HK1184710A

HK1184710A - Antigen-binding molecule binding to multiple antigen molecules repeatedly

Info

Publication number: HK1184710A
Application number: HK13112329.2A
Authority: HK
Inventors: 井川智之; 石井慎也; 前田敦彦; 中井贵士
Original assignee: 中外制药株式会社
Priority date: 2008-04-11
Filing date: 2013-11-01
Publication date: 2014-01-30

Description

Antigen binding molecules that repeatedly bind to multiple molecules of antigen

The application is a divisional application, the application date of the original application is 4 months and 10 days in 2009, the application number is 200980122466.6(PCT/JP2009/057309), and the invention name is 'antigen binding molecule repeatedly binding with multiple molecules of antigen'.

Technical Field

The present invention relates to a method for improving the pharmacokinetics of an antigen-binding molecule, a method for increasing the number of times an antigen-binding molecule binds to an antigen, an antigen-binding molecule with improved pharmacokinetics, an antigen-binding molecule with increased number of times an antigen-binding molecule binds to an antigen, and a method for producing the same.

Background

Antibodies have attracted attention as pharmaceutical products because of their high stability in plasma and few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs are currently under development (non-patent documents 1 and 2). On the other hand, as a technique applicable to 2 nd generation antibody drugs, various techniques have been developed, and techniques for improving effector functions, antigen binding ability, pharmacokinetics, and stability, techniques for reducing the risk of immunogenicity, and the like have been reported (non-patent document 3). The problems are considered to be: since antibody drugs are generally administered in very large amounts, it is difficult to prepare subcutaneous formulations; and high manufacturing cost. As a method for reducing the amount of an antibody drug to be administered, a method for improving the pharmacokinetics of an antibody and a method for improving the affinity (affinity) between an antibody and an antigen are considered.

As a method for improving the pharmacokinetics of antibodies, artificial amino acid substitutions in the constant region have been reported (non-patent documents 4 and 5). As a technique for enhancing the antigen binding ability and the antigen neutralizing ability, an affinity maturation technique has been reported (non-patent document 6), in which the binding activity to an antigen can be enhanced by introducing a mutation into an amino acid in a CDR region or the like of a variable region. By enhancing the antigen-binding ability, the biological activity in vitro can be improved, or the dose can be reduced, and the drug efficacy in vivo can be further improved (non-patent document 7).

On the other hand, the amount of antigen that can be neutralized by 1 molecule of antibody depends on affinity, and by enhancing affinity, the antigen can be neutralized with a small amount of antibody, and the affinity of the antibody can be enhanced by various methods. Further, if the antibody is covalently bound to an antigen to have infinite affinity, 1 molecule of the antigen can be neutralized with 1 molecule of the antibody (if the antibody is bivalent, two molecules of the antigen are neutralized). However, in the conventional method, the chemical quantity theory of neutralization reaction for neutralizing 1 molecule of antigen with 1 molecule of antibody (neutralizing two molecules of antigen when the antibody is bivalent) is limited, and the antigen cannot be completely neutralized with an amount of antibody equal to or less than the amount of antigen. That is, there is a limit to the effect of enhancing affinity (non-patent document 9). In the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period of time, it is necessary to administer an amount of the antibody equal to or larger than the amount of the antigen produced in vivo during the period of time.

Therefore, in order to continue the neutralizing effect of the antigen for a target period of the amount of the antibody equal to or less than the amount of the antigen, it is necessary to neutralize a plurality of antigens with 1 antibody. As a method for neutralizing a plurality of antigens with 1 antibody, there is inactivation of antigens with a catalytic antibody that imparts a catalytic function to the antibody. In the case of a protein antigen, it is considered that the antigen can be inactivated by hydrolyzing the peptide bond of the antigen, and the antigen can be repeatedly neutralized (inactivated) by catalyzing the hydrolysis reaction with the antibody (non-patent document 8). Many catalytic antibodies and catalytic antibody production techniques have been reported so far, but no catalytic antibody having sufficient catalytic activity as a pharmaceutical product has been reported. That is, in an in vivo test of an antibody against a certain antigen, a catalytic antibody that exerts an effect equal to or more than that of a normal neutralizing antibody having no catalytic function at a low dose or that can exert an effect more continuously at the same dose has not been reported so far.

Thus, there is no report on an antibody that can exhibit an in vivo effect better than that of a normal neutralizing antibody by neutralizing a plurality of antigens with 1-molecule antibody, and it is desired to develop a novel antibody production technique that can exhibit an effect more than that of a normal neutralizing antibody in vivo by neutralizing a plurality of antigens with 1-molecule antibody in order to reduce the dose and prolong the duration.

The prior art documents of the present invention are as follows.

Documents of the prior art

Non-patent document

Non-patent document 1: monoclonal antibodies in the clinic, Janic MReichert, Clark J Rosenssweig, Laura B Faden and Matthew C Dewitz, Nature Biotechnology23, 1073-.

Non-patent document 2: pavlou AK, Belsey MJ. the therapeutic antibodies marketto2008.Eur J Pharm Biopharm.2005 Apr; 59(3): 389-96.

Non-patent document 3: kim SJ, Park Y, Hong hj, antibody engineering for the purification of therapeutic antibodies, mol cells, 2005 aug31; 20(1): review.

Non-patent document 4: hinton PR, Xiong JM, Johnfs MG, Tang MT, Keller S, Tsurushita N.an engineered human IgG1antibody with ringer serum half-life.J Immunol.2006Jan 1; 176(1): 346-56.

Non-patent document 5: ghetie V, Popov S, Borvak J, Radu C, matheoi D, Medesan C, Ober RJ, Ward es.incore the serum persistence of igg fragment by random mutagenesis nat mutigenesis.nat biotechnol.1997 jul; 15(7): 637-40.

Non-patent document 6: proc Natl Acad Sci U S.2005Jun14; 102(24): 8466-71.Epub2005Jun6.A general method for making a great deal of the affinity of the antibodies by using the combined library of Rajpal A, Beyaz N, Haber L, Cappuccill G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R.

Non-patent document 7: wu H, Pfar DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. development of Motavizumab, an Ultra-potential Antibody for the prediction of Respiratory synthetic Virus Infection in the Upper and Lower Respiratory tract transfer. Jmol biol.2007, 368, 652-665.

Non-patent document 8: hanson CV, Nishiyama Y, Paul s. catalytic antibodies and hair applications. curr Opin biotechnol.2005 dec; 16(6): 631-6.

Non-patent document 9: rathanawami P, Roalsad S, Roskos L, Su QJ, Lackie S, Babcook J.Demonstroction of an in vivo generated sub-picolar affinity to intracellular human monoclonal antibody-8. Biochem Biophys ResCommun.2005Sep9; 334(4): 1004-13.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above circumstances, and an object thereof is to provide: methods for multiple binding of antigen binding molecules to antigens, methods for enhancing the pharmacokinetics of antigen binding molecules, antigen binding molecules that can be bound to antigens multiple times, antigen binding molecules with improved pharmacokinetics, pharmaceutical compositions containing the antigen binding molecules, and methods for their preparation.

Means for solving the problems

The present inventors have conducted intensive studies on a method of binding a polypeptide having an antigen-binding ability, such as an antigen-binding molecule, to an antigen multiple times and a method of improving a half-life (blood half-life) in plasma (improving pharmacokinetics). As a result, the present inventors have found that: antigen-binding molecules with weak antigen-binding activity at pH in early intranuclear bodies bind antigen multiple times and have a long half-life in plasma compared to antigen-binding activity at pH in plasma (in blood).

The present invention relates to a method for binding an antigen-binding molecule to an antigen multiple times, a method for improving pharmacokinetics of an antigen-binding molecule, an antigen-binding molecule capable of binding to an antigen multiple times, an antigen-binding molecule with improved pharmacokinetics, a method for producing an antigen-binding molecule with improved pharmacokinetics, and the like, and more specifically, the present invention provides the following [1] to [50 ].

[1] An antigen-binding molecule, wherein the ratio of KD at pH5.8 to KD at pH7.4, i.e., the value of KD (pH5.8)/KD (pH7.4), is 2 or more.

[2] [1] the antigen-binding molecule according to any one of the above aspects, wherein the value of KD (pH5.8)/KD (pH7.4) is 10 or more.

[3] [1] the antigen-binding molecule according to any one of the above aspects, wherein the value of KD (pH5.8)/KD (pH7.4) is 40 or more.

[4] The antigen-binding molecule according to any one of [1] to [3], wherein: at least 1 amino acid is substituted with histidine, or at least 1 histidine is inserted.

[5] The antigen-binding molecule according to any one of [1] to [4], wherein: the antigen binding molecules have antagonistic activity.

[6] The antigen-binding molecule according to any one of [1] to [5], wherein: the antigen binding molecule binds to a membrane antigen or a soluble antigen.

[7] The antigen-binding molecule according to any one of [1] to [6], wherein: the antigen binding molecule is an antibody.

[8] A pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to [7 ].

[9] A method of increasing the pharmacokinetics of an antigen binding molecule by making the antigen binding activity of the antigen binding molecule at ph5.8 less active than the antigen binding activity at ph 7.4.

[10] A method of increasing the number of times an antigen-binding molecule binds to an antigen by making the antigen-binding activity of the antigen-binding molecule at ph5.8 less than the antigen-binding activity at ph 7.4.

[11] A method of increasing the number of antigens to which an antigen binding molecule can bind by making the antigen binding activity of the antigen binding molecule at ph5.8 weaker than the antigen binding activity at ph 7.4.

[12] A method for dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly by making the antigen-binding activity of the antigen-binding molecule at pH5.8 weaker than the antigen-binding activity at pH 7.4.

[13] A method for releasing an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen, by making the antigen-binding activity of the antigen-binding molecule at pH5.8 weaker than the antigen-binding activity at pH 7.4.

[14] A method of increasing the antigen-elimination capacity of an antigen-binding molecule in plasma by making the antigen-binding activity of the antigen-binding molecule at ph5.8 weaker than the antigen-binding activity at ph 7.4.

[15] The method according to any one of [9] to [14], characterized in that: the ratio of KD at pH5.8 to KD at pH7.4, i.e., the value of KD (pH5.8)/KD (pH7.4), is 2 or more.

[16] The method according to any one of [9] to [14], characterized in that: the KD (pH5.8)/KD (pH7.4) value is 10 or more.

[17] The method according to any one of [9] to [14], characterized in that: the KD (pH5.8)/KD (pH7.4) value is 40 or more.

[18] A method of improving pharmacokinetics by substituting histidine for at least 1 amino acid or inserting at least 1 histidine in an antigen binding molecule.

[19] A method of increasing the number of times an antigen binding molecule binds to an antigen by substituting at least 1 amino acid of the antigen binding molecule with histidine or inserting at least 1 histidine.

[20] A method of increasing the number of antigens to which an antigen binding molecule can bind by substituting at least 1 amino acid of the antigen binding molecule with histidine or inserting at least 1 histidine.

[21] A method of intracellularly dissociating an antigen bound to an antigen binding molecule from the antigen binding molecule by substituting at least 1 amino acid of the antigen binding molecule with histidine or inserting at least 1 histidine.

[22] A method for releasing an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen, by substituting at least 1 amino acid of the antigen-binding molecule with histidine or inserting at least 1 histidine.

[23] A method of increasing the antigen-elimination capacity in the plasma of an antigen-binding molecule by substituting at least 1 amino acid of the antigen-binding molecule with histidine or inserting at least 1 histidine.

[24] The method according to any one of [18] to [23], characterized in that: by substituting histidine or inserting histidine, the ratio of the antigen-binding activity at pH5.8 to the antigen-binding activity at pH7.4, i.e., the value of KD (pH5.8)/KD (pH7.4) is increased as compared to that before histidine substitution or insertion.

[25] The method according to any one of [9] to [24], characterized in that: the antigen binding molecules have antagonistic activity.

[26] The method according to any one of [9] to [25], characterized in that: the antigen binding molecule binds to a membrane antigen or a soluble antigen.

[27] The method according to any one of [9] to [26], characterized in that: the antigen binding molecule is an antibody.

[28] A method of screening for antigen binding molecules, the method comprising the steps of:

(a) obtaining the antigen binding activity of the antigen binding molecule at pH6.7-pH10.0;

(b) obtaining the antigen binding activity of the antigen binding molecule at pH4.0-pH6.5;

(c) a step of selecting an antigen-binding molecule having an antigen-binding activity at pH6.7 to pH10.0 that is higher than the antigen-binding activity at pH4.0 to pH 6.5.

[29] [28] the screening method according to any one of the preceding methods, comprising: selecting an antibody having an antigen binding activity at pH6.7 to pH10.0 which is two times or more the antigen binding activity at pH4.0 to pH6.5.

[30] A method of screening for antigen binding molecules, the method comprising the steps of:

(a) a step of binding the antigen-binding molecule to an antigen under a condition of pH6.7 to pH 10.0;

(b) a step of subjecting the antigen-binding molecule that binds to the antigen of (a) to a pH of 4.0 to 6.5;

(c) a step of obtaining an antigen-binding molecule that dissociates at a pH of 4.0 to 6.5.

[31] A method of screening for antigen binding molecules having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(a) a step of binding an antigen-binding molecule to the column on which the antigen is immobilized under a first pH condition;

(b) a step of eluting the antigen-binding molecule bound to the column under the first pH condition from the column under the second pH condition;

(c) a step of obtaining the eluted antigen binding molecule.

[32] A method of screening for antigen binding molecules having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(a) a step of binding the antigen-binding molecule library to the column on which the antigen is immobilized under a first pH condition;

(b) a step of eluting the antigen binding molecule from the column under second pH conditions;

(c) a step of amplifying a gene encoding the eluted antigen-binding molecule;

(d) A step of obtaining the eluted antigen binding molecule.

[33] The screening method according to [31] or [32], which comprises: the first pH is pH6.7 to pH10.0, and the second pH is pH4.0 to pH 6.5.

[34] The screening method according to any one of [28] to [33], wherein the antigen-binding molecule is one in which at least 1 or more amino acids in the antigen-binding molecule are substituted with histidine or at least 1 histidine is inserted.

[35] [28] to [33], for the purpose of obtaining an antigen-binding molecule having excellent plasma retention.

[36] The screening method according to any one of [28] to [33], wherein the method is intended to obtain an antigen-binding molecule capable of binding to an antigen twice or more.

[37] The screening method according to any one of [28] to [33], wherein the number of antigens that can be bound is larger than the number of antigens that can be bound to the antigen binding site.

[38] The screening method according to any one of [28] to [33], for the purpose of obtaining an antigen-binding molecule that can dissociate an antigen bound extracellularly in a cell.

[39] The screening method according to any one of [28] to [33], wherein the antigen-binding molecule is taken into a cell in a state of being bound to an antigen and released to the outside of the cell in a state of not being bound to the antigen.

[40] [28] to [33], wherein the antigen-binding molecule having an increased ability to eliminate an antigen in plasma is obtained.

[41] The screening method according to any one of [28] to [40], wherein the antigen-binding molecule is an antigen-binding molecule used as a pharmaceutical composition.

[42] The screening method according to any one of [28] to [41], wherein: the antigen binding molecule is an antibody.

[43] A method of making an antigen binding molecule, the method comprising the steps of:

(c) selecting an antigen-binding molecule having an antigen-binding activity at pH6.7 to pH10.0 which is higher than that at pH4.0 to pH6.5;

(d) a step of obtaining a gene encoding the antigen binding molecule selected in (c);

(e) a step of preparing an antigen-binding molecule using the gene obtained in (d).

[44] A method of making an antigen binding molecule, the method comprising the steps of:

(c) A step of obtaining an antigen-binding molecule that dissociates at a pH of 4.0 to 6.5;

(d) a step of obtaining a gene encoding the antigen-binding molecule obtained in (c);

[45] A method of preparing an antigen binding molecule having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(c) a step of obtaining an eluted antigen binding molecule;

[46] A method for preparing an antigen binding molecule having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(c) A step of amplifying a gene encoding the eluted antigen-binding molecule;

(d) a step of obtaining an eluted antigen binding molecule;

(e) a step of obtaining a gene encoding the antigen-binding molecule obtained in (d);

(f) a step of preparing an antigen-binding molecule using the gene obtained in (e).

[47] [45] the production method according to [46], which is characterized in that: the first pH is pH6.7 to pH10.0, and the second pH is pH4.0 to pH 6.5.

[48] The production method according to any one of [43] to [47], which further comprises: a step of substituting at least 1 or more amino acids in the antigen-binding molecule with histidine or inserting at least 1 histidine.

[49] The production method according to any one of [43] to [48], characterized in that: the antigen binding molecule is an antibody.

[50] A pharmaceutical composition comprising the antigen-binding molecule produced by the production method according to any one of [43] to [49 ].

Effects of the invention

According to the present invention, there is provided a method of repeatedly binding 1 molecule of an antigen-binding molecule to a plurality of antigens. 1 molecule of the antigen-binding molecule can improve the pharmacokinetics of the antigen-binding molecule by binding to a plurality of antigens, and can exert an effect superior to that of a common antigen-binding molecule in vivo.

Drawings

FIG. 1 is a diagram showing the degradation pathway of an antibody that binds to a membrane-type antigen.

Figure 2 is a diagram showing the salvage mechanism of FcRn-based IgG molecules.

FIG. 3 is a schematic diagram showing the dissociation of IgG molecules from membrane-type antigens in vivo in the nucleus, thereby re-binding to new antigens.

FIG. 4 is a schematic showing the dissociation of IgG molecules from soluble antigens within the nucleus, thereby re-binding to new antigens.

FIG. 5 is a graph showing column panning with immobilized antigen.

FIG. 6 is a graph showing the results of phage ELISA of clones obtained by column panning. The upper section is WT and the lower section is CL 5.

FIG. 7 is a graph showing the biological neutralizing activity of pH-dependent binding of anti-IL-6 receptor antibodies.

FIG. 8 is a graph showing the results of Biacore sensorgrams of pH-dependent binding of anti-IL-6 receptor antibody to soluble IL-6 receptor at pH 7.4. The uppermost is WT, with the 2 nd position from the top being H3pI/L73, the 3 rd position from the top being H170/L82, and the lowermost being CLH 5/L73.

FIG. 9 is a graph showing the results of Biacore sensorgrams of pH-dependent binding of anti-IL-6 receptor antibody to soluble IL-6 receptor at pH 5.8. The uppermost is WT, with the 2 nd position from the top being H3pI/L73, the 3 rd position from the top being H170/L82, and the lowermost being CLH 5/L73.

FIG. 10 is a graph showing the results of Biacore sensorgrams of the binding (pH7.4) and dissociation (pH5.8) of a pH-dependent binding anti-IL-6 receptor antibody to a membrane-type IL-6 receptor. The uppermost is WT, with the 2 nd position from the top being H3pI/L73, the 3 rd position from the top being H170/L82, and the lowermost being CLH 5/L73.

FIG. 11 is a sensorgram showing Biacore binding of pH-dependent binding anti-IL-6 receptor antibody to SR344 repeatedly.

FIG. 12 is a graph showing the total antigen binding in pH dependent binding of anti-IL-6 receptor antibody and SR344 repeated binding assays.

FIG. 13 is a graph showing the change in antibody concentration in plasma of pH-dependent binding anti-IL-6 receptor antibody in human IL-6 receptor transgenic mice.

FIG. 14 is a graph showing the change in antibody concentration in plasma of cynomolgus monkeys with pH-dependent binding of anti-IL-6 receptor antibody.

FIG. 15 is a graph showing the change in CRP concentration in cynomolgus monkeys with pH-dependent binding of anti-IL-6 receptor antibody.

FIG. 16 is a graph showing the change in concentration of non-binding cynomolgus monkey IL-6 receptor in cynomolgus monkeys with pH-dependent binding of anti-IL-6 receptor antibody.

FIG. 17 is a graph showing the results of Biacore sensorgrams of the binding (pH7.4) and dissociation (pH5.8) of a pH-dependent binding anti-IL-6 receptor antibody to a membrane-type IL-6 receptor. WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 in this order.

FIG. 18 is a graph showing the change in antibody concentration in plasma of WT, H3pI/L73-IgG1, Fv2-IgG1, Fv4-IgG1 in human IL-6 receptor transgenic mice with pH-dependent binding of anti-IL-6 receptor antibody.

FIG. 19 is a graph showing the results of Biacore sensorgrams of the binding (pH7.4) and dissociation (pH5.8) of a pH-dependent binding anti-IL-6 receptor antibody to a membrane-type IL-6 receptor. The sequence of the above is WT, Fv4-IgG1, Fv4-IgG2, and Fv 4-M58.

FIG. 20 is a graph showing the change in antibody concentration in plasma of WT, Fv4-IgG1, Fv4-IgG2, Fv4-M58 in human IL-6 receptor transgenic mice with pH-dependent binding of anti-IL-6 receptor antibody.

FIG. 21 is a graph showing the results of Biacore sensorgrams of the binding (pH7.4) and dissociation (pH5.8) of a pH-dependent binding anti-IL-6 receptor antibody to a membrane-type IL-6 receptor. The sequence from the top is Fv1-M71, Fv1-M73, Fv3-M71 and Fv 3-M73.

FIG. 22 is a graph showing the change in antibody concentration in plasma of pH-dependent binding anti-IL-6 receptor antibody upon administration of H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, Fv4-M73, and high affinity antibody at 1.0mg/kg in cynomolgus monkeys.

FIG. 23 is a graph showing the change in CRP concentration in the groups of H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, Fv4-M73, and high affinity Ab administration, which bind to an anti-IL-6 receptor antibody in a cynomolgus monkey in a pH-dependent manner.

FIG. 24 is a graph showing the change in concentration of non-binding cynomolgus IL-6 receptor in the group of H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, Fv4-M73, and high affinity Ab that bind to an anti-IL-6 receptor antibody in a cynomolgus monkey in a pH-dependent manner.

Fig. 25 is a diagram showing FRI, FR2, FR3, FR4 and CDR1, CDR2, CDR3 of the heavy chains (VH1, VH2, VH3, VH4) and light chains (VL1, VL2, VL 3). Asterisks indicate sites where amino acid mutations are present in the aligned sequences.

FIG. 26 is a graph showing the results of Biacore sensorgrams of binding of anti-IL 6 clone 2 to IL-6 at pH7.4 and pH5.5 as a pH-dependent binding anti-IL-6 antibody. The graph at pH7.4 shows IL-6 at 100, 50, 25, 12.5 and 6.25ng/mL in this order from the top.

FIG. 27 is a graph showing the results of Biacore sensorgrams for binding of anti-IL 31R clone 1 to IL-31 receptor at pH7.4 and pH5.5 as a pH-dependent binding anti-IL-31 receptor antibody. The graph at pH5.5 shows the IL-31 receptor at 100, 50, 25, and 12.5ng/mL in this order from the top.

FIG. 28 is a graph showing the change in antibody concentration in plasma after intravenous administration of a mixed solution of SR344 and an anti-human IL-6 receptor antibody to mice.

FIG. 29 is a graph showing the change in the concentration of SR344 in plasma after intravenous administration of a mixed solution of SR344 and an anti-human IL-6 receptor antibody to mice.

Detailed Description

The present invention provides methods for increasing the number of times an antigen-binding molecule binds to an antigen. More specifically, methods are provided for increasing the number of times an antigen-binding molecule binds to an antigen by making the antigen-binding ability of the antigen-binding molecule weaker at acidic pH than at neutral pH. The present invention also provides a method of increasing the number of times an antigen-binding molecule binds to an antigen, characterized in that: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of increasing the number of times an antigen-binding molecule binds to an antigen, characterized in that: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The invention also provides methods of increasing the number of antigens to which an antigen binding molecule can bind. More specifically, methods are provided for increasing the number of antigens to which an antigen-binding molecule can bind by making the antigen-binding ability of the antigen-binding molecule weaker at acidic pH than at neutral pH. The present invention also provides a method of increasing the number of antigens to which an antigen binding molecule can bind, characterised in that: at least 1 amino acid in the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of increasing the number of antigens to which an antigen binding molecule can bind, characterised in that: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The invention also provides methods of dissociating an antigen bound to an antigen binding molecule extracellularly from the antigen binding molecule intracellularly. More specifically, a method is provided for dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly by making the antigen-binding ability of the antigen-binding molecule at acidic pH weaker than that at neutral pH. The present invention also provides a method of dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly, characterized by: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly, characterized by: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The present invention also provides a method for releasing an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen. More specifically, a method is provided in which an antigen-binding molecule taken up into a cell in a state of being bound to an antigen is released out of the cell in a state of not being bound to the antigen by making the antigen-binding ability of the antigen-binding molecule at acidic pH weaker than the antigen-binding ability at neutral pH. The present invention also provides a method for releasing an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen, characterized in that: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method for releasing an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen, characterized in that: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The invention also provides methods of increasing the antigen-elimination capacity in plasma of antigen-binding molecules. More specifically, the present invention provides methods for increasing the antigen-depleting capacity of an antigen-binding molecule in plasma by making the antigen-binding capacity of the antigen-binding molecule weaker at acidic pH than at neutral pH. The present invention also provides a method of increasing the antigen-elimination capacity in plasma of an antigen-binding molecule, characterized in that: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of increasing the antigen-elimination capacity in plasma of an antigen-binding molecule, characterized in that: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The invention also provides methods of improving the pharmacokinetics of antigen binding molecules. More specifically, methods are provided for increasing the pharmacokinetics (prolonged plasma retention) of antigen binding molecules by making the antigen binding ability of the antigen binding molecules weaker at acidic pH than at neutral pH. The present invention also provides a method of improving pharmacokinetics, which is characterized by: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of improving pharmacokinetics, which is characterized by: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

The invention also provides methods of increasing the antigen-elimination capacity in plasma of antigen-binding molecules. More specifically, methods are provided for increasing the antigen-elimination capacity in the plasma of antigen-binding molecules by making the antigen-binding ability of the antigen-binding molecule weaker at acidic pH than at neutral pH. The present invention also provides a method of increasing the antigen-elimination capacity in plasma of an antigen-binding molecule, characterized in that: at least 1 amino acid of the antigen binding molecule is substituted with or inserted into at least 1 histidine. The present invention also provides a method of increasing the antigen-elimination capacity in plasma of an antigen-binding molecule, characterized in that: substitution, deletion, addition and/or insertion of amino acids in the antibody constant region comprised by the antigen binding molecule.

In the present invention, the terms "improvement in pharmacokinetics", "improvement in pharmacokinetics" or "superior pharmacokinetics" may be used in the same sense as "improvement in retention in plasma (blood)", or "superior retention in plasma (blood)".

In the present invention, the fact that the antigen-binding ability at acidic pH is weaker than the antigen-binding ability at neutral pH means that the antigen-binding activity of the antigen-binding molecule at pH4.0 to pH6.5 is weaker than the antigen-binding activity at pH6.7 to pH 10.0. The antigen-binding activity of the antigen-binding molecule at pH5.5 to pH6.5 is preferably made weaker than the antigen-binding activity at pH7.0 to pH8.0, and the antigen-binding activity of the antigen-binding molecule at pH5.8 is particularly preferably made weaker than the antigen-binding activity at pH 7.4. Therefore, the acidic pH in the present invention is usually pH4.0 to pH6.5, preferably pH5.5 to pH6.5, and particularly preferably pH 5.8. In the present invention, the neutral pH is usually pH6.7 to pH10.0, preferably pH7.0 to pH8.0, and particularly preferably pH 7.4.

In the present invention, the description of "making the antigen-binding ability of the antigen-binding molecule at acidic pH weaker than the antigen-binding ability at neutral pH" can also be described as making the antigen-binding ability of the antigen-binding molecule at neutral higher than the antigen-binding ability at acidic pH. That is, in the present invention, the difference between the antigen binding ability of the antigen-binding molecule at acidic pH and the antigen binding ability at neutral pH may be increased (for example, as described later, the value of KD (pH5.8)/KD (pH7.4) may be increased). To increase the difference between the antigen binding capacity of the antigen binding molecule at acidic pH and the antigen binding capacity at neutral pH, for example, the antigen binding capacity at acidic pH may be decreased, the antigen binding capacity at neutral pH may be increased, or both may be increased.

In the measurement of the binding activity of the antigen, conditions other than pH can be appropriately selected by those skilled in the art, and are not particularly limited, and for example, the measurement can be performed in MES buffer at 37 ℃. The measurement of the antigen binding activity of an antigen binding molecule can be carried out by a method known to those skilled in the art, and for example, as described in the examples, measurement can be carried out using biacore (ge healthcare) or the like. When the antigen is a soluble antigen, the binding ability of the antigen-binding molecule to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip (chip) on which the antigen-binding molecule is immobilized; when the antigen is a membrane-type antigen, the binding ability of the antigen-binding molecule to the membrane-type antigen can be evaluated by allowing the antigen-binding molecule to flow as an analyte through the chip on which the antigen is immobilized.

In the present invention, as long as the antigen binding activity at acidic pH is weaker than the antigen binding activity at neutral pH, the difference between the antigen binding activity at acidic pH and the antigen binding activity at neutral pH is not particularly limited, but the ratio of KD (Dissociation constant: Dissociation constant) at pH5.8 to KD (pH5.8)/KD (pH7.4) of the antigen is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more, in terms of KD (pH5.8)/KD (pH 7.4). The upper limit of the value of KD (pH5.8)/KD (pH7.4) is not particularly limited, and any value such as 400, 1000, 10000, etc. may be used as long as it can be produced by the technique of those skilled in the art. As the value of the antigen binding activity, when the antigen is a soluble antigen, KD (dissociation constant) can be used; when the antigen is a membrane-type antigen, the Apparent KD (Apparent dissociation constant) can be used. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, and for example, biacore (ge healthcare), Scatchard plot (Scatchard plot), FACS, or the like can be used.

In the present invention, as another index indicating the difference between the antigen binding activity at acidic pH and the antigen binding activity at neutral pH, for example, the dissociation rate constant k can be used _d(Dissociation rate constant: Dissociation rate constant). As an index for indicating the difference in binding activity, when k is used_d(dissociation Rate constant) instead of KD, k at pH5.8 for antigen_d(dissociation Rate constant) and k at pH7.4_d(dissociation rate constant) ratio, i.e., k_d(pH5.8)/k_dThe value of (ph7.4) is preferably 2 or more, more preferably 5 or more, still more preferably 10 or more, and still more preferably 30 or more. To k is paired_d(pH5.8)/k_dThe upper limit of the value of (pH7.4) is not particularly limited, and may be any value such as 50, 100, 200, etc., as long as it can be made within the technical common knowledge of those skilled in the art.

As the value of the antigen binding activity, when the antigen is a soluble antigen, k can be used_d(dissociation rate constant); when the antigen is a membrane-type antigen, the apparent k can be used_d(apparent dissociation rate constant). k is a radical of_d(dissociation Rate constant) and apparent k_dThe apparent dissociation rate constant can be measured by a method known to those skilled in the art, and for example, biacore (ge healthcare), FACS, or the like can be used.

In the present invention, when the antigen binding activity of the antigen binding molecule is measured at different pH, it is preferable that the conditions other than pH are the same.

The method for making the antigen-binding activity of the antigen-binding molecule at pH5.8 weaker than that at pH7.4 (the method for imparting pH-dependent binding ability) is not particularly limited, and can be carried out by any method. Mention may be made of: for example, a method in which an amino acid in the antigen-binding molecule is substituted with histidine or histidine is inserted into the antigen-binding molecule, whereby the antigen-binding activity at pH5.8 is weaker than that at pH 7.4. It is known that pH-dependent antigen-binding activity can be imparted to an antibody by substituting histidine for an amino acid in the antibody (FEBS Letter, 309(1), 85-88 (1992)). The site at which the histidine mutation (substitution) or insertion is introduced (carried out) is not particularly limited, and any site may be used as long as the antigen-binding activity at pH5.8 is weaker than that at pH7.4 (the value of KD (pH5.8)/KD (pH7.4) is larger than that before the mutation or insertion. For example, when the antigen binding molecule is an antibody, a variable region of the antibody and the like can be mentioned. The number of mutations or insertions of histidine introduced (to be performed) can be determined as appropriate by those skilled in the art, and histidine may be substituted for only 1 site, or histidine may be inserted for only 1 site, or histidine may be substituted for a plurality of sites at 2 or more sites, or histidine may be inserted for a plurality of sites at 2 or more sites. Mutations other than histidine mutations (mutations into amino acids other than histidine) may also be introduced simultaneously. Histidine mutations and histidine insertions may also be performed simultaneously. The histidine substitution or histidine insertion can be performed randomly by a method known to those skilled in the art, such as substituting alanine in an alanine partition (scanning) for a histidine partition in histidine, and an antigen-binding molecule having a larger KD (ph5.8)/KD (ph7.4) value than before the mutation can be selected from a library of antigen-binding molecules into which a histidine mutation or insertion has been randomly introduced.

When an amino acid of the antigen-binding molecule is substituted with histidine or histidine is inserted into an amino acid of the antigen-binding molecule, although not particularly limited, the antigen-binding activity of the antigen-binding molecule after histidine substitution or insertion at ph7.4 is preferably equivalent to the antigen-binding activity of the antigen-binding molecule before histidine substitution or insertion at ph 7.4. Here, the antigen binding activity of the antigen-binding molecule after histidine substitution or insertion at ph7.4 is equivalent to the antigen binding activity of the antigen-binding molecule before histidine substitution or insertion at ph7.4, and means that the antigen-binding molecule after histidine substitution or insertion maintains 10% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more of the antigen-binding activity of the antigen-binding molecule before histidine substitution or insertion. When the antigen-binding activity of the antigen-binding molecule is lowered by histidine substitution or insertion, the antigen-binding activity can be made equivalent to that before histidine substitution or insertion by substitution, deletion, addition, and/or insertion of 1 or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules having equivalent binding activity by substitution, deletion, addition and/or insertion of 1 or more amino acids after histidine substitution or insertion.

Other methods for making the antigen-binding activity of the antigen-binding molecule at ph5.8 weaker than the antigen-binding activity at ph7.4 include: methods of substituting or inserting an amino acid in an antigen binding molecule with an unnatural amino acid. It is known that the pKa of unnatural Amino acids can be artificially controlled (Angew. Chem. int. Ed.2005, 44, 34, Chem Soc Rev.2004 Sep10; 33 (7): 422-30, Amino acids.1999; 16 (3-4): 345-79). Therefore, in the present invention, an unnatural amino acid may be used in place of histidine. The histidine substitution and/or insertion and the substitution and/or insertion of the unnatural amino acid may be performed simultaneously. The unnatural amino acid used in the present invention may be any unnatural amino acid, and any unnatural amino acid known to those skilled in the art can be used.

In addition, when the antigen-binding molecule is a substance containing an antibody constant region, as another method for making the antigen-binding activity of the antigen-binding molecule at pH5.8 weaker than the antigen-binding activity at pH7.4, there can be mentioned: methods of modifying the constant regions of antibodies contained in antigen binding molecules. Specific examples of such modifications of the antibody constant region are: such as the substitution to constant regions described in the examples.

Examples of the method for modifying the antibody constant region include: for example, a method of selecting an isotype in which the antigen-binding activity is decreased at pH5.8 (the dissociation rate is increased at pH5.8) by examining a plurality of isotypes of the constant region (IgG1, IgG2, IgG3, IgG 4). Mention may also be made of: a method in which an amino acid substitution is introduced into an amino acid sequence of a wild-type isotype (wild-type IgG1, IgG2, IgG3, IgG4 amino acid sequences), whereby the antigen-binding activity at pH5.8 is reduced (the dissociation rate at pH5.8 is increased). Since the sequence of the hinge region of the antibody constant region greatly differs depending on the isotype (IgG1, IgG2, IgG3, IgG4), and the difference in the amino acid sequence of the hinge region greatly affects the antigen-binding activity, by selecting an appropriate isotype depending on the type of antigen or epitope, it is possible to select an isotype in which the antigen-binding activity is reduced at ph5.8 (the dissociation rate at ph5.8 is increased). Since the difference in amino acid sequence of the hinge region greatly affects the antigen-binding activity, the hinge region is considered to be preferable as the amino acid substitution site of the amino acid sequence of the wild type isoform.

When the antigen-binding activity of the antigen-binding substance at pH5.8 is made weaker than that at pH7.4 (the value of KD (pH5.8)/KD (pH7.4) is made larger by the above-mentioned method or the like), although not particularly limited, it is preferable that the value of KD (pH5.8)/KD (pH7.4) is usually 2 times or more, preferably 5 times or more, and more preferably 10 times or more as compared with the original antibody.

In the present invention, "improvement of pharmacokinetics" includes not only a prolonged period of time from administration of the antigen-binding molecule to elimination from plasma (for example, until the antigen-binding molecule is degraded in cells and cannot return to plasma) but also a prolonged period of time during which the antigen-binding molecule is allowed to bind to an antigen (for example, the antigen-binding molecule is not bound to an antigen) and remains in plasma from the administration to the elimination from plasma. Even if an antigen-binding molecule is present in plasma, the antigen-binding molecule cannot bind to a new antigen when the antigen has bound to the antigen-binding molecule. Therefore, if the time during which the antigen-binding molecule does not bind to the antigen is long, the time during which the antigen-binding molecule can bind to a new antigen is long (the chance of binding to a new antigen is increased), and the time during which the antigen does not bind to the antigen-binding molecule in vivo can be reduced (in other words, the time during which the antigen-binding molecule binds to the antigen can be extended). For example, the proportion of antigen bound to the antigen binding molecule relative to the amount of antigen present in the plasma (the total amount of molecules bound to the antigen binding molecule and antigen not bound to the antigen binding molecule) generally decreases over time after administration of the antigen binding molecule. However, if the time during which the antigen-binding molecule is retained in a state in which it can bind to the antigen is long, the decrease can be suppressed (for example, the degree of decrease is reduced), and as a result, the ratio of the antigen bound to the antigen-binding molecule to the antigen present in the body becomes high after a certain period of time has elapsed since the administration of the antibody.

That is, the "improvement in pharmacokinetics" in the present invention does not necessarily mean that the time from administration of the antigen-binding molecule to elimination of the antigen-binding molecule is prolonged (lengthened). The following are also included in the "improvement of pharmacokinetics" of the present invention: even if the time from administration to elimination of the antigen-binding molecule does not vary, the time during which the antigen-binding molecule remains in the plasma in a state in which it can bind to the antigen (for example, in a state in which the antigen-binding molecule does not bind to the antigen) becomes long; and a case where the time during which the antigen in vivo is not bound to the antigen-binding molecule is reduced (in other words, the time during which the antigen-binding molecule is bound to the antigen is lengthened); or a case where the ratio of the antigen bound to the antigen-binding molecule becomes high relative to the antigen present in the body. Therefore, the "improvement in pharmacokinetics" of the present invention includes at least the following (1) to (4).

(1) The time from administration of the antigen binding molecule to elimination of the antigen binding molecule from the plasma is extended.

(2) After administration of the antigen binding molecule, the antigen binding molecule is present in the plasma in a state capable of binding to the antigen for an extended period of time.

(3) After administration of the antigen binding molecule, the time during which the antigen in vivo does not bind to the antigen binding molecule is reduced (the time during which the antigen binding molecule binds to the antigen in vivo is extended).

(4) The proportion of antigen bound to the antigen binding molecule is increased relative to the antigen present in the body.

When the antigen is a soluble antigen present in plasma, the elimination of the antigen bound by the antigen-binding molecule may be accelerated even if the pharmacokinetics (speed of elimination from plasma) of the antigen-binding molecule is equivalent. This is associated with an increase in the pharmacokinetics of the antigen-binding molecule relative to the antigen by reducing the pharmacokinetics of the antigen (faster elimination from the plasma), i.e., an increase in the time the antigen-binding molecule is present in the plasma in a state that it can bind to the antigen. Thus, as a means of "pharmacokinetic enhancement" of the antigen-binding molecules of the invention, there is also included an increase in the rate of elimination of soluble antigen from plasma (antigen elimination in plasma of the antigen-binding molecule) following administration of the antigen-binding molecule.

In the present invention, whether or not the antigen-binding molecule of 1 molecule binds to a plurality of antigens can be judged by whether or not the pharmacokinetics of the antigen-binding molecule is improved when the antigen is a membrane antigen. Whether "pharmacokinetics is improved" can be judged as follows. For example, whether the time from administration of the antigen-binding molecule to elimination of the antigen-binding molecule is prolonged can be judged by measuring any one of parameters of the half-life in plasma, the retention time in mean plasma, the clearance rate in plasma, etc. of the antigen-binding molecule (ファ - マコキネティクス demonstration による understanding (southern mountain hall)). For example, when an antigen-binding molecule is administered to a mouse, rat, monkey, rabbit, dog, human, or the like, the pharmacokinetics of the antigen-binding molecule is improved when the half-life in plasma is increased or the retention time in average plasma is increased. The above parameters can be determined by methods known to those skilled in the art, for example, by using pharmacokinetic analysis software winnonlin (pharsight) and performing a non-compartmental analysis (noncromompartmental analysis) according to the attached operating guidelines, and thus can be appropriately evaluated.

Whether the time from administration to elimination of the antigen-binding molecule in the plasma in a state of being able to bind to the antigen is prolonged or not can be determined by measuring the plasma concentration of the antigen-binding molecule not bound to the antigen, and measuring any of the parameters such as the plasma half-life, mean plasma residence time, and plasma clearance of the antigen-binding molecule not bound to the antigen. The determination of the concentration in plasma of antigen-binding molecules that are not bound to an antigen can be performed according to methods well known to those skilled in the art, for example according to the method of Clin Pharmacol.2008Apr; 48(4): 406-17.

Whether or not the time during which the antigen is not bound to the antigen-binding molecule in vivo decreases after administration of the antigen-binding molecule (the time during which the antigen-binding molecule is bound to the antigen becomes longer) can be determined by measuring the plasma concentration of the unbound antigen to which the antigen-binding molecule is not bound, and maintaining the plasma concentration of the unbound antigen or the ratio of the amount of the unbound antigen to the total amount of antigen at a low level. The plasma concentration of the unbound antigen or the ratio of the amount of unbound antigen to the total amount of antigen can be determined by methods known to those skilled in the art, for example, according to Pharm res.2006jan; 23(1): 95-103 by the methods described in. When an antigen exhibits any function in vivo, whether the antigen is bound by an antigen binding molecule that neutralizes the function of the antigen can also be evaluated based on whether the function of the antigen is neutralized. Whether the function of the antigen is neutralized can be assessed by determining any in vivo markers that reflect the function of the antigen. Whether an antigen is bound by an antigen binding molecule that activates the function of the antigen can be assessed by determining any in vivo marker that reflects the function of the antigen.

The measurement of the plasma concentration of the unbound antigen, the measurement of the ratio of the amount of antigen to the total amount of antigen of the unbound antigen, the measurement of the in vivo marker, and the like are not particularly limited, and it is preferable to perform the measurement after a certain time has elapsed since the antigen-binding substance was administered. In the present invention, the time after the antigen-binding substance is administered is not particularly limited, and those skilled in the art can appropriately determine, depending on the nature of the antigen-binding substance to be administered, for example, 1 day after the antigen-binding substance is administered, 3 days after the antigen-binding substance is administered, 7 days after the antigen-binding substance is administered, 14 days after the antigen-binding substance is administered, 28 days after the antigen-binding substance is administered, and the like.

In the present invention, it is preferable that the pharmacokinetics in a human body is improved. When it is difficult to measure the plasma retention in a human body, the plasma retention in a human body can be predicted from the plasma retention in a mouse (e.g., a normal mouse, a human antigen-expressing transgenic mouse, a human FcRn-expressing transgenic mouse, etc.) or a monkey (e.g., a cynomolgus monkey, etc.).

The method for measuring the plasma retention is not particularly limited, and the method can be performed, for example, according to the method described in examples.

Whether an antigen-binding molecule can bind to an antigen multiple times can be evaluated by determining whether an antigen bound to the antigen-binding molecule under the same neutral conditions as in plasma dissociates under the same acidic conditions as in endosomes, and how much antigen can bind again under neutral conditions. Specifically, the antigen-binding molecule-antigen complex is allowed to act under neutral conditions using an apparatus for evaluating an antigen-binding molecule-antigen reaction such as Biacore, and then exposed to acidic conditions for a certain period of time, and then whether or not the antigen-binding molecule is capable of binding to the antigen is measured again under neutral conditions, whereby the evaluation can be performed. When the antigen-binding amount of the antigen-binding molecule to which the pH-dependent binding ability is imparted is increased by two times as compared with the antigen-binding molecule before the modification, it can be said that the number of times of binding of the antigen-binding molecule to which the pH-dependent binding ability is imparted is increased by two times as compared with the antigen-binding molecule before the modification. When the antigen is a membrane-type antigen and the antigen-binding molecule bound to the antigen is taken up via the antigen and decomposed by lysosomes, and eliminated from plasma, it is possible to evaluate whether or not the number of times of binding of the antigen-binding molecule to which the pH-dependent binding ability has been imparted is increased as compared with the antigen-binding molecule before modification, by evaluating whether or not the pharmacokinetics of the antigen-binding molecule to which the pH-dependent binding ability has been imparted is increased or how much the binding period with the antigen has been increased as compared with the antigen-binding molecule before modification. For example, when the binding period of the antigen-binding molecule to which the pH-dependent binding ability is imparted is increased by two times as compared with the antigen-binding molecule before the modification, it can be said that the number of times of binding of the antigen-binding molecule to which the pH-dependent binding ability is imparted is increased by two times as compared with the antigen-binding molecule before the modification. In addition, when the plasma concentration of the unbound antigen to which the antigen-binding molecule is unbound is measured and the plasma concentration of the unbound antigen or the time period during which the ratio of the amount of antigen to the total amount of antigen is maintained at a low level is increased by two times, it can be said that the number of times of binding of the antigen-binding molecule to which the pH-dependent binding ability is imparted is increased by two times as compared with the antigen-binding molecule before modification.

When the antigen is a soluble antigen, the antigen bound to the antigen-binding molecule under neutral conditions in plasma dissociates in vivo in the nucleus, and when the antigen-binding molecule returns to plasma, the antigen-binding molecule can be bound to the antigen again under neutral conditions in plasma, so that the antigen-binding molecule having a property of dissociating the antigen under acidic conditions in the nucleus can be bound to the antigen multiple times. When the antigen bound to the antigen-binding molecule is dissociated in the intranuclear space, the antigen is transported to the lysosome and decomposed, and thus the rate of elimination of the antigen from the plasma is increased, as compared with the case where the antigen bound to the antigen-binding molecule is not dissociated in the intranuclear space (the antigen is returned to the plasma while maintaining the state of being bound to the antigen-binding molecule). That is, whether or not the antigen-binding molecule can bind to the antigen multiple times can also be determined using the rate of elimination of the antigen from plasma as an index. The rate of elimination of antigen from plasma can also be measured, for example, by administering the antigen (e.g., membrane antigen) and antigen-binding molecule to the body and measuring the concentration of antigen in the plasma after administration. When an antigen (e.g., a membrane antigen) is produced (secreted) in vivo, the antigen concentration in plasma decreases as the elimination rate of the antigen from plasma increases, and therefore whether or not the antigen-binding molecule can bind to the antigen multiple times may be determined using the antigen concentration in plasma as an index.

In the present invention, the phrase "increasing the number of times the antigen-binding molecule binds to an antigen" means that the number of times the antigen-binding molecule is administered to a human, a mouse, a monkey, or the like is increased by setting the step of binding the antigen-binding molecule to the antigen and taking it into the cell to 1 time. That is, in the present invention, "the antigen-binding molecule binds to the antigen twice" means that the antigen-binding molecule is taken into the cell in a state of being bound to the antigen, and then released to the outside of the cell in a state of being dissociated from the antigen, and the released antigen-binding molecule is taken into the cell while being bound to the antigen again.

When the antigen-binding molecule is taken into the cell, the antigen-binding molecule may be taken in a state of binding to 1 antigen, or may be taken in a state of binding to 2 or more antigens.

In the present invention, the phrase "the number of times of binding of the antigen-binding molecule to the antigen" means that the number of times of binding of the antigen-binding molecule to the antigen does not necessarily have to be increased for all the antigen-binding molecules, and may be, for example, an increase in the ratio of the antigen-binding molecules bound to the antigen twice or more among the antigen-binding molecules contained in the antigen-binding molecule composition, an increase in the average value of the number of times of binding of the antigen-binding molecules contained in the antigen-binding molecule composition, or.

In the present invention, it is preferable that the number of times of binding of the antigen-binding molecule to the antigen increases when the antigen-binding molecule is administered to a human, but when it is difficult to measure the number of times of binding of the antigen in a human, the number of times of binding of the antigen in a human can be predicted from the results of in vitro measurement and the results of in vivo measurement in a mouse (for example, a transgenic mouse expressing the antigen, a transgenic mouse expressing human FcRn, or the like) or a monkey (for example, a cynomolgus monkey, or the like).

In the present invention, the antigen-binding molecule preferably binds to the antigen twice or more, and for example, at least 10% or more, preferably 30% or more, more preferably 50% or more, and still more preferably 80% or more (for example, 90% or more, 95% or more, and the like) of the antigen-binding molecules contained in the antigen-binding molecule composition bind to the antigen twice or more.

In the present invention, the phrase "increasing the number of antigens that can be bound by an antigen-binding molecule" refers to increasing the number of antigens that can be bound by an antigen-binding molecule during the period from when the antigen-binding molecule is administered to an animal such as a human, a mouse, or a monkey to when lysosomes in the cells are decomposed.

Generally, since an antibody such as IgG has 2 binding sites, 1 antibody binds to 2 antigens at most, and the antibody bound to the antigen is taken into cells and degraded by lysosomes together with the antigen. Therefore, antibodies such as IgG can bind to 2 antigens at the maximum. By the method of the present invention, the antigen-binding activity of an antigen-binding molecule such as an antibody at the pH in vivo in the nucleus is made weaker than the antigen-binding activity at the pH in plasma, and the antigen-binding molecule such as an antibody taken into the cell dissociates the antigen in the cell, is released to the outside of the cell, and can be bound to the antigen again. That is, with the method of the present invention, it is possible to bind to antigens in a number greater than the number of antigen binding sites of the antigen binding molecule. Specifically, for example, in the case of IgG having 2 binding sites, 3 or more, preferably 4 or more antigens can be bound by the method of the present invention during the period from the administration of the antibody until the antibody is decomposed. For example, when the antibody is a neutralizing antibody, "increasing the number of antigens to which the antigen binding molecule can bind" may also refer to increasing the number of antigens to which the antigen binding molecule can neutralize. Thus, when the antibody is a neutralizing antibody, "binding" may also be interchanged with "neutralizing".

In the present invention, "increasing the number of antigens that the antigen-binding molecule can bind" means that the number of antigens that can be bound by all the antigen-binding molecules does not necessarily increase, and may be, for example, an increase in the average number of antigens that can be bound by the antigen-binding molecules contained in the antigen-binding molecule composition, an increase in the proportion of antigen-binding molecules that can bind to antigens that are larger than the number of antigen-binding sites of the antigen-binding molecules, or the like.

In the present invention, it is preferable that the number of antigens to which the antigen-binding molecule can bind is increased when the antigen-binding molecule is administered to a human, and when it is difficult to measure the number of antigens in a human body, the number of antigens that can be bound in a human body can be predicted from the results of in vitro measurement, the results of measurement of a mouse (for example, a transgenic mouse expressing an antigen, a transgenic mouse expressing human FcRn, or the like) or a monkey (for example, a cynomolgus monkey, or the like). In general, when the antibody is a neutralizing antibody, it is considered that the number of times the antigen-binding molecule binds to the antigen is correlated with the number of antigens that the antigen-binding molecule can neutralize, and therefore the number of antigens that the antigen-binding molecule can neutralize can be measured in the same manner as the number of times the antigen-binding molecule binds to the antigen.

The present invention also provides: a method of binding an antigen-binding molecule to an antigen in vivo two or more times by administering an antigen-binding molecule having antigen-binding activity at acidic pH lower than that at neutral pH.

The invention also relates to: a method of neutralizing more antigens than the number of antigen-binding sites of an antigen-binding molecule by administering an antigen-binding molecule having an antigen-binding activity at acidic pH lower than that at neutral pH, in an antigen-binding molecule having a neutralizing activity. Preferably, it relates to a method for neutralizing 3 or more, preferably 4 or more antigens by administering IgG having antigen binding activity at acidic pH lower than that at neutral pH.

The invention also relates to: a method for dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly by making the antigen-binding ability of the antigen-binding molecule at acidic pH weaker than that at neutral pH. In the present invention, the site of dissociation of the antigen from the antigen-binding molecule may be any site as long as it is intracellular, and is preferably in the early endonucleosome. In the present invention, the phrase "the antigen bound to the antigen-binding molecule outside the cell is dissociated from the antigen-binding molecule inside the cell" means that all the antigen that is not bound to the antigen-binding molecule and taken into the cell is dissociated from the antigen-binding molecule inside the cell, and the proportion of the antigen that is dissociated from the antigen-binding molecule inside the cell may be increased as compared to before the antigen-binding ability of the antigen-binding molecule at acidic pH is made lower than the antigen-binding ability at neutral pH.

The invention also relates to: a method of promoting binding of an antigen binding molecule that is not bound to an antigen to FcRn within a cell by making the antigen binding ability of the antigen binding molecule at acidic pH weaker than the antigen binding ability at neutral pH. In general, FcRn binds to an antigen-binding molecule in the nuclear body, and when the antigen-binding molecule binds to a membrane-type antigen, it is considered that it cannot bind to FcRn, and therefore, as a preferred embodiment of the present invention, when the antigen is a membrane-type antigen, the following method can be exemplified: dissociation of the antigen-binding molecule from the antigen in the intranuclear space is promoted and binding of the antigen-binding molecule to FcRn is promoted by making the antigen-binding ability of the antigen-binding molecule weaker at pH in the intranuclear space (acidic pH) than at pH in plasma (neutral pH). When the antigen is a soluble antigen, the following methods can be exemplified: an antigen-binding molecule can bind to FcRn regardless of the presence or absence of antigen binding, but if dissociation of antigen from the antigen-binding molecule can be promoted in vivo by making the antigen-binding ability of the antigen-binding molecule weaker at pH in vivo than at plasma (neutral pH), binding of the "unbound antigen-binding molecule to FcRn will be promoted.

Whether the antigen is membrane-type or soluble, the antigen binding molecule can bind to the antigen again as long as the antigen binding molecule that is not bound to the antigen can return to the plasma through FcRn, and thus the antigen binding molecule can bind to the antigen multiple times by repeating this process. In the present invention, "promoting binding of an antigen-binding molecule to FcRn in a cell" means that not all the antigen-binding molecule need bind to FcRn, and the proportion of the antigen-binding molecule that binds to FcRn in a cell but does not bind to the antigen may be increased as compared to before the antigen-binding ability of the antigen-binding molecule at pH in vivo in the nucleus is made weaker than the antigen-binding ability at pH in plasma. In the method of promoting binding of an intracellular antigen-binding molecule to FcRn of the present invention, examples of preferred antigen-binding molecules include: an antigen-binding molecule that binds to a membrane antigen (membrane antigen) such as a membrane protein. Examples of other preferred antigen binding molecules are: an antigen-binding molecule that binds to a soluble antigen such as a soluble protein.

A method of promoting binding of an antigen binding molecule to FcRn in a cell may also be referred to as a method of enhancing the binding activity of an antigen binding molecule to FcRn in a cell (e.g., in vivo in a nucleus).

The invention also relates to: a method for releasing an antigen-binding molecule, which has been taken up into a cell in a state of being bound to an antigen, out of the cell in a state of not being bound to the antigen by making the antigen-binding ability of the antigen-binding molecule at acidic pH weaker than the antigen-binding ability at neutral pH. In the present invention, "the antigen-binding molecule taken into the cell in a state of being bound to the antigen is released to the outside of the cell in a state of not being bound to the antigen" means that all the antigen-binding molecule taken into the cell in a state of being bound to the antigen is not necessarily released to the outside of the cell in a state of not being bound to the antigen, as long as the proportion of the antigen-binding molecule released to the outside of the cell is increased as compared to before the antigen-binding ability of the antigen-binding molecule at acidic pH is made lower than the antigen-binding ability at neutral pH. Preferably, the antigen binding molecules that are released outside the cell maintain antigen binding capacity. The method of releasing the antigen-binding molecule, which is taken up into the cell in a state of being bound to the antigen, out of the cell in a state of not being bound to the antigen can be said to be a method of imparting a property that the antigen-binding molecule is easily released out of the cell in a state of not being bound to the antigen in a case where the antigen-binding molecule is taken up into the cell in a state of being bound to the antigen.

The invention also relates to: a method of increasing the antigen-elimination capacity in the plasma of an antigen-binding molecule by making the antigen-binding capacity of the antigen-binding molecule weaker at acidic pH than at neutral pH. In the present invention, the term "antigen-eliminating ability in plasma" refers to an ability to eliminate an antigen present in plasma from plasma when an antigen-binding molecule is administered to the body or secreted from the body. Therefore, in the present invention, the phrase "the antigen-eliminating ability in plasma of the antigen-binding molecule is increased" means that when the antigen-binding molecule is administered in vivo, the speed of elimination of the antigen from the plasma is increased as compared to before the antigen-binding ability of the antigen-binding molecule at acidic pH is made lower than the antigen-binding ability at neutral pH. Whether or not the antigen-eliminating ability in plasma of the antigen-binding molecule is increased can be judged, for example, by administering the soluble antigen and the antigen-binding molecule in vivo and measuring the plasma concentration of the soluble antigen after administration. When the concentration of soluble antigen in plasma is decreased after administration of soluble antigen and antigen-binding molecule by making the antigen-binding ability of the antigen-binding molecule at acidic pH lower than the antigen-binding ability at neutral pH, it can be judged that the antigen-eliminating ability in plasma of the antigen-binding molecule is increased.

The invention also relates to: a method of improving the pharmacokinetics of an antigen binding molecule by substituting at least 1 amino acid of the antigen binding molecule with, or inserting, histidine or a non-natural amino acid.

The present invention also provides: methods of increasing the number of times an antigen-binding molecule binds to an antigen by substituting at least 1 amino acid of the antigen-binding molecule with histidine or a non-natural amino acid, or inserting histidine or a non-natural amino acid.

The invention also relates to: methods of increasing the number of antigens to which an antigen-binding molecule can bind by substituting at least 1 amino acid of the antigen-binding molecule with histidine or an unnatural amino acid, or inserting histidine or an unnatural amino acid.

The present invention also provides: a method of dissociating an antigen bound to an antigen-binding molecule extracellularly from the antigen-binding molecule intracellularly by substituting at least 1 amino acid of the antigen-binding molecule with histidine or an unnatural amino acid, or inserting histidine or an unnatural amino acid.

The present invention also provides: a method for releasing an antigen-binding molecule taken up into a cell in a state of being bound to an antigen out of the cell in a state of not being bound to the antigen by substituting at least 1 amino acid of the antigen-binding molecule with histidine or an unnatural amino acid, or inserting histidine or an unnatural amino acid.

The present invention also provides: a method for increasing the antigen-depleting capacity of the plasma of an antigen-binding molecule by substituting histidine or a non-natural amino acid for at least 1 amino acid of the antigen-binding molecule or inserting histidine or a non-natural amino acid.

The site for introducing a histidine or unnatural amino acid mutation (substitution, insertion, etc.) is not particularly limited, and any site may be substituted with histidine or an unnatural amino acid, or a histidine or an unnatural amino acid may be inserted at any site. Preferred examples of sites for substitution with or insertion of histidine or unnatural amino acids are: a region that affects the antigen binding ability of the antigen binding molecule. For example, when the antigen binding molecule is an antibody, the variable region or CDR of the antibody can be exemplified. The number of mutations introduced by histidine or unnatural amino acids is not particularly limited, and only 1 position may be substituted with histidine or an unnatural amino acid, or only 1 position may be inserted with histidine or an unnatural amino acid. Alternatively, histidine or an unnatural amino acid may be substituted for a plurality of positions at 2 or more positions, or may be inserted at a plurality of positions. In addition to substitution or insertion with histidine or an unnatural amino acid, deletion, addition, insertion, and/or substitution of other amino acids may be performed simultaneously.

In the present invention, as an example of the site substituted with histidine or an unnatural amino acid, when the antigen-binding molecule is an antibody, the following sites can be mentioned, for example, taking into consideration the CDR sequence of the antibody or the sequence determining the structure of the CDR as the modification site. Amino acid positions are indicated by Kabat numbering (Kabat EA et al, 1991.Sequences of Proteins of Immunological interest. NIH).

Heavy chain: h27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, H102

Light chain: l24, L27, L28, L32, L53, L54, L56, L90, L92, L94

Among these modification sites, H32, H61, L53, L90 and L94 are considered to be highly common modification sites.

Although not particularly limited, the following sites are preferred as modification sites when the antigen is an IL-6 receptor (e.g., human IL-6 receptor).

Heavy chain: h27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, H102

Light chain: l24, L27, L28, L32, L53, L56, L90, L92, L94

When combining multiple sites and substituting histidine or unnatural amino acids, specific examples of preferred combinations are: e.g., a combination of H27, H31, H35; combinations of H27, H31, H32, H35, H58, H62, H102; a combination of L32, L53; combinations of L28, L32, L53, and the like. Further, as examples of preferable combinations of the substitution sites of the heavy chain and the light chain, there can be mentioned: combinations of H27, H31, L32, L53.

Although not particularly limited, in the antigen of IL-6 (such as IL-6) cases, as a preferred modified sites, can be cited as the following sites.

Heavy chain: h32, H59, H61, H99

Light chain: l53, L54, L90, L94

Although not particularly limited, in the case where the antigen is an IL-31 receptor (e.g., human IL-31 receptor), the preferred modification site is H33.

Of these, only 1 site may be substituted with histidine or an unnatural amino acid, or a plurality of sites may be substituted with histidine or an unnatural amino acid.

The methods of the invention are applicable to any antigen binding molecule independent of the type of target antigen.

In the present invention, the antigen-binding molecule is not particularly limited as long as it has a specific binding activity to the target antigen, and preferred examples of the antigen-binding molecule include: a substance having an antigen binding region of an antibody. Examples of antigen binding regions of antibodies are: a CDR or a variable region. When the antigen binding region of the antibody is a CDR, it may contain all 6 CDRs contained in the full-length antibody, or 1 or 2 or more CDRs. When a CDR is included as a binding region of an antibody, the included CDR may be subjected to deletion, substitution, addition, and/or insertion of amino acids, or the like, or may be a part of a CDR.

The invention also relates to when an antibody constant region is comprised in an antigen binding molecule: methods for improving the pharmacokinetics of antigen binding molecules by modifying (e.g., substituting, deleting, adding, and/or inserting amino acids) the antibody constant region contained in the antigen binding molecule.

When an antibody constant region is included in the antigen binding molecule, the invention also provides: a method of increasing the number of times of binding of an antigen-binding molecule to an antigen by modifying (e.g., substituting, deleting, adding, and/or inserting an amino acid) an antibody constant region contained in the antigen-binding molecule.

The invention also relates to when an antibody constant region is comprised in an antigen binding molecule: a method of increasing the number of antigens that an antigen-binding molecule can bind to by modifying (e.g., substituting, deleting, adding, and/or inserting amino acids) the antibody constant region contained in the antigen-binding molecule.

The invention also relates to when an antibody constant region is comprised in an antigen binding molecule: a method in which an antigen bound to an antigen-binding molecule outside a cell is dissociated from the antigen-binding molecule inside the cell by modifying (e.g., substituting, deleting, adding, and/or inserting an amino acid) an antibody constant region contained in the antigen-binding molecule.

The invention also relates to when an antibody constant region is comprised in an antigen binding molecule: a method in which an antigen-binding molecule incorporated into a cell in a state of being bound to an antigen is released to the outside of the cell in a state of not being bound to the antigen by modifying (e.g., substitution, deletion, addition, and/or insertion of an amino acid) an antibody constant region contained in the antigen-binding molecule.

The invention also relates to when an antibody constant region is comprised in an antigen binding molecule: a method of increasing the ability of an antigen-binding molecule to eliminate an antigen in plasma by modifying (e.g., substituting, deleting, adding, and/or inserting an amino acid) the antibody constant region contained in the antigen-binding molecule.

A preferred embodiment of the antigen-binding substance of the present invention includes an antigen-binding substance including an FcRn binding region. Antigen binding substances comprising the FcRn binding region can be taken up intracellularly via the salvage pathway of FcRn and then returned to the plasma again. The FcRn binding region is preferably a region that binds directly to FcRn. Preferred examples of FcRn binding regions are: the Fc region of an antibody. However, since a region of albumin, IgG, or the like, which is capable of binding to a polypeptide having a binding ability to FcRn, can indirectly bind to FcRn via albumin, IgG, or the like, the FcRn binding region in the present invention may be a region that binds to such a polypeptide having a binding ability to FcRn.

The antigen recognized by an antigen-binding molecule such as an antibody to be subjected to the method of the present invention is not particularly limited, and an antibody recognizing any antigen can be subjected to the method. Examples of antibodies that can be used to improve pharmacokinetics using the methods of the invention are: for example, an antibody recognizing a membrane antigen such as a receptor protein (membrane-bound receptor or soluble receptor) or a cell surface marker, an antibody recognizing a soluble antigen such as cytokine, or the like. In the present invention, preferable examples of the membrane antigen include: a membrane protein. In the present invention, examples of the soluble antigen include: a soluble form of the protein. Specific examples of antigens recognized by antibodies whose pharmacokinetics are enhanced by the method of the present invention are: for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-31, IL-23, IL-2 receptor, IL-6 receptor, OSM receptor, gp130, IL-5 receptor, CD40, CD4, Fas, osteopontin, CRTH2, CD26, PDGF-D, CD20, monocyte chemotactic activator, CD23, TNF- α, HMGB-1, α 4 integrin, ICAM-1, CCR2, CD11a, CD3, IFN γ, yBLS, HLA-DR, TGF- β, CD52, IL-31 receptor, and the like. A particularly preferred antigen is the IL-6 receptor.

The antigen-binding molecule to be subjected to the method of the present invention includes: antigen-binding molecules having antagonistic activity (antagonistic antigen-binding molecules), antigen-binding molecules having agonistic activity (agonistic antigen-binding molecules), and the like, and preferable examples thereof include: an antagonistic antigen-binding molecule, particularly an antagonistic antigen-binding molecule that recognizes a membrane antigen such as a receptor or a soluble antigen such as a cytokine. For example, an antagonistic antigen binding molecule that recognizes a receptor is an antigen binding molecule that binds to the receptor, blocks binding of the receptor to its ligand, and inhibits signaling mediated by the receptor.

In the present invention, the antigen-binding molecule to be used is not particularly limited, and may be any antigen-binding molecule. The antigen binding molecules used in the present invention preferably have an antigen binding activity (antigen binding region) and an FcRn binding region. In the present invention, antigen binding molecules comprising a human FcRn binding region are particularly preferred. Examples of antigen binding molecules having antigen binding activity and an FcRn binding region are antibodies. A preferred example of the antibody of the present invention is an IgG antibody. When an IgG antibody is used as the antibody, the type thereof is not limited, and IgG of the same type (subclass) as IgG1, IgG2, IgG3, and IgG4 can be used. For the constant region of IgG of these isotypes, amino acid mutations may be introduced into the constant region portion as in M73. Examples of the introduced amino acid mutation include: for example, amino acid mutations that increase or decrease binding to Fc γ receptor (Proc Natl Acad Sci US A.2006Mar14; 103 (11): 4005-10), amino acid mutations that increase or decrease binding to FcRn (J Biol chem.2001Mar2; 276 (9): 6591-604), etc., but are not limited thereto. In addition, by selecting appropriate constant regions of IgG2, etc., pH-dependent binding can also be altered.

When the antigen-binding molecule to be targeted by the present invention is an antibody, the antibody may be derived from any animal such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Further, for example, a chimeric antibody, a modified antibody in which an amino acid sequence is substituted with a humanized antibody, or the like may be used. Also, the antibody may be a dual specificity antibody, a modified antibody to which various molecules are bound, a polypeptide comprising an antibody fragment, or the like.

"chimeric antibody" refers to an antibody prepared by combining sequences derived from different animals. Specific examples of chimeric antibodies are: for example, an antibody comprising the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody.

The "humanized antibody", also called a reshaped (reshaped) human antibody, is an antibody obtained by grafting a Complementarity Determining Region (CDR) of an antibody derived from a mammal other than a human, for example, a mouse antibody, to a CDR of a human antibody. Methods for identifying CDRs are well known (Kabat et al, Sequence of proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). In addition, a general gene recombination method is also known (see European patent application publication Nos. EP125023 and WO 96/02576).

A dual specificity antibody is one that has variable regions that recognize different epitopes within the same antibody molecule. The dual specific antibody may be an antibody recognizing 2 or more different antigens, or may be an antibody recognizing 2 or more different epitopes on the same antigen.

Examples of the polypeptide comprising an antibody fragment include Fab fragment, F (ab') 2 fragment, scFv (Nat Biotechnol. 2005Sep; 23 (9): 1126-36) domain antibody (dAb) (WO2004/058821, WO2003/002609), scFv-Fc (WO2005037989), dAb-Fc, and Fc fusion protein. Among these molecules, particularly those comprising an Fc region have binding activity to FcRn, and are therefore suitable for use in the method found in the present invention.

Also, the antigen binding molecules that can be used in the present invention can be antibody-like molecules. Antibody-like molecules refer to molecules that function by binding to a target molecule (Current Opinion in Biotechnology2006, 17: 653-658, Current Opinion in Biotechnology2007, 18: 1-10, Current Opinion in Structural Biology1997, 7: 463-469, Protein Science2006, 15: 14-27), such as DARPins (WO2002/020565), Affibody (WO1995/001937), Avimer (WO2004/044011, WO2005/040229), Adnectin (WO2002/032925), etc. Even these antibody-like molecules can bind to a plurality of target molecules in 1 molecule as long as they can perform pH-dependent binding with the target molecules.

The antigen-binding molecule may be a receptor protein and a receptor Fc fusion protein for targeted binding, for example, TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, CTLA4-Fc fusion protein, etc. (Nat Med.2003Jan; 9 (1): 47-52, BioDrugs.2006; 20 (3): 151-60). Even these receptor proteins and receptor Fc fusion proteins can bind to a plurality of target molecules in 1 molecule as long as they can perform pH-dependent binding with the target molecules.

The antigen-binding molecule may also be an artificial ligand protein and an artificial ligand fusion protein having a neutralizing effect on the target binding, for example, there are mutant IL-6(EMBO J.1994 Dec15; 13 (24): 5863-70) and the like. Even these artificial ligand proteins and artificial ligand fusion proteins can bind to a plurality of target molecules in 1 molecule as long as they can bind to the target molecules in a pH-dependent manner.

Furthermore, the antibody of the present invention may be an antibody with a modified sugar chain. Examples of the antibody having a modified sugar chain include: for example, glycosidation-modified antibodies (WO99/54342, etc.), fucose-deficient antibodies added to sugar chains (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913, etc.), antibodies having sugar chains with bisecting (bisecting) GlcNAc (WO02/79255, etc.), and the like.

The method of the present invention is not limited by a particular theory, and for example, the relationship between making the antigen-binding ability at acidic pH weaker than that at neutral pH, improving pharmacokinetics, and binding to antigen multiple times can be illustrated as follows.

For example, when the antibody is an antibody that binds to a membrane antigen, the antibody administered in vivo binds to the antigen, and thereafter the antibody remains bound to the antigen and taken into endosomes in the cell together with the antigen by internalization. Thereafter, the antibody moves to the lysosome while being bound to the antigen, and the antibody is decomposed by the lysosome together with the antigen. The elimination from plasma mediated by internalization is called antigen-dependent elimination and is reported in most antibody molecules (drug Discov today.2006 Jan; 11 (1-2): 81-8). When 1 molecule of IgG antibody binds to an antigen in a bivalent state, 1 molecule of the antibody binds to 2 molecules of the antigen and is internalized, and is directly decomposed by lysosomes. Therefore, in the case of a normal antibody, 1 molecule of IgG antibody cannot bind to 3 or more molecules of antigen. For example, 1 molecule of IgG antibody having a neutralizing activity cannot neutralize 3 or more molecules of antigen.

The retention in plasma of IgG molecules is long (slow elimination) due to the role of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up in endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions in the endosomes. The IgG molecules that do not bind to FcRn are taken up into the lysosome and decomposed by the lysosome, but the IgG molecules bound to FcRn migrate to the cell surface, dissociate from FcRn under neutral conditions in plasma, and return to the plasma.

When the antibody is an antibody that binds to a soluble antigen, the antibody administered in vivo binds to the antigen, and thereafter the antibody is taken into the cell while remaining bound to the antigen. The antibody taken into the cell is released to the outside of the cell most likely through FcRn, but is released to the outside of the cell while being bound to the antigen, and therefore cannot be bound to the antigen again. Therefore, in the case of a normal antibody, 1 molecule of IgG antibody cannot bind to 3 or more molecules of antigen, as in the case of an antibody that binds to a membrane antigen.

The inventors believe that: when an antibody that binds to an antigen such as a membrane antigen by internalization is taken into an endosome in a cell, the antibody that binds to the antigen migrates into lysosomes and is decomposed, whereas an IgG antibody whose antigen has been dissociated in the endosome can bind to FcRn expressed in the endosome. Namely, the present inventors have found that: an antibody that strongly binds to an antigen in plasma and weakly binds to an antigen in a nuclear body binds to an antigen in plasma, forms a complex with the antigen, is taken into the nuclear body in a cell by internalization in this state, is dissociated from the antigen in the nuclear body, then binds to FcRn and moves to the cell surface, returns to the plasma in a state of not being bound to the antigen, and can neutralize a plurality of membrane-type antigens. Further found that: an antibody having a property of strongly binding to an antigen in plasma and weakly binding to an antigen in the intranuclear space dissociates from an antigen in the intranuclear space even when bound to an antigen such as a soluble antigen, and is released into plasma again in a state of not being bound to an antigen, thereby neutralizing a plurality of soluble antigens.

In particular, the present inventors have focused on the difference between the plasma pH and the intranuclear pH, and found that an antibody that strongly binds to an antigen under the plasma pH condition and weakly binds to an antigen under the intranuclear pH condition can bind to a plurality of antigens with 1 antibody molecule, and has excellent retention in plasma.

Endosomes are one of the membrane vesicles, form a network within the cytoplasm of eukaryotic cells, and govern the metabolism of macromolecules from the cell membrane to lysosomes. The pH in the endosome is reported to be generally acidic at pH5.5 to pH6.0 (Nat Rev Mol Cell biol.2004 Feb; 5 (2): 121-32), and the pH in the plasma is known to be almost neutral (generally pH 7.4).

Therefore, an antigen-binding molecule having antigen-binding activity at acidic pH that is weaker than that at neutral pH binds to an antigen in plasma at neutral pH and is taken up into cells, and then dissociates from the antigen in the nucleus at acidic pH. The antigen-binding molecule dissociated from the antigen binds to FcRn and moves to the cell surface, and returns to the plasma again in a state of not being bound to the antigen, and as a result, the antigen can be bound to the antigen several times, and pharmacokinetics is improved.

< antigen-binding molecule substance >

The present invention also provides an antigen-binding molecule having an antigen-binding activity at pH4.0 to pH6.5 lower than that at pH6.7 to pH10.0, preferably an antigen-binding molecule having an antigen-binding activity at pH5.0 to pH6.0 lower than that at pH7.0 to 8.0. Specific examples of antigen-binding molecules having an antigen-binding activity at pH4.0-pH6.5 lower than that at pH6.7-10.0 include: an antigen binding molecule having an antigen binding activity at pH5.8 that is less than the antigen binding activity at pH 7.4. An antigen binding molecule having an antigen binding activity at pH5.8 that is lower than the antigen binding activity at pH7.4 may also be referred to as an antigen binding molecule having an antigen binding activity at pH7.4 that is higher than the antigen binding activity at pH 5.8.

The antigen-binding molecule of the present invention having an antigen-binding activity at pH5.8 lower than that at pH7.4 is not limited as long as it has an antigen-binding activity at pH5.8 lower than that at pH7.4, and the difference in the binding activities is small even if it has a low antigen-binding activity at pH 5.8.

Preferred examples of the antigen-binding molecule of the present invention having an antigen-binding activity at ph5.8 lower than that at ph7.4 include antigen-binding molecules having an antigen-binding activity at ph7.4 which is at least twice as high as that at ph 5.8; more preferably, the antigen-binding molecule has an antigen-binding activity at ph7.4 that is 10 times or more the antigen-binding activity at ph 5.8; more preferably, the antigen-binding molecule has an antigen-binding activity at pH7.4 that is 40 times or more the antigen-binding activity at pH 5.8.

Specifically, in a preferred embodiment of the antigen-binding molecule of the present invention, in which the antigen-binding activity at ph5.8 is lower than that at ph7.4, the ratio of KD at ph5.8 to KD at ph7.4, i.e., the value of KD (ph5.8)/KD (ph7.4), is 2 or more, more preferably the value of KD (ph5.8)/KD (ph7.4) is 10 or more, and still more preferably the value of KD (ph5.8)/KD (ph7.4) is 40 or more, with respect to the antigen. The upper limit of the value of KD (pH5.8)/KD (pH7.4) is not particularly limited, and any value such as 400, 1000, 10000, etc. may be used as long as it can be prepared by the technique of those skilled in the art.

Further, the antigen-binding activity at pH5.8 as the present invention is lower than that at pH7.4Other preferred embodiments of antigen binding molecules with antigen binding activity are those with k at pH5.8 for the antigen_dAnd k at pH7.4_dRatio of (i.e. k)_d(pH5.8)/k_dThe value of (pH7.4) is 2 or more, preferably k_d(pH5.8)/k_dThe value of (pH7.4) is 5 or more, and k is more preferably_d(pH5.8)/k_dThe value of (pH7.4) is 10 or more, and k is more preferably_d(pH5.8)/k_dThe value of (pH7.4) is 30 or more. To K_d(pH5.8)/k_dThe upper limit of the value of (ph7.4) is not particularly limited, and may be any value such as 50, 100, 200, etc., as long as it can be produced by the technique of those skilled in the art.

In the measurement of the binding activity of the antigen, conditions other than pH can be appropriately selected by those skilled in the art, and are not particularly limited, and for example, the measurement can be performed in MES buffer at 37 ℃. The measurement of the antigen binding activity of the antigen binding molecule can be performed by a method known to those skilled in the art, and for example, as described in the examples, measurement can be performed using Biacore T100(GE Healthcare) or the like.

It is considered that such an antigen-binding molecule weakly bound to an antigen at an acidic pH is easily dissociated from the antigen under an acidic condition in the endosome, and is considered to be bound to FcRn after being internalized in the cell and to be easily released to the outside of the cell. Antigen-binding molecules that are not broken down within the cell and released outside the cell can bind to the antigen again. Thus, for example, where the antigen binding molecule is a neutralizing antigen binding molecule, an antigen binding molecule that readily dissociates from an antigen under acidic conditions within the endosome can bind to the antigen and neutralize the antigen multiple times. As a result, the antigen-binding molecules having an antigen-binding activity at pH4.0 to pH6.5 lower than that at pH6.7 to pH10.0 become antigen-binding molecules having an excellent retention property in plasma.

As a preferred embodiment of the antigen-binding molecule having an antigen-binding activity at pH5.8 lower than that at pH7.4, there can be mentioned a case where at least 1 amino acid in the antigen-binding molecule is substituted with histidine or notA natural amino acid, or an antigen binding molecule with at least 1 histidine or non-natural amino acid inserted. The site for introducing a mutation of histidine or an unnatural amino acid is not particularly limited, as long as the antigen-binding activity at pH5.8 is weaker than that at pH7.4 (KD (pH5.8)/KD (pH7.4) as compared with that before the substitution, or the value of k is increased_d(pH5.8)/k_d(pH7.4) becomes larger), and any site can be used. For example, when the antigen binding molecule is an antibody, it may be the variable region or CDR of the antibody, or the like. The number of amino acids substituted with histidine or an unnatural amino acid or the number of amino acids to be inserted may be determined as appropriate by those skilled in the art, and 1 amino acid may be substituted with histidine or an unnatural amino acid, or 1 amino acid may be inserted, or 2 or more amino acids may be substituted with histidine or an unnatural amino acid, or 2 or more amino acids may be inserted. In addition, other amino acid deletions, additions, insertions, and/or substitutions may be made in addition to or in addition to histidine or an unnatural amino acid substitution. Substitution with histidine or an unnatural amino acid, or insertion of histidine or an unnatural amino acid can be performed randomly by, for example, substituting alanine in the alanine region with histidine in the histidine region in histidine region, as is well known to those skilled in the art, or by selecting KD (pH5.8)/KD (pH7.4) or K as compared to before mutation from an antigen-binding molecule into which a mutation with histidine or an unnatural amino acid has been randomly introduced _d(pH5.8)/k_d(pH7.4) is increased.

Preferred examples of the antigen-binding molecule which is mutated to histidine or an unnatural amino acid in this manner and has an antigen-binding activity at pH5.8 which is lower than that at pH7.4 include: an antigen binding molecule having an antigen binding activity at pH7.4 after mutation to a histidine or an unnatural amino acid that is equivalent to the antigen binding activity at pH7.4 prior to mutation to a histidine or an unnatural amino acid. In the present invention, the phrase "the antigen-binding molecule after histidine or unnatural amino acid mutation has an equivalent antigen-binding activity to the antigen-binding molecule before histidine or unnatural amino acid mutation" means that the antigen-binding activity of the antigen-binding molecule after histidine or unnatural amino acid mutation is at least 10% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more, based on 100% of the antigen-binding activity of the antigen-binding molecule before histidine or unnatural amino acid mutation. The antigen binding activity at pH7.4 after histidine or unnatural amino acid mutation can be higher than the antigen binding activity at pH7.4 before histidine or unnatural amino acid mutation. When the antigen-binding activity of the antigen-binding molecule is lowered by substitution or insertion of histidine or an unnatural amino acid, the antigen-binding activity can be made equivalent to that before substitution or insertion of histidine by substituting, deleting, adding, and/or inserting 1 or more amino acids in the antigen-binding molecule, or the like. The present invention also includes: an antigen-binding molecule having equivalent binding activity can be obtained by substitution, deletion, addition and/or insertion of 1 or more amino acids after such histidine substitution or insertion.

Further, when the antigen-binding molecule is a substance containing an antibody constant region, another preferable embodiment of the antigen-binding molecule having an antigen-binding activity at pH5.8 lower than that at pH7.4 is: methods of modifying the constant regions of antibodies contained in antigen binding molecules. Specific examples of modified antibody constant regions are: such as the constant regions described in the examples.

When the antigen-binding activity of the antigen-binding substance at pH5.8 is made weaker than that at pH7.4 (the value of KD (pH5.8)/KD (pH7.4) is made larger by the above-mentioned method or the like), it is preferable that the value of KD (pH5.8)/KD (pH7.4) is usually 2 times or more, preferably 5 times or more, and more preferably 10 times or more as compared with the original antibody, although it is not particularly limited.

The antigen-binding molecule of the present invention may have any other properties, for example, an agonistic antigen-binding molecule or an antagonistic antigen-binding molecule, as long as the antigen-binding activity at pH4.0 to pH6.5 is lower than the antigen-binding activity at pH6.7 to 10.0. Examples of preferred antigen binding molecules of the invention are: antagonizing the antigen binding molecule. Antagonistic antigen binding molecules are typically antigen binding molecules that inhibit the binding of a ligand (agonist) to a receptor, inhibiting receptor-mediated signaling into the cell.

The present invention also provides: an antibody in which an amino acid at least 1 position is substituted with a histidine or a non-natural amino acid. Amino acid positions are indicated by Kabat numbering (Kabat EA et al, 1991.Sequences of Proteins of Immunological interest. NIH).

Light chain: l24, L27, L28, L32, L53, L54, L56, L90, L92, L94

Although not particularly limited, in the case where the antigen is an IL-6 receptor (e.g., human IL-6 receptor), the following sites are preferred as modification sites.

Heavy chain: h27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, H102

Light chain: l24, L27, L28, L32, L53, L56, L90, L92, L94

When a plurality of sites are combined and substituted with histidine or an unnatural amino acid, specific examples of preferred combinations are: for example, a combination of H27, H31, and H35, a combination of H27, H31, H32, H35, H58, H62, and H102, a combination of L32 and L53, a combination of L28, L32, and L53, and the like. Also, examples of preferred combinations of substitution sites for heavy and light chains are: combinations of H27, H31, L32, L53.

Although not particularly limited, in the antigen of IL-6 (such as IL-6) cases, preferably modified sites are the following.

Heavy chain: h32, H59, H61, H99

Light chain: l53, L54, L90, L94

The antigen recognized by the antigen binding molecules of the invention may be any antigen. Specific examples of the antigen recognized by the antibody of the present invention include: the above receptor proteins (membrane-bound receptor, soluble receptor), cell surface markers and other membrane antigens or cytokines and other soluble antigens, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-31, IL-23, IL-2 receptor, IL-6 receptor, OSM receptor, gp130, IL-5 receptor, CD40, CD4, Fas, osteopontin, CRTH2, CD26, PDGF-D, CD20, single cell chemokine activator, CD23, TNF-alpha, HMGB-1, alpha 4 integrin, ICAM-1, CCR2, CD11a, CD3, IFN gamma, BLyS, HLA-DR, TGF-beta, CD52, IL-31 receptor, etc.

Particularly preferred antigens are for example: the IL-6 receptor.

The antigen binding molecules of the invention are as described above.

In the present invention, a preferred embodiment of the antigen-binding molecule includes an antibody. Examples of antibodies having antigen binding activity and an FcRn binding region are: an IgG antibody. When an IgG antibody is used as the antibody, the kind thereof is not limited, and IgG1, IgG2, IgG3, IgG4, and the like can be used.

The source of the antibody of the present invention is not particularly limited, and any antibody may be used, and for example, a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, and the like may be used. Further, for example, a modified antibody obtained by substituting an amino acid sequence with the above-mentioned chimeric antibody, humanized antibody or the like may be used. The antibody may be the above-mentioned dual-specificity antibody, a modified antibody having various molecules bound thereto, a polypeptide comprising an antibody fragment, a sugar chain-modified antibody, or the like.

For example, in the case of a human-mouse chimeric antibody, a chimeric antibody can be obtained by ligating a DNA encoding a V region of the antibody and a DNA encoding a C region of a human antibody, inserting the resulting product into an expression vector, and introducing the vector into a host to produce the antibody.

The "humanized antibody", also called a reshaped (reshaped) human antibody, is an antibody obtained by grafting Complementarity Determining Regions (CDRs) of an antibody derived from a mammal other than a human, for example, a mouse antibody, to CDRs of a human antibody. Methods for identifying CDRs are well known (Kabat et al, Sequence of Proteins of Immunological Interest (1987), National institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). In addition, a general gene recombination method is also known (see European patent application publication Nos. EP125023 and WO 96/02576). Humanized antibodies can be generated using well known methods as follows: for example, a CDR of a mouse antibody is determined, the CDR is ligated with a Framework Region (FR) of a human antibody to obtain an antibody, a DNA encoding the obtained antibody is obtained, and then a humanized antibody is produced by a system using a usual expression vector. The DNA can be synthesized by PCR using a plurality of oligonucleotides as primers, and the oligonucleotides are prepared so as to have overlapping regions in the terminal regions of the CDR and the FR (see the method described in WO 98/13388). The FRs of human antibodies linked via the CDRs are selected so that the CDRs form a good antigen binding site. Amino acids of the FRs in the variable region of an antibody can be modified as necessary to form suitable antigen binding sites for the CDRs of the reshaped human antibody (Sato et al, Cancer Res. (1993) 53: 10.01-6). Among amino acid residues in the modifiable FR, there are a portion directly binding to an antigen by a non-covalent bond (Amit et al, Science (1986) 233: 747-53), a portion affecting or acting on the CDR structure (Chothia et al, J.mol.biol. (1987) 196: 901-17), and a portion involved in the interaction of VH-VL (EP 239400A).

When the antibody of the present invention is a chimeric antibody or a humanized antibody, a C region derived from a human antibody is preferably used as the C region of the antibody. For example, C γ 1, C γ 2, C γ 3, C γ 4, etc. may be used in the H chain; in the L chain, C.kappa.C.lambda.can be used. Amino acid mutations may be introduced into the human antibody C region as necessary to increase or decrease binding to Fc γ receptor or FcRn, to improve antibody stability, or to improve antibody production. The chimeric antibody of the present invention preferably comprises a variable region derived from an antibody derived from a mammal other than human and a constant region derived from a human antibody. The humanized antibody preferably contains CDRs derived from an antibody of a mammal other than human and FRs and C regions derived from a human antibody. The constant region derived from a human antibody preferably comprises an FcRn binding region, examples of such antibodies are: IgG (IgG1, IgG2, IgG3, IgG 4). The constant region used in the humanized antibody of the present invention may be a constant region of an antibody belonging to any isotype. The constant region of human IgG is preferably used, but not limited thereto. The FR derived from a human antibody to be used for a humanized antibody is also not particularly limited, and may be of an antibody of any isotype.

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

Chimeric antibodies and humanized antibodies obtained using human-derived sequences have reduced immunogenicity in humans and are therefore useful for administration to humans for therapeutic purposes and the like.

The antibody of the present invention can be obtained by any method, for example, by artificially lowering the antigen binding activity at pH5.8 to that at pH7.4 by the histidine substitution or the like described above with respect to an antibody having an antigen binding activity originally higher at pH5.8 than that at pH7.4 or an antibody having an antigen binding activity of the same degree, or by selecting an antibody having an antigen binding activity at pH5.8 lower than that at pH7.4 from a plurality of antibodies obtained from an antibody library or hybridoma described below, and selecting the antibody of the present invention.

When an amino acid in an antibody is substituted with histidine, a known sequence may be used as the amino acid sequence of the H chain or L chain of the antibody before introduction of histidine mutation, or an amino acid sequence of a newly obtained antibody according to a method known to those skilled in the art may be used. For example, antibodies can be obtained from antibody libraries, or from monoclonal antibody-producing hybridoma clones encoding antibody genes.

Since many antibody libraries are already known as an antibody library, and a method for preparing an antibody library is also known, a person skilled in the art can obtain an appropriate antibody library. For example, with respect to antibody phage libraries, reference may be made to Clackson et al, Nature1991, 352: 624-8; marks et al, j.mol.biol.1991, 222: 581-97; waterhouses et al, Nucleic Acids Res.1993, 21: 2265-6; griffiths et al, EMBO J.1994, 13: 324.0-60; vaughan et al, Nature Biotechnology1996, 14: 309-14 and Japanese patent application laid-open No. Hei 20-504970. Further, known methods such as a method using eukaryotic cells as a library (WO 95/15393) and a ribosome-indicating method can be used. Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, a phage capable of binding to an antigen can be selected by expressing the variable region of a human antibody as a single-chain antibody (scFv) on the surface of the phage by phage display. Analysis of the genes of the selected phage allows the determination of the DNA sequence encoding the variable region of the human antibody that binds to the antigen. Once the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared based on the sequence to obtain a human antibody. The above-mentioned methods are well known and reference may be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388.

A method for obtaining a gene encoding an antibody from a hybridoma, basically, using a known technique, using a desired antigen or a cell expressing a desired antigen as a sensitizing antigen, immunizing the antigen according to a usual immunization method, fusing the obtained immunocyte with a known parent cell by a usual cell fusion method, screening a cell (hybridoma) producing a monoclonal antibody by a usual screening method, synthesizing a cDNA for an antibody variable region (V region) from the mRNA of the obtained hybridoma using a reverse transcriptase, and ligating the cDNA with a DNA encoding a constant region (C region) of the desired antibody.

More specifically, the sensitizing antigen used for obtaining the above-mentioned antibody genes encoding H chain and L chain includes, but is not particularly limited to: both immunogenic and non-immunogenic partial antigens, including haptens and the like. For example, a full-length protein or a partial peptide of the target protein, or the like can be used. It is known that a substance composed of a polysaccharide, a nucleic acid, a lipid, or the like can serve as an antigen, and the antigen of the antibody of the present invention is not particularly limited. The antigen can be prepared by a method known to those skilled in the art, for example, a method using baculovirus (for example, WO 98/46777). Hybridomas can be prepared, for example, by the method of Milstein et al (G.Kohler and C.Milstein, Methods enzymol.1981, 73: 3-46). When the immunogenicity of the antigen is low, the antigen can be immunized by binding it to a macromolecule having immunogenicity such as albumin. If necessary, the antigen may be bound to another molecule to become a soluble antigen. When a transmembrane molecule such as a membrane antigen (e.g., a receptor or the like) is used as the antigen, an extracellular region portion of the membrane antigen may be used as a fragment, or a cell expressing the transmembrane molecule on the cell surface may be used as the immunogen.

Antibody-producing cells can be obtained by immunizing an animal with the above-mentioned appropriate sensitizing antigen. Alternatively, antibody-producing lymphocytes are immunized in vitro to become antibody-producing cells. Various mammals can be used as the animal to be immunized, but rodents, lagomorphs, and primates are generally used. There can be exemplified: rodents such as mice, rats, hamsters, and the like; rabbit-shaped mesh such as rabbit; primates such as monkeys including cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees. Furthermore, transgenic animals having all the components of human antibody genes (reporters) are also known, and human antibodies can also be obtained by using such animals (see WO 96/34096; Mendez et al, nat. Genet.1997, 15: 146-56). Without using the above transgenic animal, for example, a desired human antibody having an antigen-binding activity can be obtained by sensitizing human lymphocytes with a desired antigen or cells expressing a desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese patent publication (Kokoku) No. 1-59878). The desired human antibody can also be obtained by immunizing a transgenic animal having all the components of the human antibody gene with the desired antigen (see WO93/12227, WO92/03918, WO94/02602, WO96/34096, WO 96/33735).

Immunization of animals can be carried out as follows: the sensitizing antigen is diluted and suspended in a Phosphate Buffered Saline (PBS) or a physiological saline as appropriate, mixed with an adjuvant as necessary, emulsified, and then injected into the abdominal cavity or the subcutaneous space of an animal to immunize. After that, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. Confirmation of antibody production can be carried out by determining the titer of the antibody of interest in the serum of the animal by conventional methods.

Hybridomas can be prepared as follows: antibody-producing cells obtained from animals or lymphocytes immunized with the desired antigen are fused with myeloma cells using a common fusing agent (e.g., polyethylene glycol) (Monoclonal Antibodies): Principles and Practice, Academic Press, 1986, 59-103). The hybridoma cells are cultured and proliferated as necessary, and the binding specificity of the antibody produced by the hybridoma is measured by a known analysis method such as immunoprecipitation, Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Thereafter, the hybridomas that produce the antibodies whose target specificity, affinity, or activity have been determined can be subcloned by a limiting dilution method or the like, if necessary.

Next, the gene encoding the selected antibody can be cloned from a hybridoma or an antibody-producing cell (sensitized lymphocyte or the like) using a probe capable of specifically binding to the antibody (for example, an oligonucleotide complementary to a sequence encoding the antibody constant region or the like). Cloning from mRNA can also be performed by RT-PCR. Immunoglobulins are classified into five different classes, IgA, IgD, IgE, IgG and IgM. These classes are further divided into subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1, IgA-2, etc.). In the present invention, the H chain and L chain used for the production of an antibody are not particularly limited, and may be derived from an antibody belonging to any of the above-mentioned classes and subclasses, and IgG is particularly preferable.

Here, the genes encoding the H chain and the L chain can also be modified by genetic engineering techniques. For example, an antibody such as a mouse antibody, a rat antibody, a rabbit antibody, a hamster antibody, a goat antibody, or a camel antibody may be appropriately modified to reduce its antigenicity against a human foreign antibody, and a genetically recombinant antibody such as a chimeric antibody or a humanized antibody may be appropriately prepared. A chimeric antibody is an antibody comprising the variable regions of H chain and L chain of a non-human mammal such as a mouse antibody and the constant regions of H chain and L chain of a human antibody, and can be obtained by linking DNA encoding the variable regions of a mouse antibody and DNA encoding the constant regions of a human antibody, inserting them into an expression vector, and introducing them into a host to produce an antibody. Humanized antibodies, also known as reshaped (reshaped) human antibodies, can be synthesized by PCR from a plurality of oligonucleotides that are made to have overlapping portions at the ends of DNA sequences designed to link the Complementarity Determining Regions (CDRs) of non-human mammals, such as mouse antibodies. The resulting DNA is ligated with a DNA encoding a human antibody constant region, inserted into an expression vector, and introduced into a host to produce a humanized antibody (see EP239400, WO 96/02576). FRs of human antibodies connected via CDRs are selected for which complementarity determining regions form good antigen binding sites. Amino acids of the framework regions of the antibody variable regions may be substituted as required to form appropriate antigen-binding sites for the complementarity determining regions of the reshaped human antibody (K.Sato et al, Cancer Res.1993, 53: 10.01-10.06).

In addition to the above-described humanization, it is considered that modification is performed to improve biological properties of an antibody such as binding to an antigen. The modification in the present invention can be carried out by site-specific mutagenesis (see, for example, Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, cassette mutagenesis, and the like. Generally, an antibody mutant having improved biological properties has 70% or more, more preferably 80% or more, and still more preferably 90% or more (e.g., 95% or more, 97%, 98%, 99%, etc.) amino acid sequence homology and/or similarity with respect to the amino acid sequence of the variable region of the original antibody. In this specification, sequence homology and/or similarity is defined as: sequence alignment and gap introduction are performed as necessary to maximize sequence homology, and then the proportion of amino acid residues that are identical (same residues) or similar (amino acid residues classified as the same group according to the characteristics of the general amino acid side chains) to the original antibody residues. In general, natural amino acid residues are divided into the following groups according to the nature of their side chains:

(1) hydrophobicity: alanine, isoleucine, valine, methionine and leucine;

(2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine, and serine;

(3) Acidity: aspartic acid and glutamic acid;

(4) alkalinity: arginine, histidine and lysine;

(5) residues that influence chain orientation: glycine and proline; and

(6) aromatic property: tyrosine, tryptophan and phenylalanine.

In general, a total of 6 complementarity determining regions (hypervariable regions; CDRs) present in the variable regions of the H chain and L chain interact to form the antigen binding site of an antibody. Even one of the variable regions is known to have the ability to recognize and bind to an antigen, although it has a lower affinity than when all binding sites are included. Thus, the H chain and L chain-encoding antibody gene of the present invention may encode a fragment portion including each antigen-binding site of the H chain and L chain as long as the polypeptide encoded by the gene maintains the binding property to a desired antigen.

As mentioned above, the heavy chain variable region is generally composed of 3 CDR regions and 4 FR regions. In a preferred embodiment of the present invention, the amino acid residues for "modification" may be appropriately selected from, for example, amino acid residues located in a CDR region or an FR region. In general, amino acid residues in CDR regions are modified to sometimes reduce the binding ability to an antigen. Therefore, in the present invention, the amino acid residues to be used for the "modification" are not particularly limited, and are preferably appropriately selected from the amino acid residues located in the FR region. Even in the case of CDR, when it is confirmed that the binding ability is not decreased by modification, the site can be selected. In addition, the sequence of FR that can be used as an antibody variable region in an organism such as a human or a mouse can be appropriately obtained by those skilled in the art using public databases and the like.

The present invention also provides a gene encoding the antibody of the present invention. The gene encoding the antibody of the present invention may be any gene, and may be DNA, RNA, other nucleic acid analogs, and the like.

The invention also provides a host cell carrying the gene. The host cell is not particularly limited, and examples thereof include Escherichia coli and various animal cells. Host cells can be used, for example, as production systems for making or expressing antibodies of the invention. A production system for producing a polypeptide comprising: in vitro (in vitro) and in vivo (in vivo) production systems. Examples of in vitro production systems are: production systems using eukaryotic cells and production systems using prokaryotic cells.

Examples of eukaryotic cells that can be used as host cells are: animal cells, plant cells, fungal cells. The animal cells include: mammalian cells, such as CHO (J.Exp.Med. (1995) 108: 94.0), COS, HEK293, 3T3, myeloma cells, BHK (baby hamster kidney), HeLa, Vero, etc.; amphibian cells, such as Xenopus laevis oocytes (Valle et al, Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, Tn 5. CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are suitably used for expression of the antibody of the present invention. Among animal cells, CHO cells are particularly preferable for the purpose of large-scale expression. The introduction of the vector into the host cell can be carried out, for example, by the following method: the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (manufactured by Boehringer Mannheim), the electroporation method, the lipofection method, and the like.

Examples of plant cells include tobacco-derived cells and duckweed (Lemna minor), which are known as protein production systems, and the antibody of the present invention can be produced by a method of culturing the cells using callus. Protein expression systems using fungal cells are well known and can be used as hosts for producing the antibodies of the invention, such as: yeasts such as cells of the genus Saccharomyces (Saccharomyces) (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Saccharomyces pombe), and the like); and filamentous fungi such as cells of Aspergillus (Aspergillus) (Aspergillus niger) and the like).

When prokaryotic cells are used, there are production systems using bacterial cells. As bacterial cells, in addition to the above-mentioned escherichia coli (e.coli), there is known a production system using bacillus subtilis, and these bacterial cells can be used for producing the antibody of the present invention.

< screening method >

The present invention provides a method of screening for antigen binding molecules that have a lower antigen binding activity at acidic pH than at neutral pH. The present invention also provides a method for screening an antigen-binding molecule that can bind to a plurality of antigens using 1 molecule. The present invention also provides a method for screening an antigen-binding molecule having excellent retention in plasma. The present invention also provides a method of screening for antigen binding molecules that dissociate antigens bound to the antigen binding molecule extracellularly within a cell. The present invention also provides a method for screening for an antigen-binding molecule that is taken into a cell in a state of being bound to an antigen and released to the outside of the cell in a state of not being bound to the antigen. The invention also provides methods for screening for antigen binding molecules having increased ability to eliminate an antigen in plasma. The invention also provides methods of screening for antigen binding molecules that are particularly useful as pharmaceutical compositions.

Specifically, the present invention provides a method for screening for antigen-binding molecules, which comprises the following steps.

(a) Obtaining the antigen binding activity of the antigen binding molecule at pH6.7 to pH10.0,

(b) Obtaining the antigen binding activity of the antigen binding molecule at pH4.0-pH6.5,

In the screening method of the present invention, there is no particular limitation as long as the antigen-binding activity of the antigen-binding molecule at ph6.7 to ph10.0 is an antigen-binding activity between ph6.7 and ph10.0, and a preferable antigen-binding activity is, for example, an antigen-binding activity between ph7.0 and ph 8.0; more preferred antigen-binding activity is, for example, antigen-binding activity at pH 7.4. The antigen binding activity of the antigen-binding molecule at ph4.0 to ph6.5 is not particularly limited as long as it is an antigen binding activity between ph4.0 and ph6.5, and a preferable antigen binding activity is, for example, an antigen binding activity between ph5.5 and ph 6.5; more preferred antigen-binding activity is, for example, antigen-binding activity at pH5.8 or pH 5.5.

The antigen binding activity of the antigen binding molecule can be measured by methods known to those skilled in the art, and can be determined appropriately by those skilled in the art with respect to conditions other than pH. The antigen binding activity of the antigen binding molecule can be expressed as KD (Dissociation constant), Apparent KD (Apparent Dissociation constant), Dissociation rate k_d(Dissociation rate: Dissociation rate) or apparent k_d(apparent dissociation rate) and the like. These parameters can be measured by methods known to those skilled in the art, and examples thereof include Biacore (GEhealthcare), Schachtt plot, and FACS.

In the present invention, the step of selecting an antigen-binding molecule having an antigen-binding activity at pH6.7 to pH10.0 that is higher than the antigen-binding activity at pH4.0 to pH6.5 is the same as the step of selecting an antigen-binding molecule having an antigen-binding activity at pH4.0 to pH6.5 that is lower than the antigen-binding activity at pH6.7 to pH 10.0.

The difference between the antigen binding activity at pH6.7 to pH10.0 and the antigen binding activity at pH4.0 to pH6.5 is not particularly limited as long as the antigen binding activity at pH6.7 to pH10.0 is higher than the antigen binding activity at pH4.0 to pH6.5, but the antigen binding activity at pH6.7 to pH10.0 is preferably 2 times or more, more preferably 10 times or more, and still more preferably 40 times or more the antigen binding activity at pH4.0 to pH 6.5.

The present invention also provides a method of screening for antigen binding molecules, the method comprising the steps of:

(a) a step of binding the antigen-binding molecule to the antigen at a pH of 6.7 to 10.0,

(b) A step of subjecting the antigen-binding molecule capable of binding to the antigen of (a) to a pH of 4.0 to 6.5,

(a) a step of selecting an antigen-binding molecule that does not bind to an antigen under the conditions of pH4.0 to pH6.5,

(b) A step of binding the antigen-binding molecule selected in (a) to an antigen at pH6.7 to pH10.0,

(c) A step of obtaining an antigen-binding molecule that binds to an antigen under a condition of pH6.7 to pH 10.0.

(c) A step of obtaining an antigen-binding molecule that dissociates at a pH of 4.0 to 6.5,

(d) A step of amplifying a gene encoding the dissociated antigen-binding molecule,

(e) A step of obtaining the eluted antigen binding molecule.

The steps (a) to (d) may be repeated two or more times. Accordingly, the present invention provides a method further comprising the step of repeating the steps (a) to (d) twice or more in the above-mentioned method. The number of repetitions of steps (a) to (d) is not particularly limited, and is usually 10 or less.

(c) A step for obtaining an antigen-binding molecule that binds to an antigen under a condition of pH6.7 to pH10.0,

(e) A step of obtaining the eluted antigen binding molecule.

In the screening method of the present invention, when a phage library or the like is used, the step of amplifying the gene encoding the antigen-binding molecule can also be used as the step of amplifying the phage.

In the method of the present invention, the binding of the antigen to the antigen-binding molecule may be performed in any state, and is not particularly limited. For example, an antigen binding molecule can be bound to an antigen by contacting the antigen with an immobilized antigen binding molecule; the antigen binding molecule may be bound to an antigen by contacting the antigen binding molecule with the immobilized antigen. In addition, the antigen binding molecule can be bound to an antigen by contacting the antigen binding molecule with the antigen in a solution.

The present invention also provides a method of screening for antigen binding molecules having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(a) a step of binding the antigen-binding molecule to the column on which the antigen is immobilized under a first pH condition,

(b) A step of eluting the antigen-binding molecule bound to the column under the first pH condition from the column under the second pH condition,

(c) A step of obtaining the eluted antigen binding molecule.

The present invention also provides a method of screening for antigen binding molecules having a binding activity at a first pH that is lower than the binding activity at a second pH, the method comprising the steps of:

(a) A step of passing the antigen-binding molecule through a column on which an antigen is immobilized under a first pH condition,

(b) A step of recovering the antigen-binding molecule eluted without binding to the column in the step (a),

(c) A step of binding the antigen-binding molecule recovered in (b) to the column under a second pH condition,

(d) A step of obtaining the antigen binding molecule bound to the column in step (c).

The invention also provides a method of screening for an antigen binding molecule having a binding activity at a first pH that is greater than the binding activity at a second pH, the method comprising the steps of.

(a) A step of binding the antigen-binding molecule library to the column on which the antigen is immobilized under a first pH condition,

(b) A step of eluting the antigen-binding molecule from the column at a second pH,

(c) A step of amplifying a gene encoding the eluted antigen-binding molecule,

(d) A step of obtaining the eluted antigen binding molecule.

The steps (a) to (c) may be repeated two or more times. Accordingly, the present invention provides a method further comprising the step of repeating the steps (a) to (c) twice or more in the above-mentioned method. The number of repetitions of steps (a) to (c) is not particularly limited, and is usually 10 or less.

In the present invention, any pH may be used as long as the first pH and the second pH are not the same pH. Examples of preferred combinations of the first pH and the second pH are: the first pH is a combination of pH between pH6.7 and 10.0 and the second pH is pH between pH4.0 and pH 6.5; examples of more preferred combinations are: the first pH is a combination of pH between pH7.0 and pH8.0, and the second pH is a combination of pH between pH5.5 and pH 6.5; examples of further preferred combinations are: the first pH is pH7.4, the second pH is pH5.8 or a combination of pH 5.5.

Examples of other preferred combinations of first pH and second pH are: the first pH is a combination of pH between pH4.0 and 6.5 and the second pH is pH between pH6.7 and pH 10.0; examples of more preferred combinations are: the first pH is a combination of pH between pH5.5 and pH6.5, and the second pH is a combination of pH between pH7.0 and pH 8.0; examples of further preferred combinations are: the first pH is pH5.8 or pH5.5, and the second pH is pH 7.4.

The antigen binding molecules screened using the methods of the invention may be any antigen binding molecule, for example, the antigen binding molecules described above may be used in the screening of the invention. For example, antigen binding molecules having a native sequence may be screened, or antigen binding molecules having a substituted amino acid sequence may be screened. Preferred examples of antigen binding molecules screened in the present invention are: for example, an antigen binding molecule having at least 1 amino acid of the antigen binding molecule substituted with histidine or inserted with at least 1 histidine. The site for introducing the histidine substitution or insertion is not particularly limited, and may be introduced at any site. Histidine substitutions or insertions may be introduced at 1 site, or at a plurality of sites up to 2 sites. Preferred examples of antigen binding molecules screened in the present invention are: such as antigen binding molecules comprising modified antibody constant regions.

The antigen-binding molecule to be screened by the method of the present invention may be, for example, a plurality of different antigen-binding molecules in which histidine substitution or insertion is introduced into different sites by a method such as histidine partitioning.

Accordingly, the screening method of the present invention may further comprise: a step of substituting at least 1 amino acid of the antigen binding molecule with histidine or inserting at least 1 histidine.

The screening method of the present invention may use an unnatural amino acid instead of histidine. Thus, the present invention can also be understood by interchanging the above histidine with a non-natural amino acid.

The screening method of the present invention may further comprise a step of modifying amino acids of the constant region of the antibody.

The antigen-binding substance screened by the screening method of the present invention can be prepared in any manner, for example, there can be used: a preexisting antibody, a preexisting library (e.g., phage library), a hybridoma obtained by immunizing an animal, an antibody or library prepared from B cells derived from an immunized animal, an antibody or library obtained by introducing a histidine or unnatural amino acid mutation into the antibody or library (e.g., a library in which the content of histidine or unnatural amino acids is increased, or a library in which a histidine or unnatural amino acid mutation is introduced at a specific site), and the like.

By the screening method of the present invention, antigen-binding molecules that bind to antigens many times and have excellent retention in plasma can be obtained. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule having excellent retention in plasma.

When administered to animals such as humans, mice, monkeys, etc., the screening method of the present invention can also provide antigen-binding molecules that can bind to an antigen twice or more. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule capable of binding to an antigen twice or more.

When administered to animals such as humans, mice, monkeys, etc., the screening method of the present invention can also provide antigen-binding molecules that can bind to antigens in an amount greater than the number of antigen-binding sites of the antigen-binding molecules. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule capable of binding to an antigen in a number greater than the number of antigen-binding sites of the antigen-binding molecule. For example, when the antibody is a neutralizing antibody, it can be used as a screening method for obtaining an antigen-binding molecule capable of neutralizing more antigens than the number of antigen-binding sites of the antigen-binding molecule.

When administered to animals such as humans, mice, monkeys, etc., the screening method of the present invention can also provide antigen-binding molecules capable of dissociating antigens bound extracellularly intracellularly. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that dissociates an antigen bound extracellularly in a cell.

When administered to an animal such as a human, a mouse or a monkey, the screening method of the present invention can also provide an antigen-binding molecule which is taken into a cell in a state of being bound to an antigen and released to the outside of the cell in a state of not being bound to the antigen. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that is taken into a cell in a state of being bound to an antigen and released to the outside of the cell in a state of not being bound to the antigen.

When administered to animals such as humans, mice, monkeys, etc., the screening method of the present invention can also provide antigen-binding molecules that can rapidly eliminate antigens from plasma. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule having an increased (high) ability to eliminate an antigen in plasma.

These antigen-binding molecules are considered to be particularly excellent as a pharmaceutical product because they can reduce the dose or frequency of administration to a patient and, as a result, can reduce the total dose. Therefore, the screening method of the present invention can be used as a screening method for an antigen-binding molecule as a pharmaceutical composition.

The present invention also provides libraries having an increased proportion of histidine compared to the original library. The library in which the proportion of histidines contained in the antigen-binding molecule contained in the library is increased can be used for the aforementioned screening method or the preparation method described later.

The library having an increased content of histidine can be prepared by a method known to those skilled in the art, for example, the following method. When synthesizing nucleic acids for preparing libraries, 20 kinds of 3-base codons (trinucleotides) encoding 20 kinds of amino acids were contained with equal probability by the trinucleotide method (J Mol biol. 2008Feb29; 376 (4): 1182-. In this case, the possibility of histidine appearing at the site of the library can be increased by increasing the proportion of the histidine-encoding trinucleotide to the 20 amino acids to a higher proportion than the other amino acids.

< method for producing antigen-binding molecule >

The present invention provides a method for producing an antigen-binding molecule having antigen-binding activity at a pH in vivo in the nucleus that is lower than that at a pH in plasma. The present invention also provides a method for producing an antigen-binding molecule having excellent retention in plasma. The invention also provides methods for preparing antigen binding molecules that are particularly useful as pharmaceutical compositions.

Specifically, the present invention provides a method for preparing an antigen-binding molecule, comprising the steps of:

(a) obtaining the antigen binding activity of the antigen binding molecule at pH6.7-pH10.0,

(c) A step of selecting an antigen-binding molecule having an antigen-binding activity at pH6.7 to pH10.0 higher than that at pH4.0 to pH6.5,

(d) Obtaining a gene encoding the antigen binding molecule selected in (c),

The present invention also provides a method of preparing an antigen binding molecule, the method comprising the steps of:

(d) A step of obtaining a gene encoding the antigen-binding molecule obtained in (c),

(a) a step of binding an antigen-binding molecule to an antigen at a pH of 6.7 to 10.0,

(e) A step of obtaining an eluted antigen-binding molecule,

(f) Obtaining a gene encoding the antigen-binding molecule obtained in (e),

(g) A step of preparing an antigen-binding molecule using the gene obtained in (f).

(e) A step of obtaining an eluted antigen-binding molecule,

(f) Obtaining a gene encoding the antigen-binding molecule obtained in (e),

The present invention also provides a method of preparing an antigen binding molecule having a binding activity at a first pH that is higher than the binding activity at a second pH, the method comprising the steps of:

(c) A step of obtaining an eluted antigen-binding molecule,

(c) A step of amplifying a gene encoding the eluted antigen-binding molecule,

(d) A step of obtaining an eluted antigen-binding molecule,

(e) A step of obtaining a gene encoding the antigen-binding molecule obtained in (d),

In the production method of the present invention, the step of amplifying the gene encoding the antigen-binding molecule may be used as a step of amplifying a phage, when a phage library or the like is used.

The antigen-binding substance used in the production method of the present invention may be produced in any manner, and for example,: a preexisting antibody, a preexisting library (e.g., phage library), a hybridoma obtained by immunizing an animal or an antibody or library prepared from B cells derived from an immunized animal, an antibody or library obtained by introducing a histidine or unnatural amino acid mutation into such an antibody or library (e.g., a library in which the content of histidine or unnatural amino acids is increased or a library in which a histidine or unnatural amino acid mutation is introduced into a specific site), and the like.

In the above-mentioned production method, the antigen binding activity of the antigen-binding molecule at pH6.7 to pH10.0 is not particularly limited as long as it is an antigen binding activity between pH6.7 and pH10.0, and a preferable antigen binding activity is, for example, an antigen binding activity between pH7.0 and pH 8.0; a further preferable antigen-binding activity is, for example, an antigen-binding activity at pH 7.4. The antigen binding activity of the antigen-binding molecule at ph4.0 to ph6.5 is not particularly limited as long as it is an antigen binding activity between ph4.0 and ph6.5, and a preferable antigen binding activity is, for example, an antigen binding activity between ph5.5 and ph 6.5; further preferable antigen-binding activity is, for example, antigen-binding activity at pH5.8 or pH 5.5.

The antigen binding activity of the antigen binding molecule can be measured by methods known to those skilled in the art, and can be determined appropriately by those skilled in the art with respect to conditions other than pH.

The step of selecting an antigen-binding molecule having an antigen-binding activity at pH6.7 to pH10.0 that is higher than the antigen-binding activity at pH4.0 to pH6.5 is the same as the step of selecting an antigen-binding molecule having an antigen-binding activity at pH4.0 to pH6.5 that is lower than the antigen-binding activity at pH6.7 to pH 10.0.

The antigen binding activity at ph6.7 to ph10.0 is not particularly limited as long as it is higher than the antigen binding activity at ph4.0 to ph6.5, and the difference between the antigen binding activity at ph6.7 to ph10.0 and the antigen binding activity at ph4.0 to ph6.5 is preferably 2 times or more, more preferably 10 times or more, and still more preferably 40 times or more the antigen binding activity at ph6.7 to ph10.0, compared to the antigen binding activity at ph4.0 to ph 6.5.

In the above-mentioned production method, the binding of the antigen to the antigen-binding molecule may be carried out in any state, and is not particularly limited. For example, an antigen binding molecule can be bound to an antigen by contacting the antigen with an immobilized antigen binding molecule; the antigen binding molecule may also be bound to an antigen by contacting the antigen binding molecule with an immobilized antigen. Alternatively, the antigen-binding molecule may be bound to an antigen by contacting the antigen-binding molecule with the antigen in a solution.

In the above production method, any pH may be used as long as the first pH and the second pH are each different pH. Examples of preferred combinations of the first pH and the second pH are: the first pH is a combination of pH between pH6.7 and 10.0 and the second pH is pH between pH4.0 and pH 6.5; examples of more preferred combinations are: the first pH is a combination of pH between pH7.0 and pH8.0, and the second pH is a combination of pH between pH5.5 and pH 6.5; examples of further preferred combinations are: the first pH is pH7.4, the second pH is pH5.8 or a combination of pH 5.5.

Examples of other preferred combinations of first pH and second pH are: the first pH is a combination of pH between pH4.0 and pH6.5, and the second pH is a combination of pH between pH6.7 and pH 10.0; examples of more preferred combinations are: the first pH is a combination of pH between pH5.5 and pH6.5, and the second pH is a combination of pH between pH7.0 and pH 8.0; examples of further preferred combinations are: the first pH is pH5.8 or pH5.5, and the second pH is pH 7.4.

The antigen-binding molecule prepared by the above-described preparation method may be any antigen-binding molecule, and preferable examples are, for example: an antigen binding molecule having at least 1 amino acid of the antigen binding molecule substituted with histidine or inserted with at least 1 histidine. The site for introducing such a histidine mutation is not particularly limited, and may be introduced at any site. A histidine mutation may be introduced into 1 site, or a plurality of sites including 2 or more sites.

Therefore, in the preparation method of the present invention, a step of substituting at least 1 amino acid of the antigen-binding molecule with histidine or inserting histidine may be further included.

In the preparation method of the present invention, an unnatural amino acid may be used instead of histidine. Thus, the present invention can also be understood by interchanging the above mentioned histidines with unnatural amino acids.

As another embodiment of the antigen-binding molecule produced by the above production method, there is an antigen-binding molecule comprising a modified antibody constant region, and therefore, the production method of the present invention may further comprise a step of modifying amino acids in the antibody constant region.

The antigen-binding molecule produced by the production method of the present invention is an antigen-binding molecule having excellent retention in plasma. Therefore, the production method of the present invention can be used as a production method of an antigen-binding molecule having excellent retention in plasma.

When the antigen-binding molecule prepared by the preparation method of the present invention is administered to animals such as human, mouse, monkey, etc., it is considered that the antigen-binding molecule can bind to the antigen twice or more. Therefore, the production method of the present invention can be used as a production method of an antigen-binding molecule capable of binding to an antigen twice or more.

When the antigen-binding molecule prepared by the production method of the present invention is administered to an animal such as a human, a mouse, or a monkey, it is considered that the antigen-binding molecule can bind to more antigens than the number of antigen-binding sites of the antigen-binding molecule. Therefore, the production method of the present invention can be used as a production method of an antigen-binding molecule capable of binding to an antigen in a larger number than the number of antigen-binding sites of the antigen-binding molecule.

When the antigen-binding molecule prepared by the production method of the present invention is administered to an animal such as a human, a mouse, or a monkey, it is considered that the antigen bound to the antigen-binding molecule outside the cell can be dissociated from the antigen-binding molecule inside the cell. Therefore, the production method of the present invention can be used as a production method of an antigen-binding molecule capable of dissociating an antigen bound extracellularly in a cell.

When the antigen-binding molecule prepared by the production method of the present invention is administered to an animal such as a human, a mouse, or a monkey, it is considered that the antigen-binding molecule taken into the cell in a state of being bound to the antigen can be released to the outside of the cell in a state of not being bound to the antigen. Therefore, the production method of the present invention can be used as a method for producing an antigen-binding molecule that is taken into a cell in a state of being bound to an antigen and is released to the outside of the cell in a state of not being bound to the antigen.

Furthermore, it is considered that the antigen-binding molecule prepared by the preparation method of the present invention can rapidly eliminate an antigen from plasma when administered to an animal such as a human, a mouse, or a monkey. Therefore, the production method of the present invention can be used as a production method of an antigen-binding molecule having an increased (high) ability to eliminate an antigen in plasma.

These antigen-binding molecules can reduce the number of administrations to patients, and are considered to be particularly excellent as pharmaceutical products. Therefore, the preparation method of the present invention can be used as a preparation method of an antigen-binding molecule as a pharmaceutical composition.

The gene obtained by the production method of the present invention is usually carried (inserted) on an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it is a vector that stably retains the inserted nucleic acid, and when Escherichia coli is used as a host, for example, pBluescript vector (Stratagene) is preferable as a cloning vector, but various commercially available vectors can be used. Expression vectors are particularly useful when used in order to produce the antigen binding molecules of the invention. The expression vector is not particularly limited as long as it is a vector that expresses the antigen-binding molecule in vitro, in Escherichia coli, in cultured cells, or in biological individuals, and for example, when the antigen-binding molecule is expressed in vitro, a pBEST vector (manufactured by Promega corporation) is preferable; when expressing the antigen-binding molecule in E.coli, pET vector (manufactured by Invitrogen) is preferable; for expression of antigen binding molecules in cultured cells, the pME18S-FL3 vector (GenBank accession number AB009864) is preferred; when the antigen-binding molecule is expressed in an individual organism, the pME18S vector (Mol Cell biol.8: 466-472(1988)) and the like are preferred. The insertion of the DNA of the present invention into a vector can be carried out by a conventional method, for example, by ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edge. Ausubel et al (1987) published John Wiley & sons. section 11.4-11.11).

The host cell is not particularly limited, and various host cells can be used according to the purpose. Examples of the cell for expressing the antigen-binding molecule include: bacterial cells (e.g., streptococci (Streptococcus), staphylococci (Staphylococcus), escherichia coli (e.coli), Streptomyces (Streptomyces), Bacillus subtilis (Bacillus subtilis)); fungal cells (e.g.yeast (Yeast), Aspergillus (Aspergillus)); insect cells (e.g., Drosophila S2(Drosophila S2), Spodoptera SF9(Spodoptera SF 9)); animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. The vector can be introduced into the host cell by a known method such as calcium phosphate precipitation, electric pulse electroporation (Current protocols in Molecular Biology apparatus. Ausubel et al, (1987) John Wiley & sons. section9.1-9.9), lipofection, and microinjection.

The host cell can be cultured according to a known method. For example, when animal cells are used as hosts, DMEM, MEM, RPMI1640, or IMDM can be used as the culture medium. In this case, a serum supplement such as FBS or Fetal Calf Serum (FCS) may be used in combination, or the cells may be cultured in serum-free culture. The pH during the culture is preferably about 6 to 8. Usually, the culture is carried out at about 30 to 40 ℃ for about 15 to 200 hours, and the medium may be exchanged or aerated or stirred as necessary.

Appropriate secretion signals can be inserted into the polypeptide of interest to cause secretion of the antigen binding molecule expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. The signal may be endogenous or heterologous with respect to the antigen binding molecule of interest.

On the other hand, as a system for producing a polypeptide in vivo, there are, for example, a production system using animals or a production system using plants. The target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in the animals or plants and recovered. The "host" in the present invention includes these animals and plants.

When animals are used, there are production systems using mammals and insects. As the mammal, goat, pig, sheep, mouse, cow, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals may be used.

For example, a polynucleotide encoding the antigen-binding molecule of the present invention is prepared and used as a fusion gene with a gene encoding a polypeptide inherently produced in milk such as goat β -casein. Then, a polynucleotide fragment comprising the fusion gene is injected into a goat embryo, and the embryo is transferred into a female goat. The goat which received the embryo gives rise to a transgenic goat from which, or from the milk produced by its offspring, the antigen binding molecule of interest can be obtained. In order to increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat, the transgenic goat may be administered with an appropriate hormone (Ebert et al, Bio/Technology (1994) 12: 699-702).

As the insect that produces the antigen-binding molecule of the present invention, for example, silkworm can be used. When silkworms are used, the target antigen-binding molecule can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which a polynucleotide encoding the target antigen-binding molecule is inserted.

When a plant is used for producing the antigen-binding molecule of the present invention, tobacco can be used, for example. When tobacco is used, a polynucleotide encoding an antigen-binding molecule of interest is inserted into a plant expression vector such as pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). Infection of tobacco, such as tobacco (Nicotiana tabacum), with this bacterium allows the desired antigen-binding molecule to be obtained from the leaves of this tobacco (Ma et al, Eur. J. Immunol. (1994) 24: 131-8). Duckweed (Lemna minor) is infected with the same bacteria and the desired antigen binding molecule can be obtained from the cells of duckweed after cloning (Cox KM et al nat. Biotechnol. 2006Dec; 24 (12): 1591-.

The antigen-binding molecule thus obtained can be isolated from the inside or outside of the host cell (medium, milk, etc.) and purified to be a substantially pure and homogeneous antigen-binding molecule. The separation and purification of the antigen-binding molecule can be carried out by a separation and purification method generally used for the purification of a polypeptide, but is not limited thereto. For example, the antigen-binding molecule can be separated and purified by appropriately selecting a combination of chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, and the like.

Examples of chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and characterization guide: A Laboratory CourseMicroanal. Ed Daniel R. Marshark et al (1996) Cold Spring harbor Laboratory Press). The chromatography can be performed by liquid chromatography, for example, HPLC, FPLC and the like. Columns for affinity chromatography are, for example: protein A column and protein G column. Examples of protein a columns are: hyper D, POROS, Sepharose F.F (Pharmacia), and the like.

If desired, an appropriate protein modifying enzyme may be allowed to act on the antigen binding molecule to modify or partially remove the peptide as desired, either before or after purification of the antigen binding molecule. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glycosidase.

< anti-IL-6 receptor antibody >

The present invention also provides an anti-IL-6 receptor antibody according to any one of the following items (a) to (m).

(a) An antibody comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which at least 1 of Tyr at position 27, Asp at position 31, Asp at position 32, Trp at position 35, Tyr at position 51, Asn at position 59, Ser at position 63, Met at position 106, and Tyr at position 108 is substituted with His;

(b) An antibody (H3pI) comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which Tyr at position 27, Asp at position 31 and Trp at position 35 are substituted with His;

(c) an antibody comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which His is substituted for Tyr at position 27, Asp at position 31, Asp at position 32, Trp at position 35, Asn at position 59, Ser at position 63, and Tyr at position 108;

(d) an antibody (H170) comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which the amino acid sequence of position 27 is Tyr, position 31 is Asp, position 32 is Asp, position 35 is Trp, position 59 is Asn, position 63 is Ser, position 108 is Tyr substituted with His, position 99 is Ser substituted with Val, position 103 is Thr substituted with Ile;

(e) an antibody comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which Asp at position 31, Tyr at position 51, Ser at position 63, Met at position 106 and Tyr at position 108 are substituted with His;

(f) An antibody (CLH5) comprising a heavy chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which Asp at position 31, Tyr at position 51, Ser at position 63, Met at position 106 and Tyr at position 108 are substituted with His, Ser at position 99 is substituted with Phe, Thr at position 103 is substituted with Ile;

(g) an antibody comprising a light chain variable region having the following amino acid sequence: in SEQ ID NO: 2(PF1L variable region) in which at least one of Asp at position 28, Tyr at position 32, Glu at position 53, Ser at position 56, and Asn at position 92 is substituted by His;

(h) an antibody (L73) comprising a light chain variable region having the following amino acid sequence: in SEQ ID NO: 2(PF1L variable region) in which Asp at position 28, Tyr at position 32 and Glu at position 53 are substituted with His;

(i) an antibody (L82) comprising a light chain variable region having the following amino acid sequence: in SEQ ID NO: 1(H53 variable region) in which Tyr at position 32 and Glu at position 53 are substituted with His;

(j) an antibody (CLL5) comprising a light chain variable region having the following amino acid sequence: in SEQ ID NO: 2(PF1L variable region) in which the Tyr at position 32, the Glu at position 53, the Ser at position 56 and the Asn at position 92 are substituted with His;

(k) An antibody comprising the heavy chain variable region of (b) and the light chain variable region of (h);

(l) An antibody comprising the heavy chain variable region of (d) and the light chain variable region of (i);

(m) an antibody comprising the heavy chain variable region of (f) and the light chain variable region of (h).

As a polypeptide having the sequence set forth in SEQ ID NO: 1(H53 variable region), wherein at least 1 of Tyr at position 27, Asp at position 31, Asp at position 32, Trp at position 35, Tyr at position 51, Asn at position 59, Ser at position 63, Met at position 106 and Tyr at position 108 is substituted with His in the amino acid sequence thereof, for example, the following heavy chain variable region:

a heavy chain variable region having SEQ ID NO: 3(H3pI)

A heavy chain variable region having SEQ ID NO: 4(H170) amino acid sequence

A heavy chain variable region having SEQ ID NO: 5(CLH5)

As a polypeptide having the sequence set forth in SEQ ID NO: 2(PF1L variable region), wherein at least 1 of Asp at position 28, Tyr at position 32, Glu at position 53, Ser at position 56, and Asn at position 92 is substituted with His in the amino acid sequence thereof, for example, the following light chain variable region is exemplified:

A light chain variable region having SEQ ID NO: 6(L73)

A light chain variable region having SEQ ID NO: 7(L82)

A light chain variable region having SEQ ID NO: 8(CLL5)

Amino acid positions and amino acid substitutions in each of the above antibodies of H3pI, H170, CLH5, L73, L82 and CLL5 are shown in table 1 below. Amino acid positions are indicated according to Kabat numbering.

[ Table 1]

Position of

27

31

32

33

35

50

58

61

62

63

64

65

95

99

100B

102

H3pI

H

H170

H

V

I

H

CLH5

H

F

I

H

Position of

24

27

28

32

53

55

56

90

92

94

L73

H

L82

H

CLL5

H

Position 33 of H chain and position 55 of L chain have a sequence of histidine in WT.

The present invention provides an antibody comprising at least one of the amino acid substitutions (a) to (j) described above, and a method for producing the antibody. Accordingly, the antibody of the invention further comprises: an antibody comprising an amino acid substitution other than the amino acid substitutions described in (a) to (j) above in addition to the amino acid substitution described in any one of (a) to (j) above. Examples of the amino acid substitutions other than the amino acid substitutions described in the above (a) to (j) include substitution, deletion, addition and/or insertion of an amino acid sequence of a CDR portion, substitution, deletion, addition and/or insertion of an amino acid sequence of an FR portion, and the like.

The present invention also provides an anti-IL-6 receptor antibody according to any one of the following items (1) to (28).

(1) An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21(VH1-IgG1) from the 1 st to 119 th amino acid sequences of the variable region of the heavy chain (VH1-IgG1 variable region);

(2) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22(VH2-IgG1) from position 1 to position 119 (VH2-IgG1 variable region);

(3) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23(VH3-IgG1) from the 1 st to 119 th amino acid sequences of the heavy chain variable region (VH3-IgG1 variable region);

(4) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24(VH4-IgG1) and a heavy chain variable region (VH4-IgG1 variable region) having an amino acid sequence from position 1 to position 119;

(5) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25(VL1-CK) from the amino acid sequence from position 1 to position 107 (VL1-CK variable region);

(6) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26(VL2-CK) from the amino acid sequence from position 1 to position 107 (VL2-CK variable region);

(7) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27(VL3-CK) amino acid sequence from position 1 to position 107 in the light chain variable region (VL3-CK variable region);

(8) an antibody (Fv1-IgG1) comprising the heavy chain variable region of (2) and the light chain variable region of (6);

(9) An antibody (Fv2-IgG1) comprising the heavy chain variable region of (1) and a heavy chain variable region having SEQ ID NO: the light chain variable region of the amino acid sequence of 7 (L82);

(10) an antibody (Fv3-IgG1) comprising the heavy chain variable region of (4) and the light chain variable region of (5);

(11) an antibody (Fv4-IgG1) comprising the heavy chain variable region of (3) and the light chain variable region of (7);

(12) an antibody (VH3-IgG2 Δ GK) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 33, or a heavy chain having the amino acid sequence set forth in seq id No. 33;

(13) an antibody (VH3-M58) comprising a heavy chain having SEQ ID NO: 34;

(14) an antibody (VH3-M73) comprising a heavy chain having SEQ ID NO: 35;

(15) an antibody (Fv4-IgG2 Δ GK) comprising the heavy chain of (12) and a light chain having the sequence of SEQ ID NO: 27(VL 3-CK);

(16) an antibody (Fv4-M58) comprising the heavy chain of (13) and a light chain having the amino acid sequence of SEQ ID NO: 27(VL 3-CK);

(17) an antibody (Fv4-M73) comprising the heavy chain of (14) and a light chain having the amino acid sequence of SEQ ID NO: 27(VL 3-CK);

(18) an antibody (VH2-M71) comprising a heavy chain having SEQ ID NO: 36(VH 2-M71);

(19) an antibody (VH2-M73) comprising a heavy chain having SEQ ID NO: 37(VH 2-M73);

(20) An antibody (VH4-M71) comprising a heavy chain having SEQ ID NO: 38(VH 4-M71);

(21) an antibody (VH4-M73) comprising a heavy chain having SEQ ID NO: 39(VH 4-M73);

(22) an antibody (Fv1-M71) comprising the heavy chain of (18) and a light chain having the amino acid sequence of SEQ ID NO: 26(VL 2-CK);

(23) an antibody (Fv1-M73) comprising the heavy chain of (19) and a light chain variable region having the sequence of SEQ ID NO: 26(VL 2-CK);

(24) an antibody (Fv3-M71) comprising the heavy chain of (20) and a light chain variable region having the sequence of SEQ ID NO: 25(VL 1-CK);

(25) an antibody (Fv3-M73) comprising the heavy chain of (21) and a light chain variable region having the sequence of SEQ ID NO: 25(VL 1-CK);

(26) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 25(VL 1-CK);

(27) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 26(VL 2-CK);

(28) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 27(VL 3-CK).

The present invention also provides the FR or CDR described in any one of (a) to (v) below.

(a) SEQ ID NO: 40 (VH1, 2, 3, 4) of the heavy chain CDR1,

(b) SEQ ID NO: 41 (VH1, VH 2) of the heavy chain CDR2,

(c) SEQ ID NO: 42 (VH3) of the heavy chain CDR2,

(d) SEQ ID NO: 43 in the form of a heavy chain CDR2(VH4),

(e) SEQ ID NO: 44 (VH1, VH2) of the heavy chain CDR3,

(f) SEQ ID NO: 45 (VH3, 4) of the heavy chain CDR3,

(g) SEQ ID NO: 46 of heavy chain FR1(VH1, VH2),

(h) SEQ ID NO: 47 of heavy chain FR1(VH3, 4),

(i) SEQ ID NO: heavy chain FR2(VH1, 2, 3, 4)

(j) SEQ ID NO: the heavy chain FR3(VH1) of 49,

(k) SEQ ID NO: 50 of heavy chain FR3(VH2),

(l) SEQ ID NO: 51 of the heavy chain FR3(VH3, 4),

(m) SEQ ID NO: heavy chain FR4(VH1, 2, 3, 4) according to claim 52

(n) SEQ ID NO: 53 as described in (1) and (2) the light chain CDR1(VL1, VL),

(o) SEQ ID NO: 54 light chain CDR1(VL3),

(p) SEQ ID NO: 55, light chain CDR2(VL1, VL3),

(q) SEQ ID NO: 56 as described in (1) and (2) the light chain CDR2(VL2),

(r) SEQ ID NO: the light chain CDR3(VL1, 2 and 3) of claim 57,

(s) SEQ ID NO: 58 of the light chain FR1(VL1, 2 or 3),

(t) SEQ ID NO: FR2 light chain (VL1, 2 or 3) of 59,

(u) SEQ ID NO: 60 or the light chain FR3(VL1, 2 or 3),

(v) SEQ ID NO: the light chain FR4(VL1, 2 or 3) according to 61.

The sequences (a) to (v) are shown in FIG. 25. The present invention also provides a polypeptide comprising the FR or CDR of any one of the above (a) to (v).

The anti-IL-6 receptor antibodies of the invention also include: a fragment of an antibody comprising an amino acid substitution as described in any one of the above, or a modification thereof. Examples of the antibody fragment include Fab, F (ab') 2, Fv, and single-chain Fv (scFv) in which an H chain and an L chain are linked to each other via an appropriate linker, an H chain single domain, and an L chain single domain (e.g., Nat. Biotechnol. 2005Sep; 23 (9): 1126-36), Unibody (WO2007059782A1), and SMIP (WO2007014278A 2). The source of the antibody is not particularly limited, and may be a human antibody, a mouse antibody, a rat antibody, a rabbit antibody, or the like. The antibody of the present invention may be a chimeric antibody, a humanized antibody, a fully humanized antibody, or the like.

Specifically, the antibody is treated with an enzyme such as papain or pepsin to produce an antibody fragment; alternatively, genes encoding these antibody fragments are constructed and introduced into expression vectors and then expressed in appropriate host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M. & Horwitz, A.H.Methodsin Enzymology (1989)178, 476-496; Pluckthun, A. & Skerra, A.Methodsin Enzymology (1989)178, 497-515; Lamoy, E., Methods in Enzymology (1989)121, 652-663; Rousseaux, J. & et al, Methods in Enzymology (1989)121, 663-66; rd, R.E. Bird et al, TIE 137, ECH (1991)9, 132).

Accordingly, the present invention provides a method for producing the polypeptide of the present invention or a polypeptide encoded by a gene encoding the polypeptide of the present invention, the method comprising the step of culturing a host cell comprising a vector into which a polynucleotide encoding the polypeptide of the present invention has been introduced.

More specifically, a method for producing the polypeptide of the present invention is provided, which comprises the steps of:

(a) a step of culturing a host cell comprising a vector into which a gene encoding a polypeptide of the present invention is introduced;

(b) a step of obtaining a polypeptide encoded by the gene.

scFv is obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al, Proc. Natl.Acad.Sci.USA (1988)10.0, 5879-. The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described above. As the peptide linker for connecting the V region, for example, any single-chain peptide containing 12 to 19 amino acid residues is used.

When the anti-IL-6 receptor antibody of the present invention comprises a constant region, the constant region may be any type of constant region, and for example, constant regions of IgG1, IgG2, IgG4, or the like may be used. The constant region is preferably a human antibody constant region. The modified form may be one obtained by substitution, deletion, addition and/or insertion of an amino acid sequence for a constant region of human IgG1, human IgG2, human IgG4 or the like.

The IL-6 receptor to which the anti-IL-6 receptor antibody of the present invention binds is preferably a human IL-6 receptor.

The anti-IL-6 receptor antibody of the present invention is an antibody having excellent plasma retention, and the anti-IL-6 receptor antibody is present in plasma for a prolonged period of time in a state in which it can bind to a soluble IL-6 receptor and a membrane-type IL-6 receptor as antigens, and binds to the soluble IL-6 receptor and the membrane-type IL-6 receptor in vivo for a prolonged period of time by the anti-IL-6 receptor antibody. The anti-IL-6 receptor antibody can bind to the IL-6 receptor twice or more, and it is considered that it can neutralize 3 or more IL-6 receptors.

< pharmaceutical composition >

The present invention also relates to pharmaceutical compositions comprising the antigen-binding molecules of the present invention, the antigen-binding molecules isolated using the screening methods of the present invention, or the antigen-binding molecules prepared using the preparation methods of the present invention. The antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention is excellent in plasma retention and is expected to reduce the frequency of administration of the antigen-binding molecule, and therefore is effective as a pharmaceutical composition. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease or for examination or diagnosis.

The pharmaceutical composition of the present invention may be formulated according to methods known to those skilled in the art. For example, a sterile solution or suspension with water or a pharmaceutically acceptable liquid other than water can be prepared for parenteral administration in the form of an injection. For example, a pharmacologically acceptable carrier or vehicle, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle (vehicle), preservative, binder, and the like may be suitably combined and mixed in a unit dosage form required for generally accepted pharmaceutical practice to prepare a preparation. The amount of the active ingredient in the above preparation is set to an appropriate volume to obtain the range indicated.

The sterile composition for injection can be prepared according to a usual preparation procedure using a vehicle such as distilled water for injection.

Examples of the aqueous solution for injection include physiological saline and isotonic solution containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable cosolvents such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (Polysorbate80(TM), HCO-50, etc.) may be used in combination.

The oily liquid is sesame oil or soybean oil, and benzyl benzoate and/or benzyl alcohol can be used in combination as cosolvent. It can also be mixed with buffer (such as phosphate buffer and sodium acetate buffer), demulcent (such as procaine hydrochloride), stabilizer (such as benzyl alcohol and phenol), and antioxidant. The prepared injection is usually filled in an appropriate ampoule.

The pharmaceutical compositions of the present invention are preferably administered by parenteral routes of administration. For example, the composition can be prepared into an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, systemic or local administration can be carried out by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

The administration method may be appropriately selected depending on the age and symptoms of the patient. The amount of the antigen-binding molecule-containing pharmaceutical composition to be administered can be set, for example, as follows: each time, the weight of the medicine is 0.0001 mg-1000 mg. Alternatively, the amount to be administered may be, for example, 0.001 to 100000mg per patient, but the present invention is not necessarily limited to the above values. The dose and the administration method vary depending on the body weight, age, symptoms, and the like of the patient, and one skilled in the art can set an appropriate dose and administration method in consideration of the above conditions.

Amino acids contained in the amino acid sequence described in the present invention may be modified after translation (for example, modification in which glutamine at the N-terminal is converted to pyroglutamic acid by pyroglutamylation is known to those skilled in the art), and such amino acids are naturally included in the amino acid sequence described in the present invention even when they are modified after translation.

All prior art documents cited in this specification are incorporated herein by reference.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 1 preparation of modified humanized PM1 antibody

Preparation of recombinant soluble human IL-6 receptor (SR344)

As an antigen of the human IL-6 receptor recombinant human IL-6 receptor preparation as follows. A CHO cell constant expression strain of the soluble human IL-6 receptor (hereinafter referred to as SR344) (Yamasaki et al, Science 1988; 241: 825) -828(GenBank # X12830)) comprising the amino acid sequences from position 1 to position 344 on the N-terminal side as reported in J.biochem.108, 673-676(1990) was prepared.

SR344 was purified from the culture supernatant obtained from SR 344-expressing CHO cells by 3 kinds of column chromatography, blue sepharose6FF column chromatography, affinity chromatography using a column to which an SR 344-specific antibody is immobilized, and gel filtration column chromatography. The fraction eluted as the main peak was taken as the final pure product.

Preparation of recombinant soluble cynomolgus IL-6 receptor (cIL-6R)

Oligo DNA primers Rhe6Rf1(SEQ ID NO: 16), Rhe6Rr2(SEQ ID NO: 17) were made based on the published rhesus IL-6 receptor gene sequence (Birney et al, Ensembl2006, Nucleic acids sRs.2006 Jan 1; 34(Database disease): D556-61). A DNA fragment encoding the full-length gene of cynomolgus IL-6 receptor was prepared by PCR using cDNA prepared from cynomolgus monkey pancreas as a template and primers Rhe6Rf1 and Rhe6Rr 2. Using the obtained DNA fragment as a template, a 1131bp DNA fragment (SEQ ID NO: 20) encoding a protein having 6XHis added to the C-terminus of a soluble region (Met1-Pro363) including a signal region of cynomolgus monkey IL-6 receptor gene was amplified by PCR using oligo DNA primers Cynoil6R N-EcoRI (SEQ ID NO: 18) and Cynoil6R C-NotI-His (SEQ ID NO: 19). The obtained DNA fragment was digested with EcoRI-NotI, and inserted into an animal cell expression vector, and a CHO constant expression strain (CHO cell producing cyno. sIL-6R) was prepared using the vector.

The culture broth of CHO cells producing cyno. sIL-6R was purified by HisTrap column (GEHealthcare Bioscience), followed by concentration using Amicon Ultra-15Ultracel-10k (Millipore), and further purified by Superdex200pg16/60 gel filtration column (GEHealthcare Bioscience) to obtain a final pure soluble cynomolgus IL-6 receptor (hereinafter, referred to as cIL-6R).

Preparation of recombinant cynomolgus monkey IL-6(cIL-6)

Cynomolgus IL-6 was prepared as follows. A nucleotide sequence encoding 212 amino acids registered in SWISSPROT access No. p79341 was prepared, cloned into an animal cell expression vector, and the resulting vector was introduced into CHO cells to prepare a constantly expressing cell line (CHO cells producing cyno. il-6). Culture broth of CHO cells producing cyno IL-6 was purified by SP-Sepharose/FF column (GE Healthcare Bioscience), followed by concentration using Amicon Ultra-15Ultracel-5k (Millipore), further purified by Superdex75pg26/60 gel filtration column (GE Healthcare B Bioscience), and concentrated by Amicon Ultra-15Ultracel-5k (Millipore) as a final pure product of cynomolgus IL-6 (hereinafter referred to as cIL-6).

Establishment of human gp130 expression BaF3 cell line

To obtain a cell line showing IL-6-dependent proliferation, a BaF3 cell line expressing human gp130 was established as follows.

The full-length human gp130cDNA was amplified by PCR (Hibi et al, Cell 1990; 63: 1149-1157(GenBank # NM-002184)), and then cloned into an expression vector pCOS2Zeo, which was obtained by removing the DHFR gene expression site of pCHOI (Hirata et al, FEBS Letter 1994; 356: 244-248) and inserting the Zeocin-resistance gene expression site, to construct pCOS2Zeo/gp 130. The full-length human IL-6R cDNA was amplified by PCR and then cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1 (+). Mu.g of pCOS2Zeo/gp130 were mixed with BaF3 cells (0.8X 10) suspended in PBS ⁷Cells), then, a pulse was applied at a capacity of 950. mu. FD and 0.33kV using Gene Pulser (Bio-Rad). BaF3 cells into which a gene was introduced by electroporation were cultured in RPMI1640 medium (Invitrogen) containing 0.2ng/mL of mouse interleukin-3 (Peprotech) and 10% of fetal bovine serum (hereinafter, FBS; HyClone) for a whole day and night, and 100ng/mL of human interleukin-6 (R)&D systems), 100ng/mL human interleukin-6 soluble receptor (R)&D systems) and 10% FBS in RPMI1640 medium to establish a human gp 130-expressing BaF3 cell line (hereinafter referred to as BaF3/gp 130). Because the BaF/gp130 is in human interleukin-6 (R)&D systems) and SR344, and can be used to evaluate the proliferation inhibitory activity of an anti-IL-6 receptor antibody (i.e., IL-6 receptor neutralizing activity).

Production of humanized anti-II-6 antibody

In Cancer Res.1993 Feb15; 53(4): 851-6 mutations were introduced into the framework sequence and CDR sequence of a humanized mouse PM1 antibody (hereinafter, wild type is abbreviated as WT, H chain WT is abbreviated as H (WT) (amino acid sequence SEQ ID NO: 9), and L chain WT is abbreviated as L (WT) (amino acid sequence SEQ ID NO: 10)) to prepare H53 (amino acid sequence SEQ ID NO: 1), PF1H (amino acid sequence SEQ ID NO: 11), L28 (amino acid sequence SEQ ID NO: 12), and PF1L (amino acid sequence SEQ ID NO: 2) as modified H chains. Specifically, mutants were prepared by the method described in the appendix using the QuikChange site-directed mutagenesis kit (Stratagene), and the obtained plasmid fragments were inserted into animal cell expression vectors to prepare the target H chain expression vector and L chain expression vector. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art.

Expression and purification of humanized anti-IL-6 receptor antibodies

The expression of the antibody was carried out by the following method. HEK293H strain (Invitrogen) derived from human embryonic kidney cancer cells was suspended in a DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) at 5-6X 10⁵Cell Density of Individual cells/mL 10mL of adherent cell culture plates (10 cm diameter; CORNING) were seeded with 5% CO at 37 ℃₂After culturing in the incubator of (1) for a whole day and night, the medium was aspirated, and 6.9mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid was introduced into cells by lipofection. The resulting culture supernatant was recovered, and then centrifuged at room temperature for 5 minutes at a centrifugal force of about 2000g to remove cells, followed by passing through a 0.22 μm filter MILLEX^(R)GV (Millipore) and sterilized to obtain a culture supernatant. The culture supernatant obtained was subjected to rProtein A Sepharose^TMFastFlow (Amersham biosciences), according to the technicians in this field known method for purification. Regarding the purified antibody concentration, absorbance at 280nm was measured using a spectrophotometer. The absorbance was calculated from the obtained values by the PACE method, and the antibody concentration was calculated using the absorbance (Protein Science 1995; 4: 2411-2423).

EXAMPLE 2 preparation of pH-dependent binding antibody H3pI/L73

Method for producing antibody capable of neutralizing antigen multiple times

The IgG molecule is bivalent, so when it binds to an antigen at 2 sites, 1 molecule of IgG molecule can neutralize 2 molecules of antigen at most, but not 3 or more molecules of antigen. Therefore, in the case of a neutralizing antibody, in order to maintain the neutralizing effect for a certain period of time, it is necessary to administer an amount of the antibody equal to or larger than the amount of the antigen generated within the certain period of time, and there is a limit in reducing the amount of the antibody to be administered only by a technique for improving the pharmacokinetics of the antibody or for improving the affinity. Therefore, if 1 IgG molecule can neutralize 2 or more antigens, the duration of the neutralizing effect can be improved by the same dose, and the dose necessary for achieving the same duration can be reduced.

In the case of a neutralizing antibody, as the kind of antigen of the target, there are two cases: antigens are soluble antigens present in plasma and membrane antigens where the antigens are expressed on the cell surface.

When the antigen is a membrane-type antigen, the antibody to be administered binds to a membrane antigen on the cell surface, and thereafter, the antibody remains bound to the membrane antigen, and is taken into endosomes in the cell together with the antigen by internalization, and thereafter, the antibody moves to lysosomes while remaining bound to the antigen, and the antibody and the antigen are decomposed by the lysosomes together. The internalization-mediated elimination of membrane antigens from plasma is called antigen-dependent elimination, which is reported in most antibody molecules (drug Discov today.2006 Jan; 11 (1-2): 81-8). When 1 molecule of IgG antibody binds to an antigen in a bivalent state, it binds to 2 molecules of antigen and is internalized, and is directly decomposed by lysosomes, so that 1 molecule of IgG antibody cannot neutralize 2 or more molecules of antigen in the case of a normal antibody (fig. 1).

The retention in plasma of IgG molecules is long (elimination is slow) because FcRn, which is known to be a salvage receptor for IgG molecules, is functioning (Nat. Rev. Immunol.2007Sep; 7 (9): 715-25). IgG molecules taken up in endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions in the endosomes. The IgG molecules that cannot bind to FcRn are taken up into the lysosome and are decomposed by the lysosome, but the IgG molecules that bind to FcRn migrate to the cell surface, dissociate from FcRn under neutral conditions in plasma, and return to the plasma (fig. 2).

The membrane antigen-bound IgG molecule is taken into endosomes in the cell by internalization, moves to lysosomes while remaining bound to the antigen, and is degraded, and when the IgG antibody binds to the antigen in divalent form, the antigen, which neutralizes 2 molecules, is degraded together with the antigen. When taken into endosomes in cells by internalization, the dissociated antibody binds to FcRn expressed in the endosomes, as long as the IgG antibody is dissociated from the antigen under the acidic conditions in the endosomes. IgG molecules dissociated from the antigen and bound to FcRn migrate to the cell surface, dissociate from FcRn under neutral conditions in plasma, and are thereby returned to the plasma where they can bind again to new membrane antigens. By repeating the above process, 1 molecule of IgG molecule can repeatedly bind to the membrane-type antigen, and thus 1 molecule of IgG molecule can neutralize a plurality of antigens (fig. 3).

When the antigen is a soluble antigen, the administered antibody binds to the antigen in the plasma, and is retained in the plasma as a complex of the antigen and the antibody. As described above, the retention in plasma of an antibody is usually very long (the elimination rate is very slow) due to the action of FcRn, whereas the retention in plasma of an antigen is short (the elimination rate is high), and therefore the antigen bound to the antibody has the same degree of retention in plasma as the antibody (the elimination rate is very slow). The antigen is usually produced at a constant rate in vivo, and in the absence of an antibody, the antigen is present in plasma at a concentration at which the rate of production of the antigen and the rate of elimination of the antigen are balanced. In the presence of antibodies, most of the antigen binds to the antibody, and elimination of the antigen becomes very slow, so that the concentration of the antigen in plasma increases compared to that in the absence of the antibody (Kidney Int.2003, 64, 697-. If the affinity between the antibody and the antigen is infinite, the concentration of the antigen increases, the antibody is gradually eliminated from the plasma, and the effect of neutralizing the antigen by the antibody is interrupted after the time when the concentrations of the antibody and the antigen match. As for the neutralizing effect on soluble antigens, the larger the dissociation constant (KD), the less the antibody concentration can be neutralized, but no matter how strong the affinity is, the antigen cannot be neutralized at an antibody concentration of 1/2 or less which is the existing antigen concentration (biochem Biophys Res Commun.2005Sep9; 334 (4): 1004-13). Like IgG molecules to which no antigen is bound, IgG molecules to which the antigen is bound are also taken up into endosomes by pinocytosis in plasma, and bind to FcRn expressed in the endosomes under acidic conditions in the endosomes. The IgG molecules bound to FcRn move to the cell surface while remaining bound to the antigen, and dissociate from FcRn under neutral conditions in plasma, so that the IgG molecules return to the plasma again while remaining bound to the antigen, and thus cannot bind to a new antigen in the plasma. In this case, if the IgG molecule can be dissociated from the antigen under acidic conditions in the endosome, the dissociated antigen cannot be bound to FcRn, and therefore the antigen is decomposed by lysosomes. On the other hand, IgG molecules can be returned to the plasma again by binding to FcRn. The IgG molecules in the returned plasma have dissociated the antigen in the nucleus, so they are able to bind again to the new antigen in the plasma. By repeating this process, 1 molecule of IgG molecule can repeatedly bind to the soluble antigen, and thus 1 molecule of IgG molecule can neutralize a plurality of antigens (fig. 4).

Thus, it is considered that 1 molecule of IgG molecule can repeatedly neutralize an antigen, regardless of whether the antigen is a membrane-type antigen or a soluble-type antigen, as long as the IgG antibody can be dissociated from the antigen under acidic conditions in the intranuclear body. In order to dissociate IgG antibodies from antigens under acidic conditions in the endosome, the binding of antigens to antibodies under acidic conditions must be greatly reduced compared to those under neutral conditions. Since it is necessary to neutralize membrane antigens on the cell surface, strong binding to antigens is necessary at the pH of the cell surface, i.e., pH 7.4. Since the pH in the intranuclear space is generally reported to be pH5.5 to pH6.0(Nat Rev Mol Cell biol.2004 Feb; 5 (2): 121-32), it is considered that an antibody weakly binds to an antigen at pH5.5 to pH6.0 and is dissociated from the antigen under acidic conditions in the intranuclear space. That is, if the antibody strongly binds to the antigen at pH7.4 which is the pH on the cell surface and weakly binds to the antigen at pH5.5 to pH6.0 which is the pH in the intranuclear body, it is considered that 1 molecule of IgG molecule can neutralize a plurality of antigens and the pharmacokinetics can be improved.

Generally, protein-protein interactions include hydrophobic interactions, electrostatic interactions, and hydrogen bonding, the binding strength of which is often expressed in terms of an affinity or an apparent affinity. pH-dependent binding, in which the binding strength changes under neutral conditions (pH7.4) and acidic conditions (pH5.5 to pH6.0), occurs in naturally occurring protein-protein interactions. For example, the binding of the above-mentioned IgG molecule to FcRn, which is known as a salvage receptor of the IgG molecule, is strong under acidic conditions (ph5.5 to ph6.0) and very weak under neutral conditions (ph 7.4). Among these numerous protein-protein interactions in which pH-dependent binding changes, histidine residues are involved in the interaction. Since the pKa of a histidine residue is present in the vicinity of 6.0 to 6.5, the dissociation state of the proton of the histidine residue changes between neutral conditions (pH7.4) and acidic conditions (pH5.5 to pH 6.0). That is, the histidine residue is not charged under neutral conditions (ph7.4) to function as a hydrogen atom acceptor in neutrality; under acidic conditions (pH5.5 to pH6.0), histidine residues have a positive charge and act as hydrogen atom donors. Histidine residues present on the IgG side have been reported to be involved in pH-dependent binding even in the IgG-FcRn interaction described above (Mol cell.2001Apr; 7 (4): 867-77).

Thus, pH dependence can be imparted to protein-protein interactions by substituting amino acid residues involved in protein-protein interactions with histidine residues, or by introducing histidine into the site of interaction. In the protein-protein interaction between antibody and antigen, an attempt was also made to successfully obtain an antibody mutant having reduced binding to the antigen under acidic conditions by introducing histidine into the CDR sequence of an anti-lysozyme antibody (FEBSLetter (Vol. 309, No.1, 85-88, 1992)). It has also been reported that: an antibody which specifically binds to an antigen at low pH of cancer tissue and weakly binds under neutral conditions by introducing histidine into the CDR sequence (WO 2003105757).

In this way, a method of introducing pH dependency into antigen-antibody reaction has been reported, but an antibody in which 1 molecule of IgG molecule neutralizes a plurality of antigens has not been reported so far by strongly binding to an antigen at pH7.4 which is a pH in body fluid and weakly binding to an antigen at pH5.5 to pH6.0 which is a pH in a intranuclear body. That is, there is no report on modification of an antibody in which, by introducing a modification that greatly reduces binding under acidic conditions while maintaining binding under neutral conditions, the modified antibody binds to an antigen several times in vivo as compared with the antibody before modification, thereby improving pharmacokinetics and the persistence of a neutralizing effect at the same dosage.

IL-6 receptors exist in vivo in both soluble and membrane type IL-6 receptors (Nat Clin practice Rheumatotol.2006 Nov; 2 (11): 619-26). Anti IL-6 receptor antibodies bind to both soluble IL-6 receptor and membrane type IL-6 receptor, neutralizing their biological effects. It is considered that the anti-IL-6 receptor antibody is incorporated into intracellular endosomes by internalization while being bound to the membrane-type IL-6 receptor after binding to the membrane-type IL-6 receptor, and then the anti-IL-6 receptor antibody is moved to lysosomes while being bound to the membrane-type IL-6 receptor, and is decomposed by the lysosome. It has been reported that in fact humanized anti-IL-6 receptor antibodies show a non-linear clearance rate and that antigen-dependent elimination greatly contributes to The elimination of humanized anti-IL-6 receptor antibodies (The Journal of Rheumatology, 2003, 30; 71426-. That is, the 1-molecule humanized anti-IL-6 receptor antibody binds to the 1-molecule or 2-molecule membrane type IL-6 receptor (monovalent or divalent), and is degraded by lysosomes after internalization. Therefore, if a modified antibody (pH-dependent binding anti-IL-6 receptor antibody) can be produced in which binding under acidic conditions is significantly reduced while binding of the natural humanized anti-IL-6 receptor antibody under neutral conditions is maintained, it is considered that a plurality of molecules of IL-6 receptor can be neutralized with 1 molecule of the humanized anti-IL-6 receptor antibody, and thus the duration of the neutralizing effect can be improved by the same dose of the pH-dependent binding anti-IL-6 receptor antibody in vivo as compared with the natural humanized anti-IL-6 receptor antibody.

production of pH-dependent binding humanized IL-6 receptor antibody H3pI/L73

As a method for introducing pH-dependent binding into antigen-antibody reaction, a method of introducing histidine into CDR has been reported (FEBS Letter (Vol. 309, No.1, 85-88, 1992)). To confirm the amino acid residues exposed on the surface of the variable region of H53/PF1L prepared in example 1 and the residues that may interact with antigen, a Fv region model of H53/PF1L was prepared using homologous modeling using MOE software (chemical computing Group Inc.). From the three-dimensional structure model created based on the sequence information of H53/PF1L, H27, H31, H35, L28, L32, and L53(Kabat numbering, Kabat EA et al, 1991.Sequences of proteins of Immunological interest. NIH) were selected as mutation sites, and it is considered that pH-dependent binding to an antigen can be introduced by introducing histidine into the mutation sites. A mutation in which residues of H27, H31 and H35 were substituted with histidine was introduced into H53 prepared in example 1, and the resulting chain was designated as H3pI (amino acid sequence SEQ ID NO: 3); PF1L prepared in example 1 was subjected to a mutation in which the residues of L28, L32, and L53 were substituted with histidine, and the resulting chain was designated as L73 (amino acid sequence SEQ ID NO: 6).

Preparation, expression and purification of expression vector of H3pI/L73

Amino acid modifications to the selected sites for making modified antibodies are performed. Mutations were introduced into H53 (nucleotide sequence SEQ ID NO: 13) and PF1L (nucleotide sequence SEQ ID NO: 14) prepared in example 1 to prepare H3pI (amino acid sequence SEQ ID NO: 3) and L73 (amino acid sequence SEQ ID NO: 6). Specifically, the plasmid fragment obtained was inserted into an animal cell expression vector using a QuikChange site-directed mutagenesis kit (Stratagene) according to the method described in the attached manual, and the target H chain expression vector and L chain expression vector were prepared. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art. The expression and purification of H3pI for H chain and H3pI/L73 for L73 for L chain were carried out by the method described in example 1.

[ example 3] imparting pH-dependent antigen-binding ability Using CDR His modification Using phage display technology

Production of scFv molecule of humanized PM1 antibody

scFv of a humanized PM1 antibody (Cancer Res.1993Feb15; 53 (4): 851-6) was performed as a humanized anti-IL-6R antibody. VH and VL regions were amplified by PCR to generate a humanized PM1HL scFv with a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 15) between VH and VL.

Selection of sites into which histidine can be introduced by histidine partitioning

A histidine library in which 1 amino acid of each CDR amino acid was histidine was prepared by PCR using the humanized PM1HL scFv DNA prepared as a template. A library part was constructed by PCR using a primer in which a codon of an amino acid to be made into a library was used as a CAT, which is a codon corresponding to histidine, and the other parts were prepared by ordinary PCR and constructed by ligation by the assembly PCR method. The constructed library was digested with Sfi I, then inserted into the phagemid vector pELBG lacI vector, which was also digested with Sfi I, and then XL1-blue (Stratagene) was transformed. Using the obtained colonies, antigen binding evaluation and HL scFv sequence analysis were performed by phage ELISA. According to j.mol.biol 1992; 227: 381 and 388, phage-ELISA was performed using plates coated with 1. mu.g/mL SR 344. For clones for which binding to SR344 was confirmed, sequence analysis was performed using specific primers.

Phage titer was determined by ELISA using anti-Etag antibody (GE Healthcare) and anti-M13 antibody (GEHealthcare). Using this value, based on the results of phage ELISA with SR344, sites were selected in which the binding ability was not greatly changed compared to the humanized PM1HL scFv even when the CDR residues were substituted with histidine. These sites are shown in Table 2. The numbering of the individual residues follows Kabat numbering (Kabat EA et al, 1991.Sequences of proteins of Immunological interest. NIH).

[ Table 2] histidine substitution site having no significant influence on binding ability

H31，H50，H54，H56，H57，H58，H59，H60，H61，H62，H63，H64，H65，H100a，H100b，H102

L24，L26，L27，L28，L30，L31，L32，L52，L53，L54，L56，L90，L92，L93，L94

Construction of CDR histidine modification library

Amino acids substituted for CDR residues (positions into which histidine can be introduced) shown in Table 2, which have not largely changed in the histidine-binding ability even when substituted, were subjected to design into the original sequence (natural-type sequence) or into a library of histidines. A library was constructed from the sequences of H chain PF1H and L chain PF1L prepared in example 1, and the library sites were converted to the original sequence or histidine (either of the original sequence or histidine).

The library part was constructed by PCR using primers designed so that the site to be subjected to library formation was the codon of the original amino acid or the codon of histidine, and the other positions were constructed by ordinary PCR or constructed by PCR using synthetic primers in the same manner as the library part, and ligated by the assembly PCR method to construct a library (J.mol.biol. 1996; 256: 77-88).

This library was used according to j.immunological Methods 1999; 231: 119-. For in vitro translation of the E.coli cell-free line, the SDA sequence (ribosome binding site) and the T7 promoter were added to the 5 'side, and the gene3 partial sequence was ligated to the 3' side using Sfi I as a linker for ribosome display.

By passingCandida panning for pH-dependent binding of scFv from libraries

To concentrate scfvs with binding capacity only to SR344, according to nature biotechnology2000 Dec; 18: 1287-1292, two elutions were performed using ribosome display method. The prepared SR344 was biotinylated as an antigen using NHS-PEO 4-biotin (Pierce). Panning was performed using 40nM biotinylated antigen.

Using the obtained DNA Pool (DNA Pool) as a template, PCR was performed using specific primers to recover HL scFv. Digestion with Sfi I was followed by insertion into the phagemid vector pELBG lacI vector, which was also digested with Sfi I, followed by transformation with XL1-blue (Stratagene).

Escherichia coli having the target plasmid was propagated to 0.4-0.6O.D./mL in 2 YT/100. mu.g/mL ampicillin/2% glucose medium. To this was added helper phage (Helperphage) (M13KO7, 4.5X 10)¹¹pfu), incubated at 37 ℃ for 30 minutes, further incubated at 37 ℃ for 30 minutes with shaking, then transferred to a 50mL Falcon tube, centrifuged at 3000rpm for 10 minutes, resuspended in 2 YT/100. mu.g/mL ampicillin/25. mu.g/mL kanamycin/0.5 mM IPTG, and then allowed to proliferate overnight at 30 ℃.

Regarding the phage liquid, the culture solution incubated overnight was precipitated using 2.5M NaCl/10% PEG, and then diluted with PBS as a phage library solution. 10% M-PBS (PBS containing 10% skim milk) and 1M Tris-HCl were added to the phage library solution to a final concentration of 2.5% M-PBS, pH 7.4. The panning was carried out by a general method, i.e., a panning method using an antigen immobilized on a magnetic bead (J Immunol methods.2008Mar20; 332 (1-2): 2-9; JImmunel methods.2001Jan 1; 247 (1-2): 191-203; Biotechnol prog.2002Mar-Apr; 18 (2): 212-20). Specifically, 40pmol of biotin-labeled SR344 was added to the prepared phage library, and the resulting mixture was contacted with the antigen at 37 ℃ for 60 minutes. Streptavidin-coated Candida (Dynal M-280) washed with 5% M-PBS (PBS containing 5% skim milk) was added and allowed to bind for 15 minutes at 37 ℃. Candida was washed 5 times with 0.5mL each of PBST (PBS containing 0.1% Tween-20, pH7.4) and PBS (pH7.4). Candida was suspended in 1mL of PBS (pH5.5) at 37 ℃ and phages were immediately recovered. The recovered phage solution was added to 10mL of log phase (OD6000.4-0.5) XL1-Blue and allowed to stand at 37 ℃ for 30 minutes to infect. Infected E.coli were inoculated onto 2 YT/100. mu.g/mL ampicillin/2% glucose 225mm by 225mm plates. The E.coli was cultured again, and the phage was cultured in the same manner as described above, and panning was repeated 8 times.

Evaluation by phage ELISA

The single colony was inoculated in 100. mu.L 2 YT/100. mu.g/mL ampicillin/2% glucose/12.5. mu.g/mL tetracycline and incubated overnight at 30 ℃. mu.L of this was inoculated into 300. mu.L of 2 YT/100. mu.g/mL ampicillin/2% glucose, incubated at 37 ℃ for 4 hours, after which helper phage (M13KO7) 9X 10 was added⁸pfu was cultured at 37 ℃ for 30 minutes under static conditions and then cultured at 37 ℃ for 30 minutes under agitation to infect the cells. Thereafter, the medium was replaced with a medium containing 300. mu.L of 2 YT/100. mu.g/mL ampicillin/25. mu.g/mL kanamycin/0.5 mM IPTG. Subsequently, the cells were cultured overnight at 30 ℃ and the centrifuged supernatant was collected. To 40. mu.L of the centrifugation supernatant, 360. mu.L of 50 mM-PBS (pH7.4) was added and supplied to ELISA. Strepta Well96 microtiter plates (Roche) were coated overnight with 100. mu.L PBS containing 62.5ng/mL biotin-labeled SR 344. After washing with PBST to remove antigen, the cells were blocked with 250. mu.L of 2% BSA-PBS for 1 hour or more. 2% BSA-PBS was removed, and the prepared culture supernatant was added thereto and allowed to stand at 37 ℃ for 1 hour to allow the antibody to bind. After washing, 50mM PBS (pH7.4) or 50mM PBS (pH5.5) was added thereto, and the mixture was allowed to stand at 37 ℃ for 30 minutes for incubation. After washing, detection was performed using HRP-conjugated anti-M13 antibody (Amersham Parpharmacia Biotech) diluted with 2% BSA-PBS and TMB single solution (ZYMED), and the reaction was stopped by adding sulfuric acid, followed by measurement of absorbance at 450 nm.

However, in the panning using the antigen immobilized on the magnetic bead this time, a clone having a strong pH-dependent binding ability was not obtained. For clones whose pH-dependent binding performance was confirmed although weak, sequence analysis was performed using specific primers. Among these clones, the sites that had a high probability of becoming histidines are shown in Table 3.

[ Table 3] histidine substitution sites discovered by phage library (magnetic bead panning)

H50，H58，H61，H62，H63，H64，H65，H102

L24，L27，L28，L32，L53，L56，L90，L92，L94

Obtaining pH-dependent binding scFv from libraries by column panning

In the usual panning using antigens immobilized on magnetic beads, no clones with strong pH-dependent binding properties were obtained. The reason is considered to be: in the panning using an antigen immobilized on a magnetic bead or the panning using an antigen immobilized on a plate, since all phages dissociated from the magnetic bead or the plate under acidic conditions are collected, phages of even clones with weak pH dependence are collected, and the possibility that the clones that are finally concentrated contain clones with strong pH dependence is small.

Thus, as a method of panning under more stringent conditions, panning using a column on which an antigen is immobilized was studied (fig. 5). To date, there has been no report on obtaining a clone having a pH-dependent binding ability by panning using a column on which an antigen is immobilized. In the case of elution of phage bound under neutral conditions under acidic conditions in panning using a column on which an antigen is immobilized, it is considered that a clone having a weak pH dependence will be less likely to be eluted by re-binding to the antigen in the column, whereas a clone having a strong pH dependence and less likely to be re-bound in the column will be selectively eluted from the column. In the panning using an antigen immobilized on a magnetic bead or the panning using an antigen immobilized on a plate, "all" of phages dissociated under acidic conditions are recovered, but in the panning using a column on which an antigen is immobilized, elution is started by passing a buffer solution under acidic conditions through the column, and only appropriate components are recovered, and it is considered that phages having a strong pH-dependent binding ability can be selectively recovered.

First, a column on which SR344 as an antigen was immobilized was prepared. mu.L of streptavidin Sepharose (GE healthcare) was washed with 1mL of PBS, and then suspended in 500. mu.L of PBS, and exposed to 400pmol of biotin-labeled SR344 for 1 hour at room temperature. Thereafter, the above sepharose was packed in an empty column (Amersham pharmacia Biotech), and the column was washed with about 3mL of PBS. The library phage subjected to PEG precipitation was diluted to 1/25 with 0.5% BSA-PBS (pH7.4), passed through a 0.45nm filter, and then added to the column. After washing with about 6mL of PBS (pH7.4), 50mM MES-NaCl (pH5.5) was passed through the column to reach a low pH, and the dissociated antibody was eluted. The appropriate eluate fractions were recovered, and the recovered phage solution was added to 10mL log phase (OD6000.4-0.5) XL1-Blue and allowed to stand at 37 ℃ for 30 minutes for infection.

Infected E.coli were inoculated onto 2 YT/100. mu.g/mL ampicillin/2% glucose 225mm by 225mm plates. The E.coli was cultured again, and phage culture was performed in the same manner as described above, and panning was repeated 6 times.

Evaluation by phage ELISA

Evaluation of the obtained phage was performed by phage ELISA. For clones for which pH dependence was clearly confirmed, sequence analysis was performed using specific primers. As a result, a plurality of clones showing a significant pH-dependent binding as compared with WT were obtained. As shown in FIG. 6, clone CL5(H chain CLH5, L chain CLL5) (CLH 5: amino acid sequence SEQ ID NO: 5, CLL 5: amino acid sequence SEQ ID NO: 8) confirmed a particularly strong pH-dependent binding compared to WT. Thus, it can be seen that: an antibody showing strong pH-dependent binding, which is not obtained in ordinary panning using an antigen immobilized on magnetic beads, can be obtained by panning using a column on which an antigen is immobilized, and panning using a column on which an antigen is immobilized is very effective as a method for obtaining a pH-dependent binding antibody from a library. As a result of amino acid sequence analysis of the multiple clones found to bind pH-dependently, sites that became histidine with high probability among the concentrated clones are shown in Table 4.

[ Table 4] histidine substitution sites obtained by phage library (column panning)

H31，H50，H58，H62，H63，H65，H100b，H102

L24，L27，L28，L32，L53，L56，L90，L92，L94

EXAMPLE 4 expression and purification of histidine-modified body of humanized IL-6 receptor antibody

Production of expression vector for histidine-modified antibody of humanized IL-6 receptor antibody, expression of the same, and purification of the same Transforming

In order to IgG-transform the clones whose pH dependence was clearly confirmed by phage ELISA, VH and VL were amplified by PCR, respectively, and inserted into vectors for expression of animal cells by XhoI/NheI digestion and EcoRI digestion. The nucleotide sequence of each DNA fragment was determined according to a method known to those skilled in the art. The H chain was expressed and purified as IgG using CLH5 and the L chain using CLH5/L73 of L73 obtained in example 2. Expression and purification were carried out by the method described in example 1.

Antibodies with higher pH dependence were made using combinations of mutation sites. H170(SEQ ID NO: 4) was prepared by substituting H32, H58, H62, and H102 in H3pI obtained as the H chain in example 2 with histidine, H95 with valine, and H99 with isoleucine, based on the site at which His was concentrated in the phage library, structural information, and the like. The modified body was produced by the method described in example 1. Furthermore, L82(SEQ ID NO: 7) was prepared by substituting histidine at position 28 of L73 prepared as the L chain in example 2 with aspartic acid. The modified body was produced by the method described in example 1. The H chain was expressed as IgG using H170 and the L chain was purified using H170/L82 of L82 in accordance with the method described in example 1.

EXAMPLE 5 evaluation of IL-6R-neutralizing Activity of pH-dependent binding antibodies

Evaluation of neutralizing Activity of human IL-6 receptor for IgG-modified clones

IL-6 receptor neutralizing activity was evaluated for 4 of the humanized PM1 antibody (wild type: WT) and H3pI/L73, CLH5/L73, and H170/L82 prepared in examples 2 and 4.

Specifically, IL-6 receptor neutralizing activity was evaluated using BaF3/gp130, which shows IL-6/IL-6 receptor-dependent proliferation. BaF3/gp130 was washed 3 times with RPMI1640 medium containing 10% FBS, and then suspended in RPMI1640 medium containing 60ng/mL human interleukin-6 (TORAY), 60ng/mL recombinant soluble human IL-6 receptor (SR344), and 10% FBS to 5X 10⁴At a cell/mL density, 50. mu.L of each well of a 96-well plate (CORNING) was injected. Subsequently, the purified antibody was diluted with 10% FBS-containing RPMI1640 medium, and 50 μ L of each antibody was mixed in each well. At 37 deg.C, 5% CO₂Was cultured for 3 days under the conditions described above, and WST-8 reagent (cell counting kit 8; Co., Ltd.) diluted twice with PBS was added to 20. mu.L/well, and immediately thereafter absorbance at 450nm (reference wavelength: 620nm) was measured using SUNRISE CLASSIC (TECAN). After 2 hours of incubation, the absorbance at 450nm (reference wavelength: 620nm) was measured again, and the IL-6 receptor neutralizing activity was evaluated using the change in absorbance for 2 hours as an index. As a result, as shown in FIG. 7, H3pI/L73, CLH5/L73 and H170/L82 have biological neutralizing activity equivalent to that of the humanized PM1 antibody (wild type: WT).

EXAMPLE 6 Biacore analysis of pH-dependent binding antibodies

Analysis of the binding of pH-dependent binding clones to soluble IL-6 receptor

For the humanized PM1 antibody (wild type: WT) and 4 types of H3pI/L73, CLH5/L73, and H170/L82 produced in examples 2 and 4, analysis of the rate theory of antigen-antibody reaction at pH5.8 and pH7.4 was carried out using Biacore T100(GEHealthcare) (buffer solution was 10mM MES, pH7.4, or pH5.8, 150mM NaCl, 0.05% Tween 20). Binding of various antibodies to recombinant protein A/G (Pierce) -immobilized sensor core by amine couplingOn the chip, SR344 prepared at a concentration of 9.8-400nM in the analyte form was injected therein. The binding and dissociation of the pH-dependent binding clones to SR344 was observed in real time (fig. 8 and 9). All measurements were performed at 37 ℃. The binding rate constant k was calculated using Biacore T100 evaluation software (GEHealthcare)_a(1/Ms) and dissociation rate constant k_d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 5). The affinity ratios at pH5.8 and pH7.4 were then calculated, and pH-dependent binding was evaluated. All measurements were performed at 37 ℃.

The affinity ratios at pH5.8 and pH7.4 were calculated, respectively, and as a result, the pH-dependent binding (affinity) of H3pI/L73, H170/L82, and CLH5/L73 to SR344 was 41-fold, 394-fold, and 66-fold, respectively, and all clones showed high pH-dependent binding of 15-fold or more as compared with WT.

Hitherto, there has been no report of an anti-IL-6 receptor antibody that strongly binds to an antigen at a pH of 7.4, which is a pH in plasma, and weakly binds to an antigen at a pH of 5.5 to 6.0, which is a pH in a nuclear body. In this study, an antibody was obtained which specifically reduced the affinity at pH5.8 by 10 times or more while maintaining the biological neutralizing activity equivalent to that of the humanized IL-6 receptor antibody of WT and the affinity at pH 7.4.

[ Table 5] comparison of the binding of pH-dependent binding clones for SR344 to soluble IL-6 receptor

Analysis of the binding of pH-dependent binding clones to the Membrane-type IL-6 receptor

The above-prepared pH-dependent binding clone was observed to react with an antigen-antibody of a membrane type IL-6 receptor at pH5.8 and pH7.4 using Biacore T100 (GEHealthcare). Binding to the membrane type IL-6 receptor was evaluated by evaluating binding to the IL-6 receptor immobilized on the sensor chip. SR344 is biotinylated according to methods well known to those skilled in the art, and the streptavidin is used to bind the biotinylated SR344 to the sensor chip via streptavidin, taking advantage of the streptavidin's affinity for biotin. All assays were performed at 37 ℃ in a mobile phase buffer of 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20, into which pH-dependent binding clones were injected under conditions of pH7.4 to bind to SR344 (sample injection buffer of 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20), after which pH-dependent dissociation of each clone was observed at pH5.8 of the mobile phase (FIG. 10).

The dissociation rate (k.sub.g/mL) at pH5.8 was calculated by fitting only the dissociation phase at pH5.8 when bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 and dissociated in 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20 using Biacore T100 evaluation software (GEHealthcare)_d(1/s)). Similarly, the dissociation rate constant (k.sub.g/mL) at pH7.4 was calculated by fitting only the dissociation phase at pH7.4 when bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 and dissociated in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 using Biacore T100 evaluation software (GE Healthcare)_d(1/s)). The pH-dependent dissociation rate constants of the individual clones are shown in Table 6.

[ Table 6] comparison of dissociation Rate constants for dissociation of pH-dependent binding clones for SR344 from Membrane-type IL-6 receptor

The pH-dependent magnitude of the degree of dissociation was H3pI/L73, CLH5/L73, H170/L82 in that order, and all clones showed a pH-dependent dissociation from the membrane-type IL-6 receptor which was higher than WT. However, the order of pH-dependent binding and dissociation differs between soluble and membrane type IL-6 receptors. From the results, it can be seen that: H170/L82, which showed the highest pH-dependent binding in the binding assay with soluble IL-6 receptor, showed the lowest pH-dependent binding in the binding assay with membrane-type IL-6 receptor. It is known that generally IgG molecules bind to soluble antigens with a monovalent (affinity), whereas IgG molecules bind to membrane antigens with a divalent (affinity). In such soluble and membrane antigens, it is thought that the difference in binding pattern has an effect on the pH-dependent binding of H170/L82.

Example 7 confirmation of multiple binding of pH-dependent binding antibody to antigen

As described in example 2, it is believed that the pH-dependent binding antibody can bind to the antigen multiple times. That is, the pH-dependent binding antibody bound to the antigen nonspecifically enters the intranuclear body and dissociates from the soluble antigen under acidic conditions in the intranuclear body. The antibody returns to the plasma again by binding to FcRn, and since the antigen is not bound to the antibody returned to the plasma, the antibody can bind to a new antigen again. By repeating this process, the pH-dependent binding antibody can bind to the antigen multiple times. However, since IgG antibodies that do not have pH-dependent binding do not undergo dissociation of all antigens from the antibodies under acidic conditions in endosomes, the antibodies returned to the plasma by FcRn remain bound to the antigens and cannot bind to new antigens again. Therefore, in most cases, 1 molecule of IgG antibody can neutralize only 2 antigens (in the case of binding in two valences).

Thus, 3 pH-dependent binding antibodies, H3pI/L73, CLH5/L73 and H170/L82, prepared in examples 2 and 4 were evaluated for multiple binding to SR344 as an antigen, as compared with the humanized PM1 antibody (wild type: WT).

Evaluation by biacore (ge healthcare): and (3) a case where the antigen can be bound to the antigen multiple times by binding at pH7.4 and dissociating at pH 5.8. The antibody to be evaluated was bound to a sensor chip on which recombinant protein A/G (Pierce) was immobilized by amine coupling, and a mobile phase at pH7.4 was passed (step 1). The SR344 solution adjusted to pH7.4 was flowed as the analyte, and SR344 was bound to the antibody at pH7.4 (step 2). This binding at ph7.4 mimics the binding to antigen in plasma. Thereafter, only the buffer adjusted to ph5.8 (solution not containing SR 344) was flowed in the form of an analyte, and the antigen bound to the antibody was exposed to acidic conditions (step 3). This dissociation at pH5.8 is in a state mimicking the binding of an antibody-antigen complex in the nucleosome. After that, step 2 is performed again. This is where the antibody, mimicking the return to plasma by FcRn, binds again to a new antigen. Thereafter, step 2 is performed again to expose the antibody-antigen complex to acidic conditions. By repeating "step 2 → step 3" a plurality of times at 37 ℃ in this manner, the state in vivo can be simulated: the process of entry of the antibody from plasma into the intranuclear body by pinocytosis and then back into plasma by FcRn was repeated (NatRev Immunol.2007Sep; 7 (9): 715-25).

The prepared pH-dependent binding clone was analyzed for its ability to bind to SR344 multiple times at pH5.8 and pH7.4 using Biacore T100 (GEHealthcare). Specifically, the procedure is as follows. All assays were performed at 37 ℃ by first binding the above sample antibodies to a sensor chip immobilized with recombinant protein A/G (Pierce) by amine coupling, in a mobile phase buffer of 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20 (step 1). SR344 prepared as an analyte at a concentration of about 40nM (10 mM MES pH7.4, 150mM NaCl, 0.05% Tween20 in the buffer into which SR344 was injected) was injected for 3 minutes under the condition of pH7.4 to bind it (step 2). Thereafter, the injection of SR344 was stopped, and a mobile phase at pH5.8 was allowed to flow for about 70 seconds, thereby exposing the antibody/SR 344 complex to acidic conditions (step 3). The sensorgram was observed in real time with the binding (step 2) and acidic exposure (step 3) as 1 combination (set), which was repeated continuously for 10 combinations, see fig. 11. WT the rate of antibodies that can bind to the new antigen in the next step 2 is very low, since there is little dissociation of SR344 upon acidic exposure in step 3. In contrast, it was found that the dissociation of the pH-dependent binding clones, particularly H170/L82 and CLH5/L73, upon acidic exposure in step 3 was very large, and that substantially all of the bound SR344 was dissociated, so that the antibody was able to bind to substantially all new antigens in the next step 2. From the results, it can be seen that: even when binding (step 2) and acidic exposure (step 3) were repeated for 10 combinations, H170/L82 and CLH5/L73 were able to bind to substantially the new antigen in each combination.

Using the resulting sensorgrams, SR344 binding amounts of each sample in each combination were calculated using Biacore T100 evaluation software (Biacore), and the cumulative value over time for 10 combinations is shown in fig. 12. The cumulative RU values obtained for the 10 th combination corresponded to the total amount of antigen bound in 10 cycles. The results show that: the total amount of antigen bound by the pH-dependent binding clones, particularly H170/L82 and CLH5/L73, was the largest compared to WT, and repeated binding to approximately 4 times the amount of antigen compared to WT was possible. Thus, it can be seen that: by imparting pH-dependent binding to WT, binding to an antigen can be repeated, and a plurality of antigens can be neutralized.

EXAMPLE 8 PK/PD assay for pH-dependent binding of antibodies Using human IL-6 receptor transgenic mice

IL-6 receptors exist in vivo in both soluble and membrane type IL-6 receptors (Nat Clin practice Rheumatotol.2006 Nov; 2 (11): 619-26). Anti IL-6 receptor antibodies bind to soluble IL-6 receptor and membrane type IL-6 receptor, and neutralize their biological effects. It is considered that the anti-IL-6 receptor antibody, after binding to the membrane-type IL-6 receptor, remains bound to the membrane-type IL-6 receptor and is taken up into endosomes in the cell by internalization, and thereafter, the anti-IL-6 receptor antibody, while remaining bound to the membrane-type IL-6 receptor, moves to lysosomes and is decomposed by lysosomes. The pH-dependent bound anti-IL-6 receptor antibodies evaluated in example 6, namely H3pI/L73, CLH5/L73, and H170/L82, were able to dissociate under acidic conditions in the intranuclear body and return to plasma via FcRn, and the antibodies returned to plasma could bind to the antigen again, and a plurality of membrane-type IL-6 receptors could be neutralized with 1 molecule of antibody. The pH-dependent binding of anti-IL-6 receptor antibodies that have been made enable dissociation under acidic conditions in the intranuclear body for return to plasma via FcRn, and this allows an assessment of whether the pharmacokinetics of these antibodies are improved compared to WT.

Thus, the pharmacokinetics of the humanized PM1 antibody (wild type: WT) and 4 of the H3pI/L73, CLH5/L73 and H170/L82 prepared in examples 2 and 4 in human IL-6 receptor transgenic mice (hIL-6R tg mice, Proc Natl Acad Sci U S A.1995May23; 92 (11): 4862-6) were evaluated. WT and H3pI/L73, CLH5/L73, H170/L82 were administered to hIL-6R tg mice at 25mg/kg in a single intravenous dose, and blood was collected before and after administration over time. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or lower until the measurement was performed.

The concentration in the plasma of the mice was measured by ELISA. Calibration curve samples were prepared at concentrations of 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1 μ g/mL in plasma. The calibration curve sample and the mouse plasma measurement sample were injected into a multi-well Plate (immunoplate) (Nunc-Immuno Plate, Maxisorp (Nalge Nunc International Co., Ltd.)) fixed with anti-human IgG (γ -chain specificity) F (ab') 2(Sigma Co., Ltd.), allowed to stand at room temperature for 1 hour, and after that, goat anti-human IgG-BIOT (Southern Biotechnology Associates Co., Ltd.) and streptavidin alkaline phosphatase conjugate (Roche Diagnostics Co., Ltd.) were reacted in this order, a color reaction was performed using BluePhos micro phosphatase substrate system (Kirkegaard & Perry Laboratories Co., Ltd.) as a substrate, and absorbance at 650nm was measured using a microplate reader (microplate reader). The concentration in the plasma of the mouse was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices Co., Ltd.). The plasma concentrations of WT and H3pI/L73, CLH5/L73, H170/L82 varied as shown in FIG. 13.

Compared with WT, the pharmacokinetics of H3pI/L73, CLH5/L73 and H170/L82 are all improved. Among them, the pharmacokinetics of H3pI/L73 and CLH5/L73 are greatly improved. The natural anti-IL-6 receptor antibody (WT) that binds to the membrane type IL-6 receptor is taken up into endosomes in the cell by internalization, moves to lysosomes while retaining the binding to the antigen, and is degraded, so that the retention in the plasma is short. In contrast, the pharmacokinetics of pH-dependent binding of anti-IL-6 receptor antibodies is greatly improved, and it is believed that: the pH-dependent binding anti-IL-6 receptor antibody dissociates from the membrane-type IL-6 receptor as an antigen under acidic conditions in the endosome, and thereby returns to the plasma via FcRn again.

Compared with WT, the pharmacokinetics of H3pI/L73, CLH5/L73 and H170/L82 are all improved, but the retention prolonging effect of H170/L82 in plasma is smaller than that of H3pI/L73 and CLH 5/L73. IgG molecules are thought to bind membrane-type antigens generally in a bivalent manner, and thus anti-IL-6 receptor antibodies are thought to bind membrane-type IL-6 receptors also in a bivalent (avidity) manner, and are then internalized. As shown in example 6, in the analysis by Biacore, it was found that: 170/L82 rapidly dissociated from the IL-6 receptor at pH5.8 when bound to the soluble IL-6 receptor (FIG. 9), but it dissociated from the IL-6 receptor at pH5.8 at a very slow rate when bound to the membrane type IL-6 receptor (FIG. 10). It is thus assumed that: the reason why H170/L82 has a small effect of prolonging plasma retention is that it is slow to dissociate at pH5.8 when bound to a membrane-type IL-6 receptor, and therefore, it is not sufficiently dissociated in the nuclear body after internalization. That is, 1 IgG molecule neutralizes a plurality of membrane antigens, and thus it is known that: the pH dependence of dissociation after binding (avidity) in the bivalent form is more important than the pH dependence when binding (avidity) in the monovalent form.

EXAMPLE 9 PK/PD assay for pH-dependent binding antibodies Using Macaca fascicularis

In example 8, the pharmacokinetics of pH-dependent binding of anti-IL-6 receptor antibodies is greatly improved, thus suggesting that: the pH-dependent binding anti-IL-6 receptor antibody dissociates from the membrane-type IL-6 receptor as an antigen under acidic conditions in the endosome, and thereby returns to the plasma via FcRn again. If the antibody returned to the plasma again can bind to the membrane type IL-6 receptor again, it is considered that the pH-dependent binding anti-IL-6 receptor antibody sustains neutralization of the membrane type IL-6 receptor as an antigen for a longer period of time at the same administration amount as compared with the natural type anti-IL-6 receptor antibody. In addition, soluble IL-6 receptors are also present in IL-6 receptors, and thus: as for the neutralization of the soluble type IL-6 receptor, the neutralization was continued for a longer period of time at the same dosage.

The pharmacokinetics of WT and H3pI/L73 in cynomolgus monkeys were evaluated. WT and H3pI/L73 were administered to cynomolgus monkeys at a single intravenous dose of 1mg/kg, and blood was collected before and after administration over time. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or lower until the measurement was performed.

The concentration in cynomolgus monkey plasma was measured by ELISA. First, an anti-human IgG (γ -chain specific) F (ab') 2 antibody fragment (manufactured by SIGMA) was injected into a Nunc-Immuno plate or a MaxiSorp (manufactured by Nalge Nunc International) and allowed to stand at 4 ℃ overnight to prepare an anti-human IgG immobilized plate. Calibration curve samples with plasma concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05. mu.g/mL and cynomolgus monkey plasma assay samples diluted by 100-fold or more were prepared. To 100. mu.L of these calibration curve samples and plasma measurement samples, 200. mu.L of 20ng/mL cynomolgus monkey IL-6R was added, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, each of the solutions was poured onto an anti-human IgG immobilization plate, and the plate was allowed to stand at room temperature for 1 hour. Thereafter, a biotinylated anti-human IL-6R antibody (R & D) was reacted at room temperature for 1 hour, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was reacted at room temperature for 1 hour, a color reaction was performed using TMB One Component HRP Microwell Substrate (BioFX laboratories) as a Substrate, the reaction was stopped using 1N-sulfuric acid (Showa chemical), and then absorbance at 450nm was measured using a microplate reader. The concentration in cynomolgus monkey plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices Co., Ltd.). The change in plasma concentration after intravenous administration of WT and H3pI/L73 is shown in FIG. 14. As a result, the pharmacokinetics of H3pI/L73 was significantly improved in cynomolgus monkeys, as compared with WT, as in the case of human IL-6 receptor transgenic mice. The pharmacokinetics of H3pI/L73 as a pH-dependent binding anti-IL-6 receptor antibody was greatly improved, suggesting that: h3pI/L73 dissociates from the membrane-type IL-6 receptor as an antigen under acidic conditions in the endosome, and returns to the plasma again via FcRn.

To evaluate the extent to which cynomolgus monkey membrane-type IL-6 receptors were neutralized by intravenous administration of WT and H3pI/L73, the effect of the samples on plasma C-reactive protein (CRP) induced by cynomolgus monkey IL-6 was investigated. CRP is secreted once IL-6 binds to the membrane-type IL-6 receptor, and thus CRP becomes an index for neutralization of the membrane-type IL-6 receptor. Cynomolgus monkey IL-6 (cynomolgus monkey IL-6 prepared in example 1) containing 1% of inactivated cynomolgus monkey plasma was subcutaneously administered at 5. mu.g/kg on the lumbar-dorsal side from day 3 to day 10 after administration of WT and H3 pI/L73. Blood was collected from the saphenous vein at intervals of 24 hours (day 4 to day 11) from immediately before IL-6 administration to cynomolgus monkeys (day 3) and after administration, and plasma was separated. CRP concentration of each individual was measured using Cias R CRP (Kanto chemical Co., Ltd.) using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.). The change in CRP concentration when WT and H3pI/L73 were induced by cynomolgus IL-6 is shown in FIG. 15. As a result, the CRP-inhibited period of H3pI/L73 was significantly longer than that of WT. It is thus assumed that: h3pI/L73 as a pH-dependent binding anti-IL-6 receptor antibody dissociates from the membrane-type IL-6 receptor as an antigen under acidic conditions in the intranuclear body to be returned again into the plasma via FcRn, and inhibits the production of CRP for a long time as compared with WT by binding again to the membrane-type IL-6 receptor for neutralization. That is, the results show: h3pI/L73 can bind to the membrane type IL-6 receptor multiple times with 1 molecule of antibody for neutralization. The time for which H3pI/L73 inhibited the production of CRP was prolonged compared to WT, thus suggesting that: h3pI/L73 prolonged the time for which the membrane-type IL-6 receptor as an antigen was bound by the antibody, as compared with WT.

To evaluate the extent to which cynomolgus monkey soluble IL-6 receptors are neutralized by intravenous administration of WT and H3pI/L73, the concentration of non-binding cynomolgus monkey soluble IL-6 receptors in cynomolgus monkey plasma was determined. mu.L of cynomolgus plasma was added to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter bowl (Millipore) to adsorb all IgG type antibodies (cynomolgus IgG, anti-human IL-6 receptor antibody and anti-human IL-6 receptor antibody-cynomolgus soluble IL-6 receptor complex) present in the plasma onto protein A. Thereafter, the solution was spun down (spun-down) by a high-speed centrifuge, and the solution that passed through was recovered (hereinafter referred to as "passing solution"). Since the solution does not contain the anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex bound to protein A, the concentration of non-bound soluble IL-6 receptor can be determined by measuring the concentration of protein A in the solution passing through soluble cynomolgus monkey IL-6 receptor. A monoclonal anti-human IL-6R antibody (manufactured by R & D) that was ruthenated with SULFO-TAG NHS Ester (manufactured by MesoScale Discovery Co.) and a biotinylated anti-human IL-6R antibody (manufactured by R & D Co.) were mixed with the cynomolgus IL-6 receptor calibration curve sample prepared at 4000, 2000, 1000, 500, 250, 125, 62.5pg/mL and the above-mentioned protein A-treated plasma sample, and reacted at room temperature for 1 hour. Thereafter, the resulting solutions were injected into SA-coated calibration MA240096 well plates (manufactured by Meso scale discovery). After the reaction was further carried out at room temperature for 1 hour, ReadBuffer T (. times.4) (manufactured by Meso Scale Discovery) was poured in each of the reaction vessels after washing, and immediately measured by SECTOR Imager2400 (manufactured by Meso Scale Discovery). Cynomolgus IL-6 receptor concentration was calculated from the response value of the calibration curve using analytical software SOFTMAXPRO (manufactured by Molecular Devices). The concentration of unbound cynomolgus soluble IL-6 receptor of WT and H3pI/L73 was varied as shown in FIG. 16. As a result, the neutralization period of the cynomolgus monkey-soluble IL-6 receptor was significantly prolonged in comparison with WT in H3 pI/L73. It is thus assumed that: h3pI/L73, which is a pH-dependent antibody that binds to the anti-IL-6 receptor, dissociates from the soluble IL-6 receptor as an antigen under acidic conditions in the nucleus, returns to the plasma again via FcRn, and binds to the soluble IL-6 receptor again for neutralization. The prolonged inhibition of the non-binding cynomolgus soluble IL-6 receptor by H3pI/L73 compared to WT shows: h3pI/L73 prolonged the time for which soluble IL-6 receptor as an antigen was bound by the antibody compared to WT.

It was thus found that: the time until the antibody is eliminated from the plasma and the time until the soluble IL-6 receptor and the membrane type IL-6 receptor are bound by the antibody in vivo are greatly prolonged, compared to the wild type anti-IL-6 receptor antibody, the pH-dependent binding to the antigen at the pH in plasma, that is, pH7.4, and the binding to the antigen is weakened at the pH in the intranuclear body, that is, pH 5.8. This makes it possible to reduce the dose or the frequency of administration to a patient, and as a result, to reduce the total dose, and therefore, it is considered that a pharmaceutical product in which an anti-IL-6 receptor antibody binds in a pH-dependent manner is particularly excellent as an IL-6 antagonist.

EXAMPLE 10 enhancement of pH-dependent binding to Membrane-type IL-6 receptor by optimization of variable region

Optimization of variable regions H3pI/L73 and CLH5/L82

Example 9 shows that: since an antibody having a pH-dependent binding ability exerts an excellent effect, in order to further improve the pH-dependent binding ability, mutations were introduced into the CDR sequence of CLH5 obtained in example 3 to prepare VH1-IgG1(SEQ ID NO: 21) and VH2-IgG1(SEQ ID NO: 22). Furthermore, VH3-IgG1(SEQ ID NO: 23) and VH4-IgG1(SEQ ID NO: 24) were prepared as modified H chains by introducing mutations into the framework sequence and CDR sequence of H3 pI. VL1-CK (SEQ ID NO: 25), VL2-CK (SEQ ID NO: 26) and VL3-CK (SEQ ID NO: 27) were prepared as modified L chains by introducing mutations into the CDR sequences of L73 and L82. Specifically, mutants were prepared by the method described in the attached manual using a QuikChange site-directed mutagenesis kit (Stratagene), and the obtained plasmid fragments were inserted into mammalian cell expression vectors to prepare the target H chain expression vector and L chain expression vector. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art.

An antibody using VH2-IgG1(SEQ ID NO: 22) for the H chain, VL2-CK (SEQ ID NO: 26) for the L chain was used as Fv1-IgG1, an antibody using VH1-IgG1(SEQ ID NO: 21) for the H chain, and L82 for the L chain was used as Fv2-IgG1, an antibody using VH4-IgG1(SEQ ID NO: 24) for the H chain, VL1-CK (SEQ ID NO: 25) for the L chain was used as Fv3-IgG1, an antibody using VH3-IgG1(SEQ ID NO: 23) for the H chain, and VL3-CK (SEQ ID NO: 27) for the L chain was used as Fv4-IgG 1. Among these antibodies, Fv2-IgG1 and Fv4-IgG1 were expressed and purified. Expression and purification were carried out by the method described in example 1.

Analysis of the binding of pH-dependent binding clones to soluble IL-6 receptor

The rate theory of antigen-antibody reaction at pH7.4 was analyzed using Biacore T100(GE Healthcare) for humanized PM1 antibody (wild type: WT) and 4 kinds of WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 produced in examples 2 and 10 (buffer solution 10mM MES pH7.4, 150mM NaCl, 0.05% Tween 20). Binding of various antibodies to immobilized anti-IgG gamma chain specific F (ab) by amine coupling₂(Pierce) on the sensor chip, SR344 prepared as an analyte at a concentration of 9.8-40nM was injected. The binding and dissociation of the pH-dependent binding clones to SR344 was observed in real time. All measurements were performed at 37 ℃. The binding rate constant k was calculated using Biacore T100 evaluation software (GE Healthcare) _a(1/Ms) and dissociation rate constant k_d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 7).

[ Table 7] comparison of dissociation Rate constants for pH-dependent binding of SR344 cloned from soluble IL-6 receptor

The affinity of each pH7.4 was calculated, and the dissociation constants (affinity, KD value) for SR344 WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 were 2.7nM, 1.4nM, 2.0nM, and 1.4nM, respectively, and almost the same values were obtained. The results show that: the binding ability of Fv2-IgG1 and Fv4-IgG1 to soluble IL-6 receptor was equal to or higher than that of WT.

The 4 antibodies WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 prepared were observed to react with an antigen-antibody of a membrane type IL-6 receptor at pH5.8 and pH7.4 using Biacore T100(GE Healthcare). The binding of the above antibodies to the membrane type IL-6 receptor was evaluated by evaluating their binding to the IL-6 receptor immobilized on the sensor chip. SR344 is biotinylated according to methods well known to those skilled in the art, and the streptavidin is used to bind the biotinylated SR344 to the sensor chip via streptavidin, taking advantage of the streptavidin's affinity for biotin. All measurements were performed at 37 ℃ in a mobile phase buffer of 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20, and after pH-dependent binding clones were injected at pH7.4 and bound to SR344 (sample injection buffer of 10mM MESpH7.4, 150mM NaCl, 0.05% Tween20), pH-dependent dissociation of each clone was observed at pH5.8 in the mobile phase (FIG. 17).

The sample concentration was adjusted to 0.25. mu.g/mL, and only the dissociation phase at pH5.8 when bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 and dissociated in 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20 was fitted using Biacore T100 evaluation software (GEHealthcare), whereby the dissociation rate constant (k) at pH5.8 was calculated_d(1/s)). Similarly, the dissociation rate constant (k.sub.g/mL) at pH7.4 was calculated by fitting only the dissociation phase at pH7.4 when bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 and dissociated in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 using Biacore T100 evaluation software (GEHealthcare)_d(1/s))。

The pH-dependent dissociation rate constants of the individual clones are shown in Table 8.

[ Table 8] comparison of dissociation Rate constants for pH-dependent binding of SR344 cloned from the Membrane type IL-6 receptor

As a result of calculation of the pH dependence of the antibodies, the binding of 4 antibodies, i.e., WT of SR344, H3pI/L73-IgG1, Fv2-IgG1 and Fv4-IgG1, to the membrane-type IL-6 receptor was 1.0-fold, 2.59-fold, 7.18-fold and 5.56-fold, respectively, and the pH dependence of Fv2-IgG1 and Fv4-IgG1 was higher than that of H3pI/L73-IgG1, and was released from the membrane-type IL-6 receptor.

From the above, it can be seen that: fv2-IgG1 and Fv4-IgG1 showed stronger pH-dependent binding to the membrane-type IL-6 receptor than H3pI/L73-IgG1, while maintaining the affinity for the soluble IL-6 receptor equal to or higher than that of WT.

EXAMPLE 11 PK/PD assay for variable region-optimized pH-dependent binding antibodies Using human IL-6 receptor transgenic mice

The pharmacokinetics of Fv2-IgG1 and Fv4-IgG1 and WT and H3pI/L73-IgG1 produced and evaluated in example 10 were evaluated using the human IL-6 receptor transgenic mice used in example 8. WT and H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 were administered to hIL-6R tg mice at 25mg/kg in a single intravenous dose, and the concentrations of the respective antibodies in the plasma were measured in the same manner as in example 8. The plasma concentrations of WT and H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 were varied as shown in FIG. 18.

As in example 8, the pharmacokinetics of H3pI/L73-IgG1 were improved compared to WT, and the pharmacokinetics of Fv2-IgG1 and Fv4-IgG1 were further improved compared to H3pI/L73-IgG 1. When the concentration of non-binding IL-6 receptor measured in cynomolgus monkeys in example 9 was measured in hIL-6R tg mice in this test in the same manner, it was also confirmed that: the neutralization period of the soluble IL-6 receptors of Fv2-IgG1 and Fv4-IgG1 was extended compared to H3pI/L73-IgG1 (data not shown). As shown in example 10, compared with H3pI/L73-IgG1, Fv2-IgG1 and Fv4-IgG1 have an improved pH-dependent binding to the membrane-type IL-6 receptor, thereby showing: by increasing the pH-dependent binding to membrane-type IL-6 receptors, the pharmacokinetics and the neutralization period of soluble IL-6 receptors can be further increased compared to H3pI/L73-IgG 1.

EXAMPLE 12 enhancement of pH-dependent binding to Membrane-type IL-6 receptor by optimization of the constant region

Optimization of the constant regions of Fv4-IgG1

It has been reported that: in general, binding of an antibody to a membrane-type antigen varies depending on the constant region of the antibody (J Immunol methods.1997Jun 23; 205 (1): 67-72). The constant region of the pH-dependent binding antibodies made to date is the IgG1 isotype. Thus, optimization of the constant region was investigated in order to improve pH-dependent binding to the membrane-type IL-6 receptor.

A mutation was introduced into constant region IgG2(SEQ ID NO: 28) as a natural-type constant region to prepare constant region IgG 2. delta. GK (SEQ ID NO: 29). Furthermore, mutation was introduced into constant region IgG 2. delta. GK to prepare constant region M58(SEQ ID NO: 30). Mutations were further introduced into constant region IgG2 and constant region M58 to create constant regions M71(SEQ ID NO: 31) and M73(SEQ ID NO: 32).

VH3-IgG 2. delta. GK (SEQ ID NO: 33) in which the constant region of VH3-IgG1 prepared in example 10 was substituted with IgG 2. delta. GK, VH3-M58(SEQ ID NO: 34) in which the constant region was substituted with M58, and VH3-M73(SEQ ID NO: 35) in which the constant region was substituted with M73 were prepared. Specifically, the constant region portion of VH3 used in example 10 was substituted for the target constant region by NheI/NotI digestion and ligation to construct an expression vector. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art.

The following antibodies were expressed and purified: h chain used VH3-IgG 2. delta. GK (SEQ ID NO: 33), L chain used VL3-CK (SEQ ID NO: 27) Fv4-IgG 2; h chain using VH3-M58(SEQ ID NO: 34), L chain using VL3-CK (SEQ ID NO: 27) Fv 4-M58; and Fv4-M73 in which VH3-M73(SEQ ID NO: 35) is used as the H chain and VL3-CK (SEQ ID NO: 27) is used as the L chain. Expression and purification were carried out according to the method described in example 1.

Analysis of binding of constant region optimized Fv4 to soluble IL-6 receptor

The binding and dissociation to SR344 were observed in real time in the same manner as in example 10 for Fv4-IgG1, Fv4-IgG2, Fv4-M58, Fv4-M73, and WT. The binding rate constant k was calculated by the same analysis_a(1/Ms) and dissociation rate constant k_d(1/s) according to whichThe dissociation constants kd (m) were calculated (table 9).

[ Table 9] comparison of dissociation Rate constants for pH-dependent binding of SR344 cloned from soluble IL-6 receptor

The affinity at pH7.4 was calculated, and as a result, the dissociation constants (affinity, KD value) for Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 of SR344 were 1.4nM, 1.3nM, 1.4nM, and 1.4nM, respectively, and almost the same values were shown: even though the constant region was modified, the binding capacity of the pH-dependent binding clone for SR344 to the soluble IL-6 receptor did not change. It is thus assumed that: fv1, Fv2 and Fv3 were not changed in their ability to bind to a soluble IL-6 receptor even when the constant region was modified in the same manner.

Analysis of binding of constant region optimized Fv4 to Membrane-type IL-6 receptor

The produced Fv4-IgG1, Fv4-IgG2, Fv4-M58, Fv4-M73 and WT were observed to react with an antigen-antibody of a membrane-type IL-6 receptor at pH5.8 and pH7.4 by using Biacore T100(GE Healthcare) in the same manner as in example 10. After injecting pH-dependent binding clones at pH7.4 to bind to SR344, pH-dependent dissociation of each clone was observed in a mobile phase at pH5.8, and the results are shown in FIG. 19. Further, analysis was carried out in the same manner as in example 10, and the pH-dependent dissociation rates of the respective clones are shown in Table 10.

[ Table 10] comparison of dissociation Rate constants for pH-dependent binding of SR344 cloned from the Membrane-type IL-6 receptor

The respective pH dependences were calculated, and as a result, the pH dependences of Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 for SR344 were 5.6-fold, 17.0-fold, 17.6-fold, and 10.1-fold, respectively, and Fv4-IgG2, Fv4-M58, and Fv4-M73 all showed higher pH dependence than that of Fv4-IgG1 from the membrane type IL-6 receptor.

Based on the results of the binding analysis to the soluble IL-6 receptor using the variable region of Fv4 and the results of the binding analysis to the membrane type IL-6 receptor, it was found that: by substituting the constant region with IgG1 for IgG2, M58 and M73, it was possible to improve only pH-dependent binding to membrane-type IL-6 receptor without changing the affinity to soluble-type IL-6 receptor. Fv1, Fv2, and Fv3 are also considered to be the same.

EXAMPLE 13 constant region-optimized pH-dependent antibody-binding PK/PD assay Using human IL-6 receptor transgenic mice

The pharmacokinetics of Fv4-IgG1, Fv4-IgG2, and Fv4-M58 prepared in example 13 were evaluated using the human IL-6 receptor transgenic mice (hIL-6Rtg mice) used in example 8, and the influence of the constant regions on pharmacokinetics was investigated. WT, Fv4-IgG1, Fv4-IgG2, and Fv4-M58 were administered to hIL-6R tg mice at 25mg/kg in a single intravenous dose, and the concentrations of the respective antibodies in the plasma were measured in the same manner as in example 8. The plasma concentrations of WT and Fv4-IgG1, Fv4-IgG2, and Fv4-M58 were varied as shown in FIG. 20.

As in example 11, the pharmacokinetics of Fv4-IgG1 was improved compared with that of WT, and the pharmacokinetics of Fv4-IgG2 and Fv4-M58 were also improved compared with that of Fv4-IgG 1. When the concentration of non-binding IL-6 receptor measured in cynomolgus monkeys in example 9 was measured in the hIL-6R tg mouse of this test in the same manner, it was confirmed that: the neutralization period of the soluble IL-6 receptors of Fv4-IgG2, Fv4-M58 was extended compared to Fv4-IgG1 (data not shown). As shown in example 10, Fv4-IgG2, Fv4-M58 have improved pH-dependent binding to membrane-type IL-6 receptor compared to Fv4-IgG1, thereby showing: by substituting the constant region with IgG1 to IgG2 or M58, it is possible to increase pH-dependent binding to membrane-type IL-6 receptor and to increase pharmacokinetics and the neutralization period of soluble-type IL-6 receptor. It is thus assumed that: in Fv1, Fv2 and Fv3, the constant region was replaced with IgG1 to IgG2 or M58, which not only resulted in Fv4, but also in improvement in pharmacokinetics and the neutralization period of the soluble IL-6 receptor, as compared with IgG 1.

EXAMPLE 14 preparation of pH-dependent binding antibody optimized for variable region and constant region

The same method as the conventional method was used to prepare VH2-IgG1 with constant regions of M71, VH2-M71(SEQ ID NO: 36) of M73, VH2-M73(SEQ ID NO: 37) and VH4-IgG1 with constant regions of M71, VH4-M71(SEQ ID NO: 38) of M73 and VH4-M73(SEQ ID NO: 39).

The following antibodies were expressed and purified: VH2-M71 for H chain and VL2-CK for L chain, Fv 1-M71; VH2-M73 for H chain and VL2-CK for L chain, Fv 1-M73; VH4-M71 for H chain and VL1-CK for L chain, Fv 3-M71; and VH4-M73 for the H chain and Fv3-M73 for the L chain of VL 1-CK. Expression and purification were carried out according to the method described in example 1.

Assay of pH-dependent binding antibodies optimized for variable and constant regions with soluble IL-6 receptor In combination with

The binding and dissociation to SR344 of the humanized PM1 antibody (wild type: WT) and 11 antibodies, that was, H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73, were observed in real time in the same manner as in example 10. The binding rate constant k was calculated by the same analysis_a(1/Ms) and dissociation rate constant k _d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 11).

[ Table 11] comparison of dissociation Rate constants for pH-dependent binding of SR344 cloned from soluble IL-6 receptor

From the results, it can be found that: all of the 10 pH-dependent binding clones obtained had a dissociation constant (affinity, KD) equal to or higher than that of the soluble IL-6 receptor, compared with WT.

Analysis of variable and constant region optimized pH-dependent binding antibodies to Membrane-type IL-6 receptor Bonding of

The humanized PM1 antibody (wild type: WT) and 11 antibodies, that is, H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73, which have been produced so far, were reacted with an antigen-antibody of a membrane type IL-6 receptor at pH5.8 and pH7.4 using Biacore T100(GE Health care) in the same manner as in example 10. After injection of pH-dependent binding clones at pH7.4 to bind SR344, pH-dependent dissociation of individual clones was observed at pH5.8 of the mobile phase, and the results are shown in FIG. 21 (see results for Fv1-M71, Fv1-M73, Fv3-M71, and Fv3-M73 in FIG. 21, and others in FIGS. 17 and 19). Further, analysis was carried out in the same manner as in example 10, and the pH dependence of dissociation rate constant was found in Table 12 for all 11 clones.

[ Table 12] pH-dependent binding of SR344 with the pH-dependence of the dissociation rate constant cloned from the membrane-type IL-6 receptor

The 10 pH-dependent binding clones obtained showed pH-dependent binding ability to the membrane type IL-6 receptor. Further found that: compared with H3pI/L73-IgG1, the pH-dependent binding of any one of Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58 and Fv4-M73 to the membrane type IL-6 receptor was improved, and in example 9, the time from the antibody elimination of the H3pI/L73-IgG1 in cynomolgus monkeys and the time from the binding of the soluble type IL-6 receptor and the membrane type IL-6 receptor in vivo to the antibody were found to be greatly prolonged compared with WT.

EXAMPLE 15 PK/PD assay for pH-dependent binding antibodies optimized for variable and constant regions Using Macaca fascicularis

Preparation of known high affinity anti-IL-6 receptor antibodies

In order to express a known high affinity anti-IL-6 receptor antibody, namely, a high affinity anti-IL-6 receptor antibody VQ8F11-21hIgG1 described in US2007/0280945a 1(US2007/0280945a1, amino acid sequences 19 and 27), a vector for animal cell expression was constructed. Antibody variable regions were prepared by PCR (assembly PCR) method using a combination of synthetic oligo DNAs. The constant region was amplified from the expression vector used in example 1 by PCR. The antibody variable region was bound to the constant region by assembly PCR, and then inserted into a vector for mammalian expression. The obtained H chain and L chain DNA fragments are inserted into a mammalian cell expression vector to prepare a target H chain expression vector and L chain expression vector. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art. The expression vector thus prepared was used for expression and purification. Expression and purification were carried out by the method described in example 1 to obtain a high-affinity anti-IL-6 receptor antibody (high-affinity Ab).

PK/PD assay using cynomolgus monkeys

The pharmacokinetics and efficacy of H3pI/L73-IgG1 and Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, Fv4-M73, and a known high affinity anti-IL-6 receptor antibody (high affinity Ab) as pH-dependent binding antibodies in cynomolgus monkeys were evaluated. H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73 and Fv4-M73 were administered at a single intravenous dose of 0.5mg/kg, and Ab with high affinity was administered at a single intravenous dose of 1.0mg/kg, and blood was collected before and after administration at a time. The concentration of each antibody in plasma was measured in the same manner as in example 9. The plasma concentration changes of H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, Fv4-M73 and high affinity Ab are shown in FIG. 21. To evaluate the efficacy of neutralizing the cynomolgus monkey membrane-type IL-6 receptor, the cynomolgus monkey IL-6 was subcutaneously administered at 5 μ g/kg on the day following the lumbar-dorsal junction on the 3 rd to 10 th days (from the 6 th to 10 th days in the case of the high-affinity Ab) after the administration of the antibody in the same manner as in example 9, and the CRP concentration of each individual was measured 24 hours later. The change in CRP concentration upon administration of each antibody is shown in figure 22. To evaluate the efficacy of neutralizing the cynomolgus monkey soluble IL-6 receptor, the concentration of non-binding cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured in the same manner as in example 9. The concentration of non-binding cynomolgus soluble IL-6 receptor when each antibody was administered was varied as shown in FIG. 23.

It was thus found that: compared with H3pI/L73-IgG1, the antibody concentrations in the plasma of Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73 and Fv4-M73 are all maintained at high levels, while the CRP concentration and the concentration of non-binding cynomolgus soluble IL-6 receptor are maintained at low levels. That is, these antibodies extended the time for which the membrane-type IL-6 receptor and the soluble-type IL-6 receptor were bound by the antibodies (in other words, the time for neutralization), compared with H3pI/L73-IgG 1.

These pH-dependent binding anti-IL-6 receptor antibodies were confirmed to have a neutralization effect and persistence equal to or higher than those of known high-affinity anti-IL-6 receptor antibodies (high-affinity Abs) administered at 1.0mg/kg at half the dose, i.e., 0.5mg/kg, and thus it was found that: the pH-dependent binding antibodies have superior neutralizing effects and persistence compared to known high affinity anti-IL-6 receptor antibodies.

Among the antibodies shown in Table 12, even the antibody not subjected to the PK/PD test using cynomolgus monkeys in this test was confirmed to have an improved pH-dependent binding to the membrane-type IL-6 receptor as compared with H3pI/L73-IgG1, and it was considered that: these antibodies extended the binding time (in other words, the time to be neutralized, the duration of the neutralizing effect) of the membrane-type IL-6 receptor and the soluble-type IL-6 receptor by the antibodies, as compared with H3pI/L73-IgG 1.

In example 9 it was found that: compared with WT, the time taken for H3pI/L73-IgG1 to reach the elimination of the antibody from the plasma and the time taken for the soluble IL-6 receptor and the membrane-type IL-6 receptor in vivo to be bound by the antibody (persistence of the neutralizing effect) are greatly prolonged. The persistence of the neutralizing effect was considered to be significantly improved when compared with WT in the cases of Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58 and Fv4-M73, which are superior to H3pI/L73-IgG 1.

According to the above results, it is considered that: the pH-dependent binding of the anti-IL-6 receptor antibody to an antigen, which strongly binds to the antigen at pH7.4 in plasma and attenuates the binding to the antigen at pH5.8 in vivo, can reduce the dose or frequency of administration of the anti-IL-6 receptor antibody to a patient, and as a result, the total dose can be greatly reduced, and it is considered to be extremely excellent as a pharmaceutical product of an IL-6 antagonist.

EXAMPLE 16 preparation of anti-IL-6 antibody binding pH-dependently

Expression and purification of anti-IL-6 antibodies

In the humanized anti-IL-6 receptor antibodies of examples 1 to 15, a number of antibodies were successfully developed which impart pH dependency on the binding between the humanized anti-IL-6 receptor antibody and IL-6 receptor by introducing substitutions such as histidine into the variable region of the humanized anti-IL-6 receptor antibody, centering on the CDR sequence thereof, and it was found that these antibodies repeatedly bind to IL-6 receptor, resulting in a significant improvement in PK/PD.

Thus, whether or not pH dependency can be imparted to the binding of an antigen to an antibody can be examined in the same manner as in an antibody that binds to an antigen other than an anti-IL-6 receptor antibody. An anti-IL-6 antibody (anti-IL 6 wild type) comprising an H chain (WT) (amino acid sequence SEQ ID NO: 62) and an L chain (WT) (amino acid sequence SEQ ID NO: 63) which binds to human IL-6 described in WO2004/039826 was prepared by selecting human IL-6 as a different antigen. The target H chain expression vector and L chain expression vector are prepared by inserting a gene fragment encoding the amino acid sequence of the target antibody into an animal cell expression vector according to a method known to those skilled in the art. The nucleotide sequence of the resulting expression vector is determined according to methods known to those skilled in the art. Expression and purification of anti-IL 6 wild type was performed as described in example 1.

production of pH-dependent anti-IL-6 antibody

For an anti-IL-6 antibody (anti-IL 6 wild type) comprising H chain (WT) (amino acid sequence SEQ ID NO: 62) and L chain (WT) (amino acid sequence SEQ ID NO: 63), a study was conducted to impart pH dependence on the binding of the antibody to IL-6 by introducing histidine substitutions into the amino acids of the CDRs. Histidine substitutions were studied for the amino acids of the CDRs and screened, resulting in a large reduction in binding at pH5.5 compared to pH7.4, yielding several clones that showed pH-dependent binding. Histidine substitution sites in pH dependent clones are shown in Table 13. Among them, there are, for example, anti-IL 6 clone 1 comprising H chain (c1) (amino acid sequence SEQ ID NO: 64) and L chain (c1) (amino acid sequence SEQ ID NO: 65), and anti-IL 6 clone 2 comprising H chain (c1) (amino acid sequence SEQ ID NO: 64) and L chain (c2) (amino acid sequence SEQ ID NO: 66). The expression and purification of anti-IL 6 clone 1 and anti-IL 6 clone 2 were carried out according to the method described in example 1.

[ Table 13] histidine substitution site in pH-dependent cloning

H32、H59、H61、H99

L53、L54、L90、L94

Analysis of pH dependent binding clones to human IL-6 binding

For 3 of the anti-IL 6 wild type, anti-IL 6 clone 1, and anti-IL 6 clone 2 prepared above, analysis of the rate theory of antigen-antibody reaction at ph5.5 and ph7.4 was performed using Biacore T100(GE Healthcare) (the buffer was DPBS (-) ph7.4 or ph5.5, 150mm nacl). Various antibodies were bound to a sensor chip on which recombinant protein A/G (Pierce) was immobilized by amine coupling, and human IL-6(TORAY) prepared in an analyte form at an appropriate concentration was injected thereto. All measurements were performed at 37 ℃. The binding rate constant k was calculated using Biacore T100 evaluation software (GEHealthcare)_a(1/Ms) and dissociation rate constant k_d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 14). The affinity ratios at pH5.5 and pH7.4 were then calculated, and pH-dependent binding was evaluated.

[ Table 14] comparison of the binding of pH-dependent binding clones for IL-6 to IL-6

The affinity ratios (KD (pH5.5)/KD (pH7.4)) at pH5.5 and pH7.4 were respectively calculated, and as a result, the pH-dependent binding was 0.8-fold, 10.3-fold and 13.5-fold for human IL-6 anti-IL 6 wild-type, anti-IL 6 clone 1 and anti-IL 6 clone 2, respectively, and all clones showed 10-fold or more high pH-dependent binding compared with WT. The sensorgram for anti-IL 6 clone 2 at pH7.4 and pH5.5 is shown in FIG. 26.

This shows that: not only the anti-IL-6 receptor antibody but also the anti-IL-6 antibody can be produced by introducing amino acid substitutions such as histidine into the CDR sequence, thereby producing an antibody having a pH-dependent binding property in which the antibody strongly binds to an antigen under neutral conditions in plasma and the binding to the antigen is reduced under acidic conditions in endosomes. As shown in examples 1 to 15, the anti-IL-6 receptor antibody having pH-dependent binding repeatedly binds to the IL-6 receptor, and PK/PD is greatly improved, and it is considered that: compared with the wild type anti-IL 6, the anti-IL 6 clone 1 and the anti-IL 6 clone 2 with pH-dependent combination repeatedly combined with more antigens, and the PK/PD was greatly improved.

EXAMPLE 17 preparation of anti-IL-31 receptor antibody binding pH-dependently

Expression and purification of anti-IL-31 receptor antibodies

In examples 1 to 15, in the humanized anti-IL-6 receptor antibody, a plurality of antibodies were successfully prepared in which a pH dependency was imparted to the binding between the humanized anti-IL-6 receptor antibody and IL-6 receptor by introducing a substitution such as histidine into the variable region of the humanized anti-IL-6 receptor antibody centering on the CDR sequence thereof, and it was found that these antibodies repeatedly bind to IL-6 receptor, and PK/PD was greatly improved.

Thus, whether or not pH dependency can be imparted to the binding of an antigen to an antibody can be examined in the same manner as in an antibody that binds to an antigen other than an anti-IL-6 receptor antibody. A mouse IL-31 receptor was selected as a different antigen, and an anti-IL-31 receptor antibody (anti-IL 31R wild type) comprising an H chain (WT) (amino acid sequence SEQ ID NO: 67) and an L chain (WT) (amino acid sequence SEQ ID NO: 68) that binds to the mouse IL-31 receptor described in WO2007/142325 was prepared. The target H chain expression vector and L chain expression vector are prepared by inserting a gene fragment encoding the amino acid sequence of the target antibody into an animal cell expression vector according to a method known to those skilled in the art. The nucleotide sequence of the resulting expression vector is determined according to methods known to those skilled in the art. Expression and purification of anti-IL 31R wild type was performed as described in example 1.

production of pH-dependent anti-IL-31 receptor antibody

For an anti-IL-31 receptor antibody (anti-IL 31R wild type) comprising an H chain (WT) (amino acid sequence SEQ ID NO: 67) and an L chain (WT) (amino acid sequence SEQ ID NO: 68), studies were conducted to impart pH dependence on the binding of the antibody to the IL-31 receptor by introducing histidine substitutions into the amino acids of the CDRs thereof. Histidine substitutions were studied for the amino acids of the CDRs and screened, resulting in a large reduction in binding at pH5.5 compared to pH7.4, yielding several clones that showed pH-dependent binding. Histidine substitution sites in pH dependent clones are shown in Table 15. One of the sites is: anti-IL 31R clone 1 comprising H chain (c1) (amino acid sequence SEQ ID NO: 69) and L chain (WT). Expression and purification of anti-IL 31R clone 1 was performed as described in example 1.

[ Table 15] histidine substitution site in pH-dependent cloning

H33

Analysis of the binding of pH-dependent binding clones to soluble IL-31 receptor

For 2 of the anti-IL 31R wild-type and anti-IL 31R clone 1 prepared above, the rate theory analysis of antigen-antibody reaction at pH5.5 and pH7.4 was performed using Biacore T100(GE Healthcare) (the buffer was DPBS (-) pH7.4 or pH5.5, 150mM NaCl, 0.01% Tween20, 0.02% NaN) ₃). Various antibodies were bound to a sensor chip on which recombinant protein a/g (pierce) was immobilized by amine coupling, and soluble mouse IL-31 receptor (prepared by the method described in WO 2007/142325) prepared at an appropriate concentration in the form of an analyte was injected thereto. All measurements were performed at 25 ℃. The binding rate constant k was calculated using Biacore T100 evaluation software (GEHealthcare)_a(1/Ms) and dissociation rate constant k_d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 16). The affinity ratios at pH5.5 and pH7.4 were then calculated, and pH-dependent binding was evaluated.

[ Table 16] comparison of the binding of pH-dependent binding clones to mouse IL-31 receptor with that of mouse IL-31 receptor

The affinity ratios (KD (pH5.5)/KD (pH7.4)) at pH5.5 and pH7.4 were respectively calculated, and as a result, the pH-dependent binding of anti-IL 31R wild type and anti-IL 31R clone 1 to mouse IL-31 receptor was 3.2-fold and 1000-fold, respectively, and clone 1 showed about 300-fold higher pH-dependent binding compared with WT. The sensorgram for anti-IL 31R clones at pH7.4 and pH5.5 is shown in FIG. 27.

This shows that: not only the anti-IL-6 receptor antibody and the anti-IL-6 antibody, but also the anti-IL-31 receptor antibody can be prepared to have a pH-dependent binding that strongly binds to an antigen under neutral conditions in plasma and reduces the binding to an antigen under acidic conditions in endosomes by introducing amino acid substitutions such as histidine centered on the CDR sequence. As shown in examples 1 to 15, the anti-IL-6 receptor antibody having pH-dependent binding repeatedly binds to the IL-6 receptor, and PK/PD is greatly improved, and it is considered that: compared with the wild type anti-IL 31R, anti-IL 31R clone 1 with pH-dependent binding repeatedly bound more antigen and PK/PD was greatly improved.

EXAMPLE 18 iterative binding of pH-dependent binding antibodies to antigens

Expression and purification of mouse-administered antibodies

As humanized IL-6 receptor antibodies, the following 4 antibodies were made: general antibodies showing NO pH-dependent binding to the IL-6 receptor, i.e., WT-IgG1 comprising H (WT) (amino acid sequence SEQ ID NO: 9) and L (WT) (amino acid sequence SEQ ID NO: 10) and H54/L28-IgG1 comprising H54 (amino acid sequence SEQ ID NO: 70) and L28 (amino acid sequence SEQ ID NO: 12), and antibodies showing pH-dependent binding to the IL-6 receptor, i.e., H170/L82-IgG1 comprising H170 (amino acid sequence SEQ ID NO: 4) and L82 (amino acid sequence SEQ ID NO: 7) produced in example 3 and Fv4-IgG1 comprising VH3-IgG1(SEQ ID NO: 23) and VL3-CK (ID NO: 27) produced in example 10. The expression and purification of these 4 antibodies was carried out according to the method shown in example 1.

Analysis of binding of various antibodies to soluble IL-6 receptor

For the 4 antibodies WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 prepared, analysis of the velocity theory of antigen-antibody reactions at pH7.4 and pH5.8 was performed using Biacore T100(GE Healthcare) (buffer 10mM MES pH7.4 or pH5.8, 150mM NaCl, 0.05% surfactant-P20). Various antibodies were bound to a sensor chip on which recombinant protein A/G (Pierce) was immobilized by amine coupling, and SR344 prepared in the form of an analyte at an appropriate concentration was injected thereto. Binding and dissociation of various antibodies to SR344 was observed in real time. All measurements were performed at 37 ℃. The binding rate constant k was calculated using Biacore T100 evaluation software (GE Healthcare) _a(1/Ms) and dissociation rate constant k_d(1/s), and the dissociation constant KD (M) was calculated from the values (Table 17).

[ Table 17] comparison of binding rate (ka), dissociation rate (KD), and dissociation constant (KD) for SR344 with respect to each antibody to soluble IL-6 receptor

The affinity (KD value) ratios at pH5.8 and pH7.4 were calculated, respectively, and as a result, the pH-dependent binding (KD value ratios) for WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 of SR344 were 1.6-fold, 0.7-fold, 61.9-fold, and 27.3-fold, respectively. In addition, the respective dissociation rate (kd value) ratios at pH5.8 and pH7.4 were calculated, and as a result, the pH-dependent dissociation rates (kd value ratios) for WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 for SR344 were 2.9 times, 2.0 times, 11.4 times, and 38.8 times, respectively. Thereby confirming that: WT-IgG1 and H54/L28-IgG1, which are common antibodies, showed almost no pH-dependent binding, while H170/L82-IgG1 and Fv4-IgG1 showed pH-dependent binding. In addition, since the affinity (KD value) of these antibodies at ph7.4 was almost equal, it was considered that they were bound to SR344 in plasma to the same extent.

In vivo kinetic assay using mice

In vivo kinetics of SR344 and anti-human IL-6 receptor antibodies were evaluated after administration of SR344 (human IL-6 receptor: prepared in example 1) alone or in combination with SR344 and anti-human IL-6 receptor antibodies to mice that did not express human IL-6 receptor (C57 BL/6J; these anti-human IL-6 receptor antibodies did not bind to mouse IL-6 receptor). SR344 solution (5. mu.g/mL) or a mixed solution of SR344 and anti-human IL-6 receptor antibody (5. mu.g/mL, 0.1mg/mL, respectively) was administered to the tail vein in a single dose of 10 mL/kg. At this time, a sufficient amount of anti-human IL-6 receptor antibody was present in excess of SR344, and it was considered that: almost all of SR344 binds to the antibody. Blood was collected 15 minutes, 2 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or lower until the measurement was performed. As anti-human IL-6 receptor antibodies, the above-mentioned WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 were used.

Determination of the concentration of anti-human IL-6 receptor antibody in plasma by ELISA

The concentration of anti-human IL-6 receptor antibody in the plasma of mice was measured by ELISA. First, an anti-human IgG (γ -chain specific) F (ab') 2 antibody fragment (SIGMA) was injected into a Nunc-Immuno plate and a maxisorp (nalge Nunc international), and the mixture was allowed to stand at 4 ℃ overnight to prepare an anti-human IgG immobilized plate. Calibration curve samples with plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125. mu.g/mL and mouse plasma assay samples diluted 100-fold or more were prepared. To 100. mu.L of these calibration curve samples and plasma measurement samples, 200. mu.L of 20ng/mL SR344 was added and allowed to stand at room temperature for 1 hour. Thereafter, each of the solutions was poured into an anti-human IgG immobilization plate, and the plate was allowed to stand at room temperature for 1 hour. Thereafter, a biotinylated anti-human IL-6R antibody (R & D) was reacted at room temperature for 1 hour, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was reacted at room temperature for 1 hour, a color reaction was performed using TMB One Component HRP Microwell Substrate (BioFXlaboratories) as a Substrate, the reaction was stopped using 1N-sulfuric acid (Showa Chemical), and then absorbance at 450nm was measured using a microplate reader. The concentration in the plasma of the mice was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). The change in antibody concentration in plasma after intravenous administration as determined by this method is shown in FIG. 28.

Determination of SR344 concentration in plasma by electrochemiluminescence

The SR344 concentration in the plasma of the mice was determined by the electrochemiluminescence method. A SR344 calibration curve sample adjusted to 2000, 1000, 500, 250, 125, 62.5, 31.25pg/mL and a mouse plasma assay sample diluted 50 times or more were prepared, and a monoclonal anti-human IL-6R antibody (R & D) which was ruthenated with SULFO-TAGNHS Ester (Meso Scale Discovery), a biotinylated anti-human IL-6R antibody (R & D) and a WT-IgG1 solution were mixed and reacted at 37 ℃ overnight. In this case, the final concentration of WT-IgG1 was 333. mu.g/mL more than the concentration of anti-human IL-6 receptor antibody contained in the sample, and the aim was to bring about a state in which almost all of SR344 in the sample was bound to WT-IgG 1. Thereafter, the cells were individually injected into MA400PR streptavidin plates (Meso Scale Discovery). After the reaction was further carried out at room temperature for 1 hour, Read Buffer T (. times.4) (Meso Scale Discovery) was poured in each of the reaction vessels after washing, and the reaction was immediately measured by using SECTOR PR400reader (Meso Scale Discovery). SR344 concentration was calculated from the response values of the calibration curve using analytical software SOFTMax PRO (Molecular Devices). The change in plasma SR344 concentration after intravenous administration as determined by this method is shown in figure 29.

Effect of pH-dependent binding

With respect to the change in antibody concentration of the antibodies showing no pH-dependent binding, i.e., WT-IgG1 and H54/L28-IgG1, and the antibodies showing pH-dependent binding, i.e., H170/L82-IgG1 and Fv4-IgG1, WT-IgG1, H54/L28-IgG1 and Fv4-IgG1 were almost equal, while H170/L82-IgG1 was eliminated slightly more rapidly. The data of concentration change in plasma were analyzed using the pharmacokinetic analysis software WinNonlin (Pharsight), and as a result, the half-lives in plasma of WT-IgG1, H54/L28-IgG1, Fv4-IgG1, and H170/L82-IgG1 were 21.0 days, 28.8 days, 26.2 days, and 7.5 days, respectively.

When the antigen is a soluble antigen, the administered antibody binds to the antigen in the plasma and is retained in the plasma as a complex of the antigen and the antibody, as described in example 2. In general, the retention in plasma of an antibody is very long (the elimination rate is very slow) due to the function of FcRn, whereas the retention in plasma of an antigen is short (the elimination rate is high), and therefore the antigen bound to the antibody has a long retention in plasma (the elimination is very slow) to the same extent as the antibody. When SR344 (soluble human IL-6 receptor), an antigen of the humanized IL-6 receptor antibody, was administered alone, SR344 also showed extremely rapid elimination (half-life in plasma of 0.2 days). When SR344 and WT-IgG1 or H54/L28-IgG1, which is a normal antibody showing no pH-dependent binding, were administered simultaneously, the elimination rate of SR344 decreased significantly, showing long retention in plasma (half-life in plasma: WT-IgG15.3 days, H54/L28-IgG16.3 days). This is because almost all of SR344 binds to the antibody administered at the same time, and as described above, SR344 bound to the antibody has a long plasma retention property to the same extent as that of the antibody due to the function of FcRn.

When SR344 and an antibody showing pH-dependent binding, i.e., H170/L82-IgG1 or Fv4-IgG1 were administered simultaneously, the elimination of SR344 was significantly faster (half-life in plasma: H170/L82-IgG11.3 days, Fv4-IgG10.6 days) than when WT-IgG1 or H54/L28-IgG1 were administered simultaneously. This tendency is particularly significant in Fv4-IgG 1. Fv4-IgG1 was found to have an affinity at pH7.4 that was equal to or greater than that of WT-IgG1 and H54/L28-IgG1, and thus: SR344 binds almost exclusively to Fv4-IgG 1. Although Fv4-IgG1 showed equivalent or slightly longer retention in plasma and slower elimination compared to WT-IgG1 and H54/L28-IgG1, the elimination of SR344 in conjunction with Fv4-IgG1 was significantly faster. This can be illustrated by the concept of the present technology (concept) shown in fig. 4. With respect to a general antibody that does not exhibit pH-dependent binding, an antibody-soluble antigen complex is taken up into endosomes by pinocytosis in plasma, binds to FcRn expressed in the endosomes under acidic conditions in the endosomes, and the antibody-soluble antigen complex bound to FcRn moves directly to the cell surface and returns to the plasma again, so that the antigen bound to the antibody has a long retention in plasma to the same extent as the antibody (the elimination is very slow). On the other hand, since the antibody showing pH-dependent binding dissociates the antigen under acidic conditions in the endosome, only the antibody binds to FcRn and returns to plasma again, and the antigen dissociated from the antibody is not returned to plasma but is decomposed by lysosomes, so that the elimination of the antigen is significantly faster than in the case of the antibody not showing pH-dependent binding. That is, when SR344 and an antibody not showing pH-dependent binding, that is, WT-IgG1 or H54/L28-IgG1 were administered simultaneously, in vivo SR344 was bound to WT-IgG1 or H54/L28-IgG1 in plasma and thus the elimination of SR344 slowed down to the same extent as that of the antibody, but when SR344 and an antibody showing pH-dependent binding, that is, H170/L82-IgG1 or Fv4-IgG1 were administered simultaneously, SR344 was dissociated from the antibody in a low pH environment in vivo, and thus the elimination of SR344 was extremely rapid. That is, with respect to H170/L82-IgG1 or Fv4-IgG1, which are antibodies showing pH-dependent binding, SR344 was dissociated under a low pH environment in the intranuclear body, and thus it was considered that: most of the H170/L82-IgG1 or Fv4-IgG1 returned to plasma again by FcRn did not bind to SR 344. This indicates that: as shown in fig. 4, the antibody showing pH-dependent binding dissociates the antigen in the low pH environment in the endosome, returns to the plasma through FcRn in a state of not binding to the antigen, and can bind to a new antigen again in the plasma, and by repeating this process, the antibody showing pH-dependent binding can repeatedly bind to the antigen many times. As shown in example 7, this reflects that pH-dependent binding clones can repeatedly bind to antigen in Biacore, enhancing pH-dependent binding of antibody to antigen, and thus the number of times antibody and antigen are repeatedly bound can be increased.

When the antigen is a soluble type antigen, if the antigen bound to the antibody under the neutral condition in plasma is dissociated in the intranuclear and the antibody is returned to the plasma through FcRn, the antibody can be bound to the antigen again under the neutral condition in plasma, so that the antibody having the property of dissociating the antigen under the acidic condition in the intranuclear can be bound to the antigen many times. When the antigen bound to the antibody is dissociated in the intranuclear space, the antigen is transported to the lysosome and decomposed, and thus the rate of elimination of the antigen from the plasma is increased, as compared with the case where the antigen bound to the antibody is not dissociated in the intranuclear space (the antigen is returned to the plasma while being bound to the antibody). That is, whether or not an antibody can bind to an antigen multiple times can also be determined using the rate of elimination of the antigen from plasma as an index. The measurement of the rate of elimination of an antigen from plasma is performed, for example, by administering an antigen and an antibody to the body and measuring the antigen concentration in plasma after administration, as shown in this example.

Since 1 antibody of the antibody showing pH-dependent binding can repeatedly bind to an antigen several times as compared with a normal antibody not showing pH-dependent binding, it is considered that the administration amount of the antibody can be greatly reduced and the administration interval can be greatly extended.

Since repeated binding to an antigen based on this mechanism is based on pH-dependent antigen-antibody reactions, 1 antibody can be repeatedly bound to an antigen multiple times if an antibody that binds at pH7.4 in plasma and dissociates from the antigen at an acidic pH in the endosome and shows pH-dependent binding can be produced for any antigen. That is, the present technology is useful not only for IL-6 receptor, IL-6 receptor and IL-31 receptor, but also for antibodies against any antigen independent of the type of antigen.

Claims

1. A process for the preparation of a medicament, the process comprising the steps of:

(a) a step of measuring the antigen binding activity of the antibody at pH6.7 to pH 10.0;

(b) a step of measuring the antigen binding activity of the antibody at pH4.0 to pH 6.5;

(c) a step of selecting an antibody having an antigen binding activity at pH6.7 to pH10.0 higher than that at pH4.0 to pH 6.5;

(d) a step of obtaining a gene encoding the antibody selected in (c); and

(e) a step of preparing an antibody using the gene obtained in (d),

wherein the pharmaceutical product comprises any one of:

(i) an antibody having excellent retention in plasma;

(ii) an antibody capable of binding to an antigen more than twice when measured in an animal comprising FcRn-expressing cells;

(iii) an antibody that binds more antigens than its antigen binding site when measured in an animal comprising FcRn expressing cells;

(iv) antibodies that dissociate antigens bound extracellularly within cells;

(v) an antibody that binds to an antigen and is taken into the cell, and is released to the outside of the cell in a form not bound to the antigen; or

(vi) An antibody having an increased antigen-eliminating ability in plasma.

2. A process for the preparation of a medicament, the process comprising the steps of:

(a) A step of binding the antibody to the antigen at pH6.7 to pH 10.0;

(b) a step of subjecting the antigen-binding antibody of (a) to a condition of pH4.0 to pH 6.5;

(c) a step of collecting the antibody dissociated at pH4.0 to pH 6.5;

(d) a step of obtaining a gene encoding the antibody obtained in (c); and

(e) a step of preparing an antibody using the gene obtained in (d)

Wherein the pharmaceutical product comprises any one of:

(i) an antibody having excellent retention in plasma;

(iv) antibodies that dissociate antigens bound extracellularly within cells;

(vi) An antibody having an increased antigen-eliminating ability in plasma.

3. A process for the preparation of a medicament, the process comprising the steps of:

(a) a step of binding the antibody to the column on which the antigen is immobilized under a first pH condition;

(b) a step of eluting the antibody bound to the column under the first pH condition from the column under the second pH condition;

(c) A step of collecting the eluted antibody;

(d) a step of obtaining a gene encoding the antibody obtained in (c);

(e) a step of preparing an antibody using the gene obtained in (d),

wherein the pharmaceutical product comprises an antibody having a binding activity at a first pH that is higher than the binding activity at a second pH, wherein the first pH is between 6.7 and 10.0 and the second pH is between 4.0 and 6.5, and the antibody is any one of the following:

(i) an antibody having excellent retention in plasma;

(iv) antibodies that dissociate antigens bound extracellularly within cells;

(vi) An antibody having an increased antigen-eliminating ability in plasma.

4. A process for the preparation of a medicament, the process comprising the steps of:

(a) a step of binding the antibody library to the column on which the antigen is immobilized under a first pH condition;

(b) a step of eluting the antibody from the column under a second pH condition;

(c) A step of amplifying a gene encoding the eluted antibody;

(d) a step of collecting the eluted antibody;

(e) a step of obtaining a gene encoding the antibody obtained in (d); and

(f) a step of preparing an antibody using the gene obtained in (e),

(i) an antibody having excellent retention in plasma;

(iv) antibodies that dissociate antigens bound extracellularly within cells;

(vi) An antibody having an increased antigen-eliminating ability in plasma.

5. The method of any one of claims 1-4, further comprising: a step of substituting at least 1 amino acid in the antibody with histidine or inserting at least 1 histidine into the antibody.