derived sugar chain receptor d , that is, the value of k (pH acidic range) / k d (Dissociation rate constant) in the pH acidic range and k d (Dissociation rate constant) in the pH neutral range, which is k (pH acidic range) / k d (pH neutral range), is preferably 2 or more, more preferably 5 d or more, even more preferably 10 or more, and still more preferably 30 or more. The upper limit of the value of k (pH acidic range) / k d (pH acidic range) / k d (pH neutral range) is not particularly limited and can be set to any value such as 50, 100, 200, etc., as long as it can be prepared based on the common technical knowledge of those skilled in the art.

[0228] For example, the interaction between asialoglycoprotein receptor, which is one of the sugar chain receptors, and galactose is pH-dependent, showing high binding activity in the pH neutral range and low binding activity in the pH acidic range (J Biol Chem Vol. 274, No. 50, pp. 35400-3540 6, 1999). Similarly, the interaction between mannose receptor, which is one of the sugar chain receptors, and mannose is pH-dependent, showing high binding activity in the pH neutral range and low binding activity in the pH acidic range (J Biol Chem Vol. 274, No. 50, pp. 35400-3540 6, 1999). Similarly, the interaction between mannose receptor, which is one of the sugar chain receptors, and mannose is pH-dependent, showing high binding activity in the pH neutral range and low binding activity in the pH acidic range ​It is known that (J Biol Chem. 1994 Nov 11;269(45):28405-13.). Therefore, in the present invention, the glycan / glycan receptor is galactose / asialoglycop Rothene receptors and mannose / mannose receptors can be suitably used.

[0229] Also, galactose / asialoglycoprotein receptor and mannose / mannose The binding activity of the North receptor is not only pH-dependent but also calcium ion concentration-dependent. Therefore, since most glycan receptors are C-type lectins, the binding of glycan receptors to glycans is It is calcium ion concentration dependent. That is, similar to the binding of antigen-binding molecules to antigens, The binding of glycan receptors to glycans may also be calcium ion concentration dependent, and high calcium levels The binding at the ion concentration should be higher than the binding at the low calcium ion concentration.

[0230] In this invention, we use glycosylation receptors of antigen-binding molecules at different pH levels, particularly those derived from human glycosylation receptors. When measuring the binding activity to the body, it is preferable to keep all conditions except pH the same.

[0231] antigen binding molecule This invention comprises an antigen-binding domain, an FcRn-binding domain, and one or more binding domains to a glycosylated receptor. An antigen-binding molecule having binding domains, wherein the number of binding domains to the glycan receptor The antigen-binding molecules increased, and the binding activity to the antigen in the acidic pH range increased in the neutral pH range. The present invention provides an antigen-binding molecule further comprising an antigen-binding domain with lower antigen-binding activity than that of the antigen. The present invention also provides a method for producing the antigen-binding molecule and a pharmaceutical composition containing the antigen-binding molecule. The present invention provides a product. Furthermore, the present invention provides the antigen-binding molecule to cells expressing a glycosylation receptor. This includes contact with the antigen-binding molecule and / or the antigen-binding molecule, either inside or outside the body. A method for taking an antigen bound to an antigen into a cell, and the amount of the antigen-binding molecule per molecule A method for increasing the number of antigens that bind, a method for decreasing the amount of antigens present outside the cell, the antigen A method for improving the pharmacokinetics of a binding molecule, and for promoting the dissociation of the antigen from the antigen-binding molecule. The present invention provides a method for promoting this. Furthermore, the present invention relates to an antigen-binding domain, an FcRn-binding domain, and a glycan. In antigen-binding molecules having two or more binding domains to the receptor, the glycosylated receptor This includes increasing the number of binding domains for the antigen in vivo or in vitro. To promote the intracellular uptake of the binding molecule and / or the antigen to which the antigen-binding molecule binds. The method, the number of antigens to which the antigen-binding molecule binds per molecule in vivo or in vivo. Methods to increase the antigen-binding molecule's ability to eliminate antigens in vivo or in vitro. Methods for causing, methods for improving the pharmacokinetics of the antigen-binding molecule, and the antigen-binding molecule This invention provides a method for promoting the dissociation of antigens from synthetic molecules.

[0232] Method for producing antigen-binding molecules This invention relates to an antigen-binding domain, an FcRn-binding domain, particularly a human FcRn-binding domain, and one or more glycosylation receptors. An antigen-binding molecule having a body-binding domain, which binds to a glycosylation receptor in the pH neutral range. It possesses synergistic activity, and its binding activity to glycan receptors in the acidic pH range is similar to that of glycan receptors in the neutral pH range. Its binding activity is lower than that of the antigen in the acidic pH range, and its antigen binding activity in the neutral pH range is lower than that of the antigen binding activity in the neutral pH range. The present invention provides a method for producing antigen-binding molecules with lower efficacy than the present invention. Furthermore, the present invention provides a method for producing antigen-binding molecules by administration An antigen-binding compound that has an excellent effect in reducing the antigen concentration in plasma and exhibits excellent pharmacokinetics. The present invention provides a method for producing this product. Furthermore, the present invention is particularly useful when used as a pharmaceutical composition. This invention provides a method for producing antigen-binding molecules.

[0233] Specifically, the present invention provides a method for producing an antigen-binding molecule, comprising the following steps: (a) Provide polypeptide sequences of antigen-binding molecules including antigen-binding domains and FcRn-binding domains. The process of supplying, (b) Candidate motifs for the glycosylation receptor binding domain in the polypeptide sequence The process of identifying the amino acid sequence, (c) An amino acid sequence in which at least one amino acid is different from the amino acid sequence identified in (b) The process of designing motifs of glycosylation receptor-binding domains, (d) A polypeptide of an antigen-binding molecule containing the glycosylated receptor-binding domain motif designed in (c) The process of creating the gene that codes for cide, (e) Antigen-binding molecules from the culture medium of host cells transformed by the genes obtained in (d) The recovery process.

[0234] Note that 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. Also, as will be described later, the result obtained in (e) The manufacturing method of the present invention also includes a step of further treating the antigen-binding molecule with an enzyme. It is possible.

[0235] Antigen-binding domains contained in antigen-binding molecules produced by the manufacturing method provided by the present invention The substance can be provided by the method described in the "antigen-binding domain" section.

[0236] Furthermore, the FcRn contained in the antigen-binding molecule produced by the manufacturing method provided by the present invention is particularly The FcRn-binding domain having binding activity to human FcRn is located in the aforementioned "FcRn-binding domain". It can be provided by the described method, that is, the FcRn binding domain in the acidic pH range. The binding activity to FcRn, particularly human FcRn, is not particularly limited, and can be direct or indirect. A domain that has binding activity to FcRn, particularly human FcRn, may be used. As for the domain, for example, an IgG type that directly has binding activity to FcRn, especially human FcRn. Fc region of immunoglobulin, albumin, albumin domain 3, anti-human FcRn antibody, anti-human FcR n-peptides, anti-human FcRn scaffold molecules, etc., or indirectly binding activity to human FcRn Examples include molecules that bind to IgG or albumin and have such properties. polypeptides with domains that directly or indirectly have binding activity to FcRn, particularly human FcRn. The sequence can be provided as a polypeptide sequence of the antigen-binding domain.

[0237] Glycan receptor binding included in antigen-binding molecules produced by the manufacturing method provided by the present invention. A suitable example of a domain is a glycan receptor-binding domain to which a desired glycan is bound. Desired glycans include O-linked glycans or N-linked glycans, but glycan receptors A known method can be used to attach the sugar chain to the body-binding domain. One method involves using recombinant gene techniques to add a motif sequence (suna These are naturally occurring or artificially produced molecules into which a glycosylated receptor-binding domain motif has been introduced. The gene encoding the glycosylation receptor-binding domain contained in the antigen-binding molecule is introduced into the host cell. By introducing it, the desired glycan is bound to the glycan receptor from the culture medium of the host cell. The antigen-binding molecule of the present invention, which includes a domain, can be produced.

[0238] As described above, in the polypeptide sequence provided, the glycosylation receptor binding domain For example, the following methods can be used to identify candidate amino acid sequences for the protein. It is possible. Motif sequences for attaching O-linked glycans can be determined using publicly known databases, etc. Therefore, it can be identified. Also, if the hinge portion of the antibody contains the antigen-binding molecule of the present invention. This allows for the identification of which isotype of antibody the hinge portion originates from. Such hinge regions were identified as candidate motifs for the O-linked glycosylation receptor-binding domain. Obtain. Also, if the sugar chain is an N-linked sugar chain, the motif sequence to which the N-linked sugar chain is attached (sand The motif of the N-linked glycosylation receptor-binding domain consists of three amino acids: Asn-X-Ser / Thr It is known to be a continuous motif. Therefore, by recombinant gene techniques, etc., N Designed to encode Asn-X-Ser / Thr, a motif of the glycosylated receptor binding domain. In order to design a glycosylation receptor binding domain, in the provided polypeptide sequence The presence or absence of sequences identical or similar to Asn-X-Ser / Thr can be identified.

[0239] The amino acid sequence identified above has at least one different amino acid sequence. Motifs of glycosylated receptor-binding domains containing columns can be designed. For example, known databases By using such as, the motif sequence to which the O-linked glycans identified above are attached can be used. By substituting amino acids in candidate polypeptide sequences, O-linked glycosylation is achieved. A sequence can be designed. Also, if the hinge portion of the antibody contains the antigen-binding molecule of the present invention. If the hinge portion does not include a hinge portion derived from IgA antibodies, then the hinge By substituting the sequence of a portion with the sequence of a hinge portion derived from an IgA antibody, O-linked glycan mo A chief sequence can be designed. On the other hand, if the glycan is an N-linked glycan, the provided poly If no sequence identical or similar to Asn-X-Ser / Thr is identified in the peptide sequence, By inserting the Asn-X-Ser / Thr sequence at the appropriate location in the provided polypeptide sequence, This allows for the addition of a new N-linked glycosylation receptor-binding domain motif. Furthermore, the provided The amino acid residues of sequences similar to the Asn-X-Ser / Thr sequence found in the polypeptide sequence were substituted. By doing so, the Asn-X-Ser / Thr sequence, which is the motif of the N-linked glycosylation receptor binding domain, The similar sequence may be modified.

[0240] The antigen-binding molecule of the present invention binds to one or more glycosylation receptors, particularly human glycosylation receptors. It has a domain. The binding domain to glycosylation receptors, particularly human glycosylation receptors, is for antigen binding. The molecule has binding activity to glycan receptors, particularly human glycan receptors, in the pH neutral range, and The binding activity to glycan receptors in the pH range is lower than the binding activity to glycan receptors in the pH neutral range. The type and number of these receptors are not particularly limited. Also, directly or indirectly, glycan receptors, especially hyphens A domain having binding activity to a glycosylation receptor may be used. For example, sugars that have binding activity to glycan receptors, particularly human glycan receptors. Chain; Fc domain of IgG-type immunoglobulin; antibodies against glycan receptors, especially human glycan receptors. ; Binding peptides to anti-glycan receptors, especially human glycan receptors; Glycan receptors, especially human glycans Examples of receptors include scaffold molecules. In the present invention, sugars in the pH neutral range It has binding activity to chain receptors, particularly human glycan receptors, and in the acidic pH range, it binds to glycan receptors. The binding activity of the glycosylated receptor binding domain is lower than the binding activity to glycosylated receptors in the pH neutral range. This is preferable. The domain is pre-treated in a pH neutral range to form a glycosylation receptor, particularly a human glycosylation receptor. It has binding activity to the substance, and its binding activity to the glycan receptor in the pH acidic range is in the pH neutral range. If the glycan receptor binding domain has lower binding activity than the glycan receptor, it can be used as is. A glycan receptor binding domain having an N-linked glycan whose terminus is galactose, and the glycan The asialoglycoprotein receptor, a glycosylation receptor that binds to glycans, in the acidic pH range. The binding activity is preferably lower than the binding activity in the pH neutral range. A glycan receptor-binding domain having an N-linked glycan whose end is mannose, and a binding domain to said glycan The binding activity with the mannose receptor, a sugar chain receptor, in the acidic pH range is also in the neutral pH range. A suitable example of a binding activity lower than that of [the given example] is [the given example].

[0241] The glycan receptor binding domain to glycan receptors, particularly human glycan receptors, in the pH neutral range. If the binding activity is absent or weak, the amino acids in the antigen-binding molecule can be modified. Therefore, binding activity to glycan receptors, particularly human glycan receptors, can be acquired. Also, In the pH neutral range, it has binding activity to glycan receptors, especially human glycan receptors. By modifying the amino acids in the enzyme, it is possible to target glycosylation receptors, particularly human glycosylation receptors. Synergistic activity may be enhanced. The amino of the binding domain to glycan receptors, particularly human glycan receptors. Acid modification affects glycan receptors, particularly human glycan receptors, in the pH neutral range before and after amino acid modification. The desired modification can be identified by comparing the binding activity to the target.

[0242] Binding of the glycosylation receptor binding domain to glycosylation receptors, particularly human glycosylation receptors, in the acidic pH range. If the activity is not lower than the binding activity to glycan receptors, especially human glycan receptors, in the pH neutral range. This involves modifying the amino acids in antigen-binding molecules to enable glycosylation receptors in the pH acidic range. In particular, the binding activity to human glycan receptors in the pH neutral range, especially human glycan receptors A binding activity lower than that of the receptor can be acquired. Glycan receptors, especially human glycan receptors. Amino acid modification of the binding domain to the substance is performed in the acidic pH range before and after amino acid modification. Binding activity to glycan receptors, especially human glycan receptors, and glycan receptors in the neutral range, especially The desired modifications can be identified by comparing the binding activity to human glycosylation receptors.

[0243] In the present invention, unless the binding of the antigen by the antigen-binding domain is inhibited, the glycosylated receptor The binding domain can be introduced into any part of the structure of the antigen-binding molecule. This part is the antigen. Unless the binding domain inhibits the binding to the antigen, it can be introduced into the antigen-binding domain as well. Furthermore, it can be introduced into other sites as well. In another embodiment, the glycosylation receptor binding domain is Unless the binding of the antigen-binding molecule's FcRn-binding domain to FcRn, particularly human FcRn, is inhibited, the antigen binding will not be inhibited. It can be introduced into any part of the structure of the composite molecule. For example, the hinge region of an IgA antibody is O-linked. It could be a candidate amino acid sequence for the glycosylation receptor binding domain for attaching glycans, and Asn- The X-Ser / Thr motif sequence, which adds N-linked glycans, is for attaching N-linked glycans. These amino acid sequences could be candidates for the glycosylation receptor binding domain. From the culture medium of host cells into which a gene encoding an antigen-binding molecule has been introduced, as described below... The antigen-binding molecule of the present invention, which includes a glycan receptor-binding domain to which the desired glycan is bound, can be produced. ru.

[0244] In the present invention, not inhibiting the binding of the antigen to the antigen by the antigen-binding domain means that the glycosylation receptor The introduction of a receptor-binding domain enhances the antigen-binding activity of antigen-binding molecules, and the glycosylation receptor-binding domain The antigen-binding activity of the antigen-binding molecule before the introduction of the agent is increased by 20% or more, preferably 50% or more, and further... This refers to maintaining a concentration of preferably 80% or more, and more preferably 90% or more. Glycan receptor If the antigen-binding activity of the antigen-binding molecule is reduced by the introduction of a binding domain, the antigen binding activity of the antigen-binding molecule will decrease. By substitution, deletion, addition and / or insertion of one or more amino acids in the compound molecule, the sugar chain The antigen-binding activity can be altered to be equivalent to that before the introduction of the receptor-binding domain. In this invention, Following the introduction of such a glycosylation receptor-binding domain, one or more amino acids may be substituted, deleted, or added. This also includes antigen-binding molecules whose binding activity is equivalent through and / or insertion. Furthermore, the fact that it does not inhibit the binding of the glycosylation receptor domain to FcRn, particularly human FcRn, means that The introduction of a glycosylation receptor-binding domain enhances the binding activity of antigen-binding molecules to FcRn, particularly human FcRn. The sex of the antigen-binding molecule before the introduction of the glycosylation receptor-binding domain is particularly related to FcRn, especially human FcRn. 20% or more of the binding activity, preferably 50% or more, more preferably 80% or more, even more preferably This means that it maintains a level of 90% or higher. Antigen binding occurs through the introduction of a glycosylation receptor binding domain. When the binding activity of a molecule to FcRn, particularly human FcRn, decreases, one of the antigen-binding molecules This involves the substitution, deletion, addition, and / or insertion of multiple amino acids, thereby creating glycosylation receptor-binding domains. The binding activity of FcRn, particularly human FcRn, can be altered to be equivalent to that of FcRn before introduction. Therefore, after the introduction of such a glycosylation receptor-binding domain, one or more amino acid substitutions or deletions occur. This also includes antigen-binding molecules whose binding activity is equivalent through addition and / or insertion. It can be done.

[0245] Furthermore, the glycosylated receptor-binding domain is the antigen-binding domain and FcRn-binding domain that make up the antigen-binding molecule. It can also be introduced into parts other than the domain.

[0246] The structure of the antigen-binding molecule targeted in this invention is not particularly limited, and any antigen of any structure is used. Binding molecules can also be suitably used, but antigen-binding domains, FcRn in particular binding to human FcRn, are preferred. As a preferred example of an antigen-binding molecule having a main and two or more glycosylated receptor domains Examples of antibodies include antibodies. A preferred example of the antibody of the present invention is an IgG antibody. When using IgG antibodies, the type is not limited to IgG1, IgG2, IgG3, IgG4, etc. IgG of a specific type (subclass) may be used. Furthermore, the antigen-binding molecule of the present invention may contain a specific antibody. A constant region may be included, and amino acid mutations may be introduced into that constant region. These amino acid mutations, for example, increase or decrease binding to the Fcγ receptor (Pr Examples include *oc Natl Acad Sci US A. 1(2006) 103 (11), 4005-10), This is not limited to these examples. Furthermore, an appropriate constant region, such as the IgG2 constant region, should be selected. This also makes it possible to alter pH-dependent bonding.

[0247] When the antigen-binding molecule targeted by this invention is an antibody, the antibody may be a mouse antibody, a human antibody, or a rat antibody. Antibodies derived from any animal, such as rabbit antibodies, goat antibodies, and camel antibodies, can be used. Furthermore, for example, modified antibodies in which the amino acid sequence has been substituted, such as chimeric antibodies, especially humanized antibodies. The body is also suitable for use. In addition, two types of specific antibodies, antibody modifiers to which various molecules are attached, and antibodies Polypeptides containing fragments may also be used.

[0248] A "chimeric antibody" is an antibody created by combining sequences derived from different animals. Specific examples of Mella antibodies include, for example, the variable (V) region of the heavy and light chains of mouse antibodies and Examples of antibodies consisting of a heavy chain and a constant (C) region of the light chain include antibodies.

[0249] "Humanized antibodies," also known as reshaped human antibodies, are antibodies from mammals other than humans. The complementarity determining region (CDR) of the derived antibody, for example, a mouse antibody. This involves transplanting a region)) into a human antibody CDR. The method for identifying the CDR is publicly 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). Furthermore, the general genetic engineering techniques are also publicly known (EP125023). (WO1996 / 002576).

[0250] Bispecific antibodies have variable regions that recognize different epitopes within the same antibody molecule. This refers to antibodies. A two-specific antibody may be an antibody that recognizes two or more different antigens, or it may recognize the same antigen. It could also be an antibody that recognizes two or more different epitopes on an antigen.

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

[0252] The antigen-binding domain, FcRn-binding domain, and glycosylation receptor domain are designed as described above. Genes encoding the synthetic domain can be constructed. The method for constructing these genes is publicly known and involves chemical synthesis. It can be produced by, or by methods such as PCR, which can produce polynucleotides. It can also be produced by linking via an enzymatic reaction in the presence of a template such as a primer. This is possible. The antigen-binding domain, FcRn-binding domain, and glycans are designed as described above. The receptor-binding domains are expressed in the culture medium after each domain is expressed separately in the host cells as described later. Polypeptides recovered from these sources are linked by a chemical reaction in the presence of a crosslinking agent. This is possible. Synthetic chemical linkers (chemical crosslinking agents) are commonly used for crosslinking peptides. Crosslinking agents, such as N-hydroxysuccinimide (NHS) and disuccinimidyl beret β(DSS), bis(sulfosuccinimidyl)sverate(BS3), dithiobis(sulfosuccinimidyl) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), di Succinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (Sulfo-DST) , bis[2-(succinimoidoxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone(sulfo-B) Examples include SOCOES, and these crosslinking agents are commercially available.

[0253] Furthermore, the antigen-binding domain, FcRn-binding domain, and glycan-binding domain are designed as described above. The ligand was designed so that the encapsulation domain is linked in-frame by a peptide bond. The gene can also be obtained by expressing it in host cells as described later and then recovering it from the culture medium, etc. It is possible that when linked in-frame by peptide bonds, each domain is directly linked. It can be obtained and linked via a linker having a specific peptide sequence. Each domain is joined. The linker can be any peptide linker that can be introduced by genetic engineering, or a synthetic linker. Open compound linkers (see, for example, Protein Engineering (1996) 9 (3), 299-305) While linkers such as those shown can be used, peptide linkers are preferred in this 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. While this is possible, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 3). It is 0 amino acids or less, preferably 20 amino acids or less, and particularly preferably 15 amino acids. .

[0254] For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·SerSer·Gly·Gly Gly·Gly·Gly·Ser(Sequence ID: 18) Ser·Gly·Gly·Gly (Sequence ID: 19) Gly·Gly·Gly·Gly·Ser(Sequence ID: 20) Ser·Gly·Gly·Gly·Gly(Sequence ID: 21) Gly·Gly·Gly·Gly·Gly·Ser(Sequence code: 22) Ser·Gly·Gly·Gly·Gly·Gly(Sequence ID:23) Gly·Gly·Gly·Gly·Gly·Gly·Ser(Array No.: 24) Ser·Gly·Gly·Gly·Gly·Gly·Gly(Sequence ID:25) (Gly·Gly·Gly·Gly·Ser(Sequence ID:20))n (Ser·Gly·Gly·Gly·Gly(Sequence ID: 21))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker are A linker that can be appropriately selected by a person skilled in the art depending on the purpose may also be used in this invention. ru.

[0255] The gene obtained by the manufacturing method of the present invention is usually loaded (inserted) into a suitable vector. The vector is then introduced into the host cell. The vector is designed to stably hold the inserted nucleic acid. If available, there are no particular restrictions; for example, if E. coli is used as the host, a cloning vector For this purpose, pBluescript vectors (manufactured by Stratagene) are preferred, but various commercially available vectors are also available. A vector may be used. A vector is used for the purpose of producing the antigen-binding molecule of the present invention. In some cases, expression vectors are particularly useful. Expression vectors include those used in vitro and in E. coli. There are no particular restrictions as long as the vector expresses antigen-binding molecules within cultured cells or living organisms. However, for example, in vitro expression would use the pBEST vector (Promega), and in E. coli, it would be pET. Vector (manufactured by Invitrogen), or for cultured cells, pME18S-FL3 vector (GenBank Accessi (on No. AB009864), if it is a living organism, pME18S vector (Mol Cell Biol. 8:466-472) (1988)) and others are preferably used, but are not limited to these. The gene insertion of the invention is performed by conventional methods, for example, by a ligase reaction using restriction enzyme sites. (Current protocols in Molecular Biology edit. Ausubel et al.) al. (1987) Publish. John Wiley & Sons.Section 11.4-11.11).

[0256] There are no particular restrictions on the host cells mentioned above; various host cells can be used depending on the purpose. Examples of cells used to express the primordial molecule include bacterial cells (e.g., Streptococcus). (e.g., 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 include the following: Vector introduction into host cells can be performed, for example, by calcium phosphate precipitation, electrophoresis. Pulse drilling (Current protocols in Molecular Biology edit. Ausubel et al.) (1987) Published by John Wiley & Sons. Sections 9.1-9.9, Lipofection This can be done using known methods such as microinjection.

[0257] Host cell culture can be carried out according to known methods. For example, using animal cells as a host. In this case, DMEM, MEM, RPMI1640, and IMDM may be used as culture media. Furthermore, cultures using media containing serum replacement fluids such as fetal bovine serum (FCS) were also performed. Cells can also be cultured using serum-free medium. The pH during culture is approximately 6-8. The following can be appropriately selected from among them, but are not limited to this, for each culture, or throughout the culture period. Different pH levels may be selected depending on the time of year. Culturing is typically done at approximately 30-40°C for about 15-200 hours. The culture medium is replaced, aerated, and stirred as needed.

[0258] Antigen-binding molecules expressed in host cells are transferred to the lumen of the endoplasmic reticulum, the pericellular lumen, or the cell itself. To induce secretion into the external environment, an appropriate secretion signal can be incorporated into the desired polypeptide. These signals may be endogenous signals for the target antigen-binding molecule, or heterologous signals. It could also be a signal.

[0259] On the other hand, as a system for producing polypeptides in vivo, for example, using animals... Examples include production systems using plants or other organisms. A vector into which the target gene is inserted is then introduced. The polypeptide encoded by the gene produced within the cells of the animal or plant is , and are recovered from their bodily fluids or living organisms. In this invention, "host" refers to these animals and plants. To include.

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

[0261] For example, the polynucleotide encoding the antigen-binding molecule of the present invention is similar to that of goat β-casein. It was prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in eel milk. It is then produced. Next, the polynucleotide fragment containing this fusion gene is injected into a goat embryo. This embryo is transplanted into a female goat. A transgenic goat is born from the goat that received the embryo. Alternatively, the target antigen-binding molecule can be recovered from the milk produced by its offspring. To increase the amount of milk containing antigen-binding molecules produced from goats, hormones are used as appropriate. 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 that produce the antigen-binding molecules of the present invention. When silkworms are used, a polynucleotide encoding the target antigen-binding molecule is inserted. By infecting silkworms with baculovirus, the target antigen can be obtained from the silkworms' bodily fluids. The binding molecules can be recovered.

[0263] Furthermore, when plants are used to produce the antigen-binding molecules of the present invention, for example, tobacco can be used. To obtain. When using tobacco, the polynucleotide encoding the target antigen-binding molecule is inserted. The plant expression vector, for example pMON 530, is used in Agrobacterium tumefacier. It is introduced into bacteria such as Agrobacterium tumefaciens. Tobacco infected with the virus, for example, from the leaves of Nicotiana tabacum, The desired antigen-binding molecule can be recovered (Ma et al., Eur. J. Immunol. (1994) 24, 131-8). Also, clones of duckweed (Lemna minor) infected with similar bacteria. Desired antigen-binding molecules can be recovered from cells (Cox KM et al. Nat. Biotechnol.). (2006) (12), 1591-1597).

[0264] As an example of a glycosylation receptor binding domain included in the antigen-binding molecule in the present invention, a desired sugar When a glycosylated receptor-binding domain is used, sugar is attached to the glycosylated receptor-binding domain. A known method can be used to join the chains. For example, an infertile with protein secretion ability A cell extract is prepared from cultured cells of a dead mammalian cell line, and an antibody is encoded in the extract. Post-translationally modified antibodies in a cell-free protein synthesis system characterized by the addition of mRNA. A series of cell-free enzyme methods, such as the manufacturing method (Japanese Patent Publication No. 2006-141241), can produce a desired sugar chain. This can be used in methods for producing bound glycosylation receptor-binding domains. Such antigen-binding molecules The manufacturing method of the present invention may also include a step of further processing using an enzyme.

[0265] As described above, the sugar chain structure of the antigen-binding molecule produced may be single, but it may also consist of multiple sugars. It can also be produced as a mixture of linked chains. In this invention, such mixtures are also It can be suitably used. Also, antigen-binding components in which a specific sugar chain is linked to its sugar chain-binding domain. Children can also be suitably used in the present invention.

[0266] In the present invention, a specific glycan is linked to the glycan receptor binding domain contained in the antigen-binding molecule. Several known methods can be employed as ways to achieve this, and one such known method is: The sugar chains of antigen-binding molecules, whether naturally occurring or artificially produced by recombinant gene techniques, etc. One method involves utilizing the properties of antigen-binding molecules that have specific sugar chains to purify them. Antibodies with high mannose-type glycans were found using affinity chromatography with ConA-sepharose. It is known to be purified using Graph (Millward (Biologicals (2008) 36, 49-60)). Such purification methods are used in the present invention for N-linked types where the non-reducing end is mannose. It can be used to create antigen-binding molecules that contain sugar chains.

[0267] If necessary, the antigen-binding molecule may be treated with an appropriate protein-modifying enzyme before or after purification. By doing so, modifications are arbitrarily added, and modified molecules such as peptides are partially or completely modified. It can be removed. Examples of protein-modifying enzymes include trypsin, chymotrypsin, and lycopherol. Sylendopeptidase, protein kinase, glucosidase, etc., can be used. The manufacturing method of this invention also includes a step of further treating antigen-binding molecules, such as those mentioned above, with an enzyme. It may be included.

[0268] In the present invention, a specific glycan is linked to the glycan receptor binding domain contained in the antigen-binding molecule. Several known methods can be employed as a means to achieve this, but the antigen-binding molecule having a specific sugar chain is selected. Enzymatic treatment may also be employed as appropriate for the purpose of obtaining benefits. As will be described later in the examples, the ends are gas An antigen-binding molecule having an N-linked sugar chain that is lactose, and a complex sugar whose terminal is sialic acid. It can be produced from antigen-binding molecules with chains by sialidase treatment. Antibodies containing glycans are treated with sialidase and β-galactosidase to convert galactose into galactose. It is known that it is produced in a form in which it is removed from its sugar chain (Newkirk(Clin.Exp.Immu nol. (1996) 106, 259-264). These sialidase and β-galactosidase processes The manufacturing method, which includes the principle, is an antigenic chain having an N-linked sugar chain whose terminus is mannose, in the present invention. It can be used to create composite molecules. Further enzymes can be applied to such antigen-binding molecules. The manufacturing method of this invention may also include a process of using and processing the material.

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

[0270] In a different embodiment, for the purpose of obtaining an antigen-binding molecule having a specific sugar chain of the present invention, the antibody A recombinant gene encoding the original binding molecule was introduced, causing a specific sugar chain to accumulate. Its glycosidase activity (including but not limited to genetic or recombinant gene techniques) Methods for recovery from the culture medium of altered host cells can also be employed as appropriate. High-mannose type N-tubules An antibody having a combined glycan is transduced into N-acetylcholine, which has a recombinant gene encoding the antibody. Culture medium of a Lec1 mutant cell derived from CHO cells lacking luglucosaminyltransferase I activity. It is known to be recovered more often (Wright and Morrison (J. Exp. Med. (1994) 180) , 1087-1096)). The present invention has an N-linked glycan whose non-reducing end is mannose. The antigen-binding molecule is cultured in a Lec1 mutant strain into which the recombinant gene encoding the molecule has been transduced. It can be recovered from the liquid.

[0271] In yet another embodiment, with the aim of obtaining an antigen-binding molecule having a specific sugar chain of the present invention, Inhibition of a specific glycosidase reaction during the culture of cells that produce the antigen-binding molecule. By adding the agent, antigen-binding molecules with specific sugar chains accumulated in the culture medium can be recovered. Methods such as the following may be adopted as appropriate. These methods include having high-mannose type N-linked glycans that do not contain fucose. The antibody is produced by adding kifunesine during the culture of CHO cells that produce that antibody. It is known to be recovered from the culture medium of the cells in question (Zhou(Biotechnol. Bioeng. (2 008) 99, 652-665)). In the present invention, N is characterized by not having fucose and having a mannose terminus. Antigen-binding molecules with linked sugar chains, for example, have a recombinant gene encoding that molecule that exhibits a specific trait. The introduced CHO cells were harvested from the culture medium by adding kifunesine and culturing them. This is possible. Also, for example, when culturing host cells in which the glycosidase activity described above has been altered, By combining these methods of adding inhibitors, the specific glycans of the present invention can also be controlled. It is possible to obtain antigen-binding molecules through such combinations. It is known that antibodies possessing this property can be recovered (Kanda et al. (Glycobiology (2007) 17, 104- 118)) In the present invention, for the purpose of obtaining antigen-binding molecules having a specific sugar chain, a similar set A combination may be adopted as appropriate.

[0272] As described above, the sugar chain structure of the antigen-binding molecule produced may be single, but it may also consist of multiple sugars. It can also be produced as a mixture of linked chains. In this invention, such mixtures are also It can be suitably used. Also, antigen-binding components in which a specific sugar chain is linked to its sugar chain-binding domain. Children can also be suitably used in the present invention.

[0273] The antigen-binding molecules obtained in this way are found inside or outside the host cell (in culture medium, milk, etc.). It can be isolated from and purified as a substantially pure and homogeneous antigen-binding molecule. The separation and purification methods used in separation and purification are the same as those used for the purification of typical polypeptides. The separation and purification methods used are combined and appropriately selected according to the purpose. However, it is not limited to a specific method. For example, chromatography columns, Filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamine Midgel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc., are selected and combined as appropriate to detect the antigen. 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, absorption Examples include chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatography - Using liquid-phase chromatography, such as HPLC or FPLC It can be done. For affinity chromatography, the column used is protein Examples include a Protein A column and a Protein G column. For example, a column using Protein A. Examples include Hyper D, POROS, and Sepharose FF (manufactured by Pharmacia).

[0275] The antigen-binding molecule produced as described above exhibits antigen-binding activity, particularly towards FcRn and human FcRn. Whether or not it possesses the desired properties in terms of synergistic activity, glycosylation receptor binding activity, etc., is as described above. The antigen-binding activity, FcRn-binding activity, or glycosylation receptor-binding activity were evaluated using the methods described. It is valued. As a result of such evaluation, the process of designing the motif of the glycosylation receptor binding domain, And encoding polypeptides of antigen-binding molecules containing a glycosylated receptor-binding domain motif. The process of creating the gene for which the desired trait is obtained is repeated one or more times to obtain the desired trait. Antigen-binding molecules are produced.

[0276] Antigen-binding molecule containing a glycosylation receptor-binding domain of the present invention, particularly human-derived glycosylation receptor-binding molecule. Antigen-binding molecules containing domains bind to glycosylation receptors in a pH-dependent manner, and, , or, in the pH neutral range, glycan receptor binding activity, particularly human-derived glycan receptor binding activity It possesses binding activity to glycan receptors in the acidic pH range and binding activity to glycan receptors in the neutral pH range. If the activity level can be reduced to a lower level, it will promote the uptake of antigens into cells by antigen-binding molecules. Therefore, the administration of antigen-binding molecules promotes a decrease in the antigen concentration in the plasma, and the drug of antigen-binding molecules It is possible to improve dynamics and increase the number of antigens that a single antigen-binding molecule can bind to. ru.

[0277] The antigen-binding molecule produced by the manufacturing method of the present invention, upon administration, reduces the antigen concentration in plasma. It is an antigen-binding molecule that promotes the reduction of [something]. Therefore, the manufacturing method of the present invention, by administering it This method can be used as a method for producing antigen-binding molecules that promote a decrease in antigen concentration in plasma.

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

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

[0280] Furthermore, the antigen-binding molecules produced by the manufacturing method of the present invention are suitable for humans, mice, monkeys, etc. When administered to animals, the antigen bound to the antigen-binding molecule outside the cell is then transferre...

Claims

1. A method for increasing the in vivo or in vitro antigen elimination ability of an antigen-binding molecule, comprising increasing the number of binding domains to a glycan receptor in an antigen-binding molecule that includes an antigen-binding domain, an FcRn-binding domain, and one or more binding domains to a glycan receptor, The antigen-binding domain exhibits higher binding activity to the antigen under high calcium ion concentration conditions of 2 mM than under low calcium ion concentration conditions of 3 μM. The aforementioned glycan receptor binding domain is a glycan, The aforementioned glycan receptor is an asialoglycoprotein receptor or a mannose receptor. A method characterized in that the antigen-binding molecule is an antibody.

2. The method according to claim 1, wherein at least one amino acid of the antigen-binding domain comprises at least one amino acid whose side chain pKa is 4.0-8.

0.

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

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

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

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

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

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

9. The method according to claim 8, wherein the binding activity of the glycan receptor binding domain to the glycan receptor under neutral pH conditions is higher than the binding activity under acidic pH conditions.

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

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

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

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

14. The method according to claim 13, wherein the N-linked sugar chain has three or more terminal galactoses.

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

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

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

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