RU2810471C1 - ANTIBODIES WITH MODIFIED AFFINITY TO FcRn, WHICH INCREASE ANTIGEN CLEARANCE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is presented: a method of producing an antibody which involves selecting an antibody that contains an antigen-binding domain and a human FcRn-binding domain, wherein the antibody has binding activity to human FcRn at pH 5.8 and 7.4 and lower antigen-binding activity at pH 5.8 than at pH 7.4, where the binding activity to human FcRn at pH 7.4 is higher than that of intact human IgG. Then, the host cell is cultivated, the said cell contains a vector into which a polynucleotide encoding the antibody selected in the previous step is introduced, and the antibody is isolated from the host cell.

EFFECT: invention makes it possible to obtain antibodies that have an activity to bind with human FcRn at plasma pH and lower antigen-binding activity at early endosome pH than at plasma pH; such antibodies can increase the number of antigens to which one antibody molecule can bind.

10 cl, 35 dwg, 15 tbl, 17 ex

Description

Technical field

The present invention relates to:

methods facilitating antigen binding molecule-mediated uptake of antigen into cells;

methods for increasing the number of antigens to which one antigen-binding molecule can bind;

methods that accelerate the decrease in plasma antigen concentration through the introduction of antigen-binding molecules;

methods that improve the pharmacokinetics of antigen-binding molecules;

methods for reducing total antigen concentration or free antigen concentration in plasma;

antigen-binding molecules, which increase the uptake of antigen by cells;

antigen-binding molecules, which have the ability to bind to an increased number of antigens;

antigen-binding molecules capable of accelerating the decrease in plasma antigen concentration through the administration of molecules;

antigen-binding molecules with improved pharmacokinetics;

pharmaceutical compositions containing antigen-binding molecules;

methods for obtaining those described above; and so on.

A priority

The present invention claims the benefit of Japanese Patent Application No. 2010-079667, filed on March 30, 2010, and Japanese Patent Application No. 2010-250830, filed on November 9, 2010, the contents of which are incorporated herein by reference in their entirety.

Prior Art

Antibodies have attracted attention as drugs because they are highly stable in plasma and have few side effects. Currently, a number of similar IgG drugs are commercially available and many antibody-based drugs (NPL 1 and 2) are being developed. Meanwhile, various methods applicable to second generation antibody drugs have been described, including those that improve effector function, antigen-binding capacity, pharmacokinetics and stability, and those that reduce the risk of developing immunogenicity (NPL 3). Generally, the required dose of the antibody drug is very high. This, in turn, has led to problems such as high production costs as well as difficulties in preparing subcutaneous formulations. In theory, the dose of an antibody pharmaceutical can be reduced by improving the pharmacokinetics of the antibody or improving the affinity between antibodies and antigens.

The literature describes methods for improving the pharmacokinetics of antibodies using artificial amino acid substitutions in constant regions (NPL 4 and 5). Similarly, affinity maturation has been described as a method of improving antigen-binding capacity or antigen-neutralizing activity (NPL 6). This method makes it possible to increase the antigen-binding ability by introducing an amino acid mutation in the CDR region of the variable region or the like. Increased antigen-binding capacity provides improved in vitro biological activity or allows for dose reduction and further provides increased in vivo efficacy (NPL 7).

The antigen-neutralizing ability of a single antibody molecule depends on its affinity. As affinity increases, the antigen can be neutralized with less antibody. Various methods can be used to increase the affinity of an antibody (NPL 6). In addition, if the affinity can be made very high by covalently linking an antibody to an antigen, one molecule of antibody can neutralize one molecule of antigen (a divalent antibody can neutralize two molecules of antigen). However, the stoichiometry of neutralization of one antibody against one antigen (one divalent antibody against two antigens) is a limiting factor for previous methods, and thus it is impossible to completely neutralize an antigen with an amount of antibody less than the amount of antigen. In other words, the affinity enhancing effect is of marginal significance (NPL 9). To increase the duration of the neutralizing effect of a neutralizing antibody over a certain period, the antibody must be administered at a dose higher than the amount of antigen produced in the body during the same period. When improving the pharmacokinetics of antibodies or only the affinity maturation technology described above, there is thus a limiting factor in reducing the required dose of the antibody. Thus, in order to maintain the antigen-neutralizing effect of antibodies over a target period of time, using antibodies in an amount less than the amount of antigen, one antibody must neutralize multiple antigens. An antibody that binds to an antigen in a pH-dependent manner has recently been described as a new method to achieve the object described above (PTL 1). pH-dependent antigen-binding antibodies, which bind strongly to antigen under neutral conditions in plasma and dissociate from antigen under acidic conditions in the endosome, can dissociate from antigen in the endosome. If a pH-dependent antigen-binding antibody dissociates from an antigen that is recycled in the plasma via FcRn, it can rebind to another antibody. Thus, a single pH-dependent antigen-binding antibody can bind repeatedly to a number of antigens.

In addition, the retention time of the antigen in the plasma is very short compared to the retention time of antibodies recycled in the plasma through binding to FcRn. If an antibody with such a long plasma retention time binds an antigen, the plasma retention time of the antigen-antibody complex is increased by the same period as the plasma retention time of the antibody. Thus, by binding to the antibody, the retention time of the antigen is increased, and thus the concentration of the antigen in the plasma increases.

The IgG antibody has a longer retention time in plasma as a result of binding to FcRn. Binding between IgG and FcRn is observed only under acidic conditions (pH 6.0). In contrast, virtually no binding is observed under neutral conditions (pH 7.4). The IgG antibody is taken up by the cell in a nonspecific manner. The antibody returns to the cell surface by binding to endosomal FcRn under acidic conditions in the endosome and then dissociates from FcRn under neutral conditions in the plasma. If binding to FcRn under acidic conditions is not possible due to the introduction of mutations in the Fc domain of IgG, the absence of recycling of the antibody into the plasma from the endosome markedly reduces the period of time the antibody is retained in the plasma. The method describing increasing the retention time of an IgG antibody is a method that increases binding to FcRn under acidic conditions. To improve binding under acidic conditions, amino acid mutations are introduced into the Fc domain of the IgG antibody. This increases the efficiency of recycling into plasma from the endosome, resulting in increased plasma retention time. An important requirement when replacing amino acids is that there is no increase in the efficiency of binding to FcRn under neutral conditions. If an IgG antibody binds to FcRn under neutral conditions, the antibody that returns to the cell surface by binding to FcRn under acidic endosomal conditions does not dissociate from FcRn under neutral plasma conditions. In this case, the retention time is likely to decrease since the IgG antibody is not recycled into the plasma. For example, as described in J Immunol. (2002) 169(9): 5171-80, an IgG1 antibody modified by amino acid substitutions to result in an antibody capable of binding to mouse FcRn under neutral conditions (pH 7.4) is reported to exhibit very low retention time value when administered to mice. Additionally, as described in J Immunol. (2009) 182(12): 7663-71; J Biol Chem. 2007 Jan 19; 282(3): 1709-17; and J Immunol. 2002 Nov 1; 169(9): 5171-80, an IgG1 antibody has been modified by introducing amino acid substitutions such that the resulting antibody exhibits increased ability to bind to human FcRn under acidic conditions (pH 6.0), while at the same time becoming capable of binding with human FcRn under neutral conditions (pH 7.4). The resulting antibody is described to exhibit no increase in retention time or changes in retention time when administered to cynomolgus monkeys. Thus, antibody engineering technology to improve antibody function focuses only on the plasma retention time of antibodies by increasing human FcRn binding under acidic conditions, without increasing the retention time under neutral conditions (pH 7.4). To date, there are no reports describing the benefits obtained by increasing binding to human FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions into the Fc domain of an IgG antibody. Even if the affinity of the antigen for the antibody is increased, the elimination of the plasma antigen cannot be increased. It has been published that the pH-dependent antigen-binding antibodies described above are more effective as a method of increasing plasma antigen elimination than typical antibodies (PTL 1).

Thus, one pH-dependent antigen-binding antibody binds a number of antigens and is capable, compared with typical antibodies, of facilitating the elimination of antigen in plasma. Thus, pH-dependent antigen-binding antibodies have an effect that cannot be achieved by typical antibodies. However, to date, there have been no reports of methods for engineering antibodies to further increase the ability of pH-dependent antigen-binding antibodies to rebind to antigens and have the effect of increasing antigen clearance in plasma. The prior art documents related to the present invention are presented below:

LIST OF LINKS

Patent literature

[PTL 1] WO 2009/125825, ANTIGEN-BINDING MOLECULE CAPABLE OF BINDING TO TWO OR MORE ANTIGEN MOLECULES REPEATEDLY

Non-patent literature

[NPL 1] Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology 23, 1073-1078 (2005)

[NPL 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59(3): 389-96

[NPL 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review

[NPL 4] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176(1): 346-56

[NPL 5] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. July 1997; 15(7): 637-40

[NPL 6] Proc Natl Acad Sci U S A. 2005 Jun 14; 102(24): 8466-71. Epub 2005 Jun 6. A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R

[NPL 7] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract. J Mol Biol. (2007) 368:652-665

[NPL 8] Hanson CV, Nishiyama Y, Paul S. Catalytic antibodies and their applications. Curr Opin Biotechnol. 2005 Dec; 16(6): 631-6

[NPL 9] Rathanaswami P, Roalstad S, Roskos L, Su QJ, Lackie S, Babcook J. Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8. Biochem Biophys Res Commun. 2005 Sep 9; 334(4): 1004-13

The essence of the invention

Technical problem

The present invention is carried out taking into account the circumstances described above. The object of the present invention is to develop methods that facilitate the uptake of antigen into cells through the use of antigen-binding molecules, methods for increasing the number of antigens to which one antigen-binding molecule can bind, methods for accelerating the decrease in the concentration of antigen in plasma through the introduction of antigen-binding molecules, methods for improving the pharmacokinetics of antigen-binding molecules, antigen-binding molecules. molecules that facilitate the uptake of antigen by cells, antigen-binding molecules that have the ability to bind to an increased amount of bound antigens, antigen-binding molecules capable of accelerating the decline in plasma antigen concentration through administration, antigen-binding molecules with improved pharmacokinetics, pharmaceutical compositions containing antigen-binding molecules, and methods for producing those described above.

Solution

The inventors of the present invention have conducted targeted studies on methods for facilitating the uptake of antigen into cells through antigen-binding molecules (molecules such as polypeptides that have antigen-binding ability), methods for allowing antigen-binding molecules to repeatedly bind to antigens, methods for accelerating the reduction of antigen concentration in plasma through the administration of antigen-binding molecules , and methods for increasing the retention time of antigen-binding molecules. As a result, the present inventors discovered that antigen-binding molecules that have the ability to bind human FcRn at low endosomal pH and that have a higher binding activity to human FcRn than the binding activity of intact human immunoglobulin IgG at plasma pH could promote antigen uptake into cells. The present inventors have also discovered that antigen binding molecule-mediated cellular uptake of antigen can be further improved and the number of antigens to which one molecule can bind can be increased by using an antigen binding molecule that has weaker antigen binding activity at low endosomal pH values. than at plasma pH; by administering such an antigen-binding molecule, the decrease in plasma antigen concentration can be accelerated; and the pharmacokinetics of the antigen-binding molecule can be improved.

Specifically, the present invention relates to:

methods facilitating antigen binding molecule-mediated uptake of antigen into cells;

methods for increasing the number of antigens to which one antigen-binding molecule can bind;

methods that accelerate the decrease in plasma antigen concentration through the introduction of antigen-binding molecules;

methods that improve the pharmacokinetics of antigen-binding molecules;

methods for reducing total antigen concentration or free antigen concentration in plasma;

antigen-binding molecules, which increase the uptake of antigen by cells;

antigen-binding molecules, which have the ability to bind to an increased number of antigens;

antigen-binding molecules capable of accelerating the decrease in plasma antigen concentration through the administration of molecules;

antigen-binding molecules with improved pharmacokinetics;

pharmaceutical compositions containing antigen-binding molecules;

methods for obtaining those described above; and so on. More specifically, the present invention relates to:

[1] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in the acidic and neutral pH ranges, wherein the human FcRn binding activity in the neutral pH range is represented by a micromolarity value greater than 3.2;

[2] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has binding activity to human FcRn in the neutral pH range, wherein the binding activity to human FcRn in the neutral pH range is 28 times greater than for intact human IgG;

[3] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is represented by a micromolarity value greater than 2.3;

[4] an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, which has binding activity to human FcRn in the neutral pH range, wherein binding activity to human FcRn in the neutral pH range is represented by a value 38 times greater than for intact human IgG;

[5] the antigen-binding molecule according to any one of claims [1] to [4], wherein the range of neutral pH values is represented by pH values from 7.0 to 8.0;

[6] an antigen-binding molecule comprising an antigen-binding domain and a human FcRn-binding domain, for which the total plasma antigen concentration after administration of the antigen-binding molecule to a non-human animal is a value less than the plasma antigen concentration after administration of a control antigen-binding molecule to a non-human animal an animal containing the same antigen binding domain and an intact human IgG Fc domain as the human FcRn binding domain;

[7] an antigen-binding molecule for which the plasma antigen concentration after administration of the antigen-binding molecule to a non-human animal is a value less than the total plasma antigen concentration obtained for a non-human animal to which the antigen-binding molecule is not administered;

[8] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen/antigen binding molecule molar ratio (C) is for the antigen binding molecule. calculated as follows:

C=A/B,

is a value less than the antigen/antigen binding molecule molar ratio (C') for an antigen binding molecule containing the same antigen binding domain and an intact human IgG Fc domain as the human FcRn binding domain, calculated as follows:

C'=A'/B',

Where:

A is the total plasma antigen concentration after administration of an antigen-binding molecule to a non-human animal,

B is the plasma concentration of the antigen binding molecule following administration of the antigen binding molecule to a non-human animal,

A' is the total plasma antigen concentration after administration of a control antigen-binding molecule to a non-human animal,

B' is the plasma concentration of the antigen binding molecule after administration of the control antigen binding molecule to the non-human animal;

[9] the antigen binding molecule of any one of claims [6] to [8], wherein the non-human animal is a transgenic mouse with human FcRn;

[10] the antigen-binding molecule according to any one of claims [6] to [9], wherein the plasma antigen concentration is a constant total plasma antigen concentration;

[11] the antigen-binding molecule according to any one of claims [6] to [9], wherein the plasma antigen concentration is a temporary total plasma antigen concentration;

[12] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has binding activity to human FcRn in the acidic and neutral pH ranges, wherein binding activity to human FcRn in the neutral pH range is represented by a value higher than that for intact human IgG;

[13] the antigen binding molecule according to any one of claims [1] to [11], wherein the antigen binding activity of the antigen binding domain in the acidic pH range is a value less than the antigen binding activity of the antigen binding domain in the neutral pH range;

[14] the antigen-binding molecule according to claim [12] or [13], wherein the ratio of antigen binding activity in the acidic and neutral pH range is at least 2 for the KD (acidic pH range)/KD (neutral pH range) value;

[15] the antigen-binding molecule according to any one of claims [12]-[14], which contains an amino acid mutation of the antigen-binding domain, which contains a replacement of histidine with at least one amino acid for the antigen-binding domain or contains an insertion of at least one histidine;

[16] an antigen binding molecule according to any one of claims [12] to [14], wherein the antigen binding domain is obtained from a library of antigen binding domains;

[17] the antigen-binding molecule according to any one of claims [1] to [16], which contains, as a human FcRn-binding domain, an Fc domain obtained by replacing various amino acids with at least one amino acid in the parent Fc domain IgG;

[18] the antigen binding molecule according to any one of claims [1] to [17], wherein the human FcRn binding domain is a human FcRn binding domain comprising an amino acid sequence with different amino acids replaced by at least one amino acid selected from amino acids, located in positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 312 , 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the Fc domain of the parental IgG ;

[19] an antigen binding molecule according to any one of claims [1] to [18], which comprises a human FcRn binding domain comprising an amino acid substitution in the Fc domain of a parent IgG, which includes at least one amino acid substitution selected from:

replacing the amino acid Met with Gly at position 237;

replacing the amino acid Ala with Pro at position 238;

replacement of amino acid Lys with Ser at position 239;

replacement of the amino acid Ile with Lys at position 248;

replacing the amino acid Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr with Thr at position 250;

replacing the amino acid Phe, Trp or Tyr with Met at position 252;

replacing the amino acid Thr with Ser at position 254;

replacement of the amino acid Glu with Arg at position 255;

replacing the amino acid Asp, Glu or Gln with Thr at position 256;

replacing the amino acids Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val with Pro at position 257;

replacement of the amino acid His with Glu at position 258;

replacing the amino acid Ala with Asp at position 265;

replacing the amino acid Phe with Asp at position 270;

replacing the amino acid Ala or Glu with Asn at position 286;

replacing the amino acid His with Thr at position 289;

replacement of amino acid Ala with Asn at position 297;

replacement of the amino acid Gly with Ser at position 298;

replacing the amino acid Ala with Val at position 303;

replacing the amino acid Ala with Val at position 305;

replacing the amino acids Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr with Thr at position 307;

replacing the amino acids Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr with Val at position 308;

replacing the amino acids Ala, Asp, Glu, Pro or Arg with Leu or Val at position 309;

replacement of amino acids Ala, His or Ile with Gln at position 311;

replacement of amino acids Ala or His with Asp at position 312;

replacement of amino acids Lys or Arg with Leu at position 314;

replacement of amino acids Ala or His with Asn at position 315;

replacement of amino acid Ala with Lys at position 317;

replacing the amino acid Gly with Asn at position 325;

replacing the amino acid Val with Ile at position 332;

replacement of the amino acid Leu with Lys at position 334;

replacement of amino acid His with Lys at position 360;

replacing the amino acid Ala with Asp at position 376;

replacing the amino acid Ala with Glu at position 380;

replacement of amino acid Ala with Glu at position 382;

replacing the amino acid Ala with Asn or Ser at position 384;

replacing the amino acid Asp or His with Gly at position 385;

replacement of amino acid Pro with Gln at position 386;

replacing the amino acid Glu with Pro at position 387;

replacing the amino acid Ala or Ser with Asn at position 389;

replacement of amino acid Ala with Ser at position 424;

replacing the amino acids Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr with Met at position 428;

replacement of the amino acid Lys with His at position 433;

replacement of amino acids Ala, Phe, His, Ser, Trp or Tyr with Asn at position 434;

and replacing the amino acid His or Phe with Tyr at position 436 in EU numbering;

[20] the antigen binding molecule according to any one of claims [1] to [18], wherein the human FcRn binding domain comprises at least one amino acid selected from:

Met at amino acid position 237;

Ala at amino acid position 238;

Lys at amino acid position 239;

Ile at amino acid position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr at amino acid position 250;

Phe, Trp or Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

Asp, Glu or Gln at amino acid position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Phe at amino acid position 270;

Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Gly at amino acid position 298;

Ala at amino acid position 303;

Ala at amino acid position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr at amino acid position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr at amino acid position 308;

Ala, Asp, Glu, Pro or Arg at amino acid position 309;

Ala, His or Ile at amino acid position 311;

Ala or His at amino acid position 312;

Lys or Arg at amino acid position 314;

Ala or His at amino acid position 315;

Ala at amino acid position 317;

Gly at amino acid position 325;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr at amino acid position 428;

Lys at amino acid position 433;

Ala, Phe, His, Ser, Trp or Tyr at amino acid position 434;

and His or Phe at amino acid position 436 (EU numbering) in the Fc domain of the parental IgG;

[21] the antigen binding molecule of any one of claims [18] to [20], wherein the parent IgG is selected from IgG obtained from a non-human animal;

[22] the antigen-binding molecule according to any one of claims [18]-[20], wherein the parent IgG is human IgG;

[23] an antigen-binding molecule according to any one of claims [1] to [22], which has antagonistic activity;

[24] the antigen-binding molecule of claims [1] to [23], which binds to a membrane antigen or a soluble antigen;

[25] the antigen binding molecule according to any one of claims [1] to [24], wherein the antigen binding domain comprises an artificial ligand that binds to a receptor;

[26] the antigen binding molecule according to any one of claims [1] to [24], wherein the antigen binding domain comprises an artificial receptor that binds to a ligand;

[27] the antigen-binding molecule according to any one of claims [1] to [24], which is an antibody;

[28] an antigen-binding molecule [27], where the antibody is selected from a chimeric antibody, a humanized antibody or a human antibody;

[29] pharmaceutical compositions containing any of the antigen-binding molecules according to claims [1] to [28];

[30] a method of facilitating antigen binding molecule-mediated uptake of antigen into cells by increasing its human FcRn binding activity in the neutral pH range, where the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;

[31] a method of facilitating antigen binding molecule-mediated uptake of antigen into a cell by increasing the binding activity of the antigen binding molecule to human FcRn in the neutral pH range and reducing its antigen binding activity in the acidic pH range to a value less than in the neutral pH range where the antigen binding molecule contains an antigen-binding domain and a human FcRn-binding domain and has binding activity to human FcRn in the acidic pH range;

[32] a method of increasing the number of antigens to which one antigen binding molecule can bind by increasing its binding activity to human FcRn in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has binding activity to human FcRn in acidic pH range;

[33] a method of increasing the number of antigens to which one antigen-binding molecule can bind by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen-binding activity in the acidic pH range to a value less than the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;

[34] a method of increasing the ability of an antigen binding molecule to eliminate antigen from plasma by increasing its binding activity to human FcRn in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has binding activity to human FcRn in the acidic range pH;

[35] a method of increasing the ability of an antigen-binding molecule to eliminate antigen from plasma by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen-binding activity in the acidic pH range to a value less than in the neutral pH range, where the antigen-binding the molecule contains an antigen-binding domain and a human FcRn-binding domain and has binding activity to human FcRn in the acidic pH range;

[36] a method for improving the pharmacokinetics of an antigen binding molecule by increasing its binding activity to human FcRn in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has binding activity to human FcRn in the acidic pH range;

[37] a method for improving the pharmacokinetics of an antigen-binding molecule by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen-binding activity in the acidic pH range to a value less than in the neutral pH range, where the antigen-binding molecule contains an antigen-binding domain and Human FcRn-binding domain and has binding activity to human FcRn in the acidic pH range;

[38] a method that facilitates the intracellular dissociation of an antigen bound to an antigen-binding molecule outside the cell from an antigen-binding molecule by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen-binding activity in the acidic pH range to a value less than than in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;

[39] a method that facilitates the extracellular release of an antigen-binding molecule in an antigen-free form taken up by a cell in an antigen-bound form by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen-binding activity in the acidic pH range to a value less than in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;

[40] a method of reducing the plasma total antigen concentration or the concentration of free antigen by increasing its binding activity to human FcRn in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has binding activity to human FcRn in the acidic range pH values;

[41] a method of reducing the total antigen concentration or the concentration of free antigen in plasma by increasing its binding activity to human FcRn in the neutral pH range and reducing its antigen binding activity in the acidic pH range to a value less than in the neutral pH range, where the antigen binding molecule contains an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;

[42] the method according to any one of claims [30] to [41], wherein the acidic pH range is pH values from 5.5 to 6.5, and the neutral pH range is pH values from 7.0 to 8 ,0;

[43] the method according to any one of claims [30]-[41], wherein the increase in binding activity to human FcRn in the neutral pH range is an increase by replacing various amino acids with at least one amino acid in the parent Fc domain of the IgG FcRn- human binding domain;

[44] the method according to any one of claims [30]-[41], wherein the increase in human FcRn binding activity in the neutral pH range is an increase by replacing various amino acids with at least one amino acid selected from the amino acids present at the positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314 , 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the parent Fc domain of the IgG FcRn-binding human domain;

[45] the method according to any one of claims [31], [33], [35], [37]-[39] and [41], wherein the antigen-binding activity of the antigen-binding molecule in the acidic pH range is reduced to a value less than in the neutral pH range, by replacing histidine with at least one amino acid in the antigen-binding molecule or by inserting at least one histidine;

[46] the method according to any one of claims [31], [33], [35], [37]-[39] and [41], wherein the antigen binding domain is obtained from a library of antigen binding domains;

[47] method according to any one of claims [31], [33], [35], [37]-[39] and [41], where the decrease in antigen-binding activity is represented by an increase in the KD value (in the acidic pH range)/KD (in the neutral pH range), which is the ratio of antigen binding activity in the acidic pH range and in the neutral pH range relative to this value before the introduction of histidine substitution or histidine insertion;

[48] a method for producing an antigen-binding molecule, which includes the steps of:

(a) selecting an antigen binding molecule that has binding activity to human FcRn in the neutral pH range greater than 3.2 micromolar, obtained by replacing at least one amino acid in the human FcRn binding domain of the antigen binding molecule;

(b) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in step (a) are linked; And

(c) obtaining an antigen binding molecule using the gene obtained in step (b);

[49] a method for producing an antigen-binding molecule, which includes the steps of:

(a) selecting an antigen binding molecule that has human FcRn binding activity in the neutral pH range that is greater than before making changes to at least one amino acid of the human FcRn binding domain in the antigen binding molecule having human FcRn binding activity in the pH range acidic pH values;

(b) changing at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule that has higher antigen binding activity in the neutral pH range than in the acidic pH range;

(c) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) are linked; And

(d) obtaining an antigen binding molecule using the gene obtained in step (c); And

[50] a method for producing an antigen-binding molecule, which includes the steps of:

(a) selecting an antigen binding molecule that has higher binding activity to human FcRn in the neutral pH range than binding activity prior to changing at least one amino acid in the FcRn binding domain of the human antigen binding molecule having binding activity to human FcRn in the acidic range pH values;

(b) selecting an antigen-binding molecule that has higher antigen-binding activity in the neutral pH range than in the acidic pH range;

(c) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) are linked; And

(d) obtaining an antigen binding molecule using the gene obtained in step (c);

[51] an antigen-binding molecule obtained by the production method according to any one of claims [48] to [50];

[52] a method for screening an antigen binding molecule, which includes the steps of:

(a) selecting an antigen binding molecule that has binding activity to human FcRn in a neutral pH range higher than 3.2 micromolar, where the antigen binding molecule is obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;

(b) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in step (a) are linked; And

(c) obtaining an antigen binding molecule using the gene obtained in step (b);

[53] a method for screening an antigen binding molecule, which comprises the steps of:

(a) selecting an antigen binding molecule that has binding activity to human FcRn in the neutral pH range, higher than before changing at least one amino acid in the FcRn binding domain of the human antigen binding molecule having binding activity to human FcRn in the acidic range pH;

(b) changing at least one amino acid in the antigen-binding domain of the antigen-binding molecule and selecting an antigen-binding molecule that has antigen-binding activity in the neutral pH range that is higher than in the acidic pH range;

(c) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) are linked; And

(d) producing antigen binding molecules using the gene obtained in step (c);

[54] a method for screening an antigen binding molecule, which includes the steps of:

(a) selecting an antigen binding molecule that has binding activity to human FcRn in the neutral pH range, higher than before changing at least one amino acid in the human FcRn binding domain of the antigen binding molecule having binding activity to human FcRn in the acidic range pH;

(b) selecting an antigen binding molecule that has higher antigen binding activity in the neutral pH range than in the acidic pH range;

(c) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) are linked; And

(d) obtaining an antigen binding molecule using the gene obtained in step (c);

[55] the method of any one of claims [30] to [54], wherein the antigen binding domain comprises an artificial ligand that binds to a receptor;

[56] the method of any one of claims [30] to [54], wherein the antigen binding domain comprises an artificial receptor that binds to a ligand; And

[57] the method according to any one of claims [30] to [54], wherein the antigen binding molecule is an antibody.

Advantages of the invention

The present invention relates to:

methods facilitating antigen binding molecule-mediated uptake of antigen into cells; methods for increasing the number of antigens to which one antigen-binding molecule can bind; and methods that accelerate the decrease in plasma antigen concentration through the administration of antigen-binding molecules. By facilitating antigen binding molecule-mediated cellular uptake of antigen, the reduction in plasma antigen concentration can be accelerated by administration of such antigen binding molecules, and the pharmacokinetics of the antigen binding molecule can be improved to increase the number of antigens to which one antigen binding molecule can bind. Thus, antigen-binding molecules may produce better effects in vivo than conventional antigen-binding molecules.

Brief description of drawings

[Figure 1] Figure 1 shows in graphical form, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor after administration of an antibody against the human IL-6 receptor to transgenic mice with human FcRn (line 276), where the concentration in plasma, the soluble form of the human IL-6 receptor is constant (stationary infusion model).

[FIG. 2] FIG. 2 is a schematic representation that shows that dissociation of an IgG antibody molecule from a soluble antigen in an endosome results in increased elimination of the antigen, resulting in a new round of binding to another antigen.

[FIG. 3] FIG. 3 graphically shows, as a function of time, the plasma concentration of antibody in human FcRn transgenic mice.

[Figure 4] Figure 4 graphically presents, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor in human FcRn transgenic mice.

[FIG. 5] FIG. 5 graphically shows, as a function of time, the plasma concentration of antibody in a normal mouse.

[FIG. 6] FIG. 6 graphically shows, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor in a normal mouse.

[FIG. 7] FIG. 7 graphically represents, as a function of time, the plasma concentration of the unbound soluble form of the human IL-6 receptor in a normal mouse.

[FIG. 8] FIG. 8 graphically presents, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor in human FcRn transgenic mice.

[Fig.9] Fig.9 graphically shows, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor after administration of Fv4-IgG1-F14 at a low dose (0.01 mg/kg) or 1 mg /kg.

[FIG. 10] FIG. 10 graphically shows, as a function of time, the plasma antibody concentration after administration of Fv4-IgG1-F14 at a low dose (0.01 mg/kg) or 1 mg/kg.

[FIG. 11] FIG. 11 presents in graphical form, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor after administration of an antibody against the human IL-6 receptor to a normal mouse, where the plasma concentration of the soluble form of the IL-6 receptor 6 people is permanent.

[FIG. 12] FIG. 12 graphically shows, as a function of time, the plasma antibody concentration after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (line 276).

[FIG. 13] FIG. 13 graphically presents, as a function of time, the plasma concentration of the soluble form of the human IL-6 receptor after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (line 276).

[Figure 14] Figure 14 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the plasma concentration of hsIL-6R on day 1 after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into transgenic mice with human FcRn (line 276).

[Fig. 15] Fig. 15 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the concentration of antibody in plasma on day 1 after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into transgenic mice with Human FcRn (line 276).

[FIG. 16] FIG. 16 shows the antigen/antibody molar ratio (C value) as a function of time after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (line 276).

[FIG. 17] FIG. 17 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the antigen/antibody molar ratio (C value) on day 1 after co-injection of hsIL-6R and anti-IL-receptor antibody. 6 human transgenic mice with human FcRn (line 276).

[FIG. 18] FIG. 18 graphically presents, as a function of time, the plasma concentration of hsIL-6R after administration of Fv4-IgG1-F14 at low doses (0.01 or 0.2 mg/kg) or 1 mg /kg transgenic mice with human FcRn (line 276), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[FIG. 19] FIG. 19 shows, as a function of time, the concentration of hsIL-6R in the plasma of transgenic mice with human FcRn line 276 and line 32 after co-injection of transgenic mice with human FcRn (line 276 and 32) with hsIL-6R and antibodies against human IL-6 receptor.

[Figure 20] Figure 20 shows, as a function of time, the concentration of antibody in the plasma of human FcRn transgenic mice line 276 and line 32 after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice ( line 276 and 32).

[FIG. 21] FIG. 21 graphically presents, as a function of time, the plasma concentration of hsIL-6R after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 32), where the plasma concentration of hsIL- 6R is constant (steady-state infusion model).

[FIG. 22] FIG. 22 is a graphical representation of the plasma antibody concentration as a function of time following administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 32), wherein the plasma concentration of hsIL-6R is constant (stationary infusion model).

[FIG. 23] FIG. 23 shows, as a function of time, the antigen/antibody molar ratio (C value) after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 32), where the plasma concentration of hsIL- 6R is constant (steady-state infusion model).

[FIG. 24] FIG. 24 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the antigen/antibody molar ratio (C value) on day 1 after administration of anti-human IL-6 receptor antibody to transgenic mice with Human FcRn (line 32), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[FIG. 25] FIG. 25 graphically shows, as a function of time, the plasma antibody concentration after administration of anti-human IL-6 receptor antibodies having Fc variants F11, F39, F48 and F264 to human FcRn transgenic mice. (line 32), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[Fig. 26] Fig. 26 graphically presents, as a function of time, the plasma concentration of hsIL-6R after administration of anti-human IL-6 receptor antibodies having the Fc variant F11, F39, F48 and F264 to transgenic mice with Human FcRn (line 32), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[FIG. 27] FIG. 27 graphically shows, as a function of time, the plasma antibody concentration after administration of anti-human IL-6 receptor antibodies having the Fc variant F157, F196 and F262 to human FcRn transgenic mice (line 32), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[FIG. 28] FIG. 28 graphically shows, as a function of time, the plasma concentration of hsIL-6R after administration of anti-human IL-6 receptor antibodies having Fc variants F157, F196 and F262 to human FcRn transgenic mice. (line 32), where the plasma concentration of hsIL-6R is constant (steady-state infusion model).

[FIG. 29] FIG. 29 shows a pharmacokinetic model used for in silico studies of conventional and antigen-eliminating antibodies.

[FIG. 30] FIG. 30 graphically presents, as a function of time, the plasma concentration of human IL-6 after co-injection of a normal mouse with human IL-6 and anti-human IL-6 antibody.

[FIG. 31] FIG. 31 graphically shows, as a function of time, the plasma antibody concentration after co-injection of a normal mouse with human IL-6 and an anti-IL-6 antibody.

[FIG. 32] FIG. 32 shows human IgA binding sensograms to CD89-Fc fusion protein at pH 7.4 and pH 6.0 using Biacore.

[FIG. 33] FIG. 33 graphically presents, as a function of time, the plasma concentration of human IgA after co-injection of normal mouse human IgA and CD89-Fc fusion protein.

[FIG. 34] FIG. 34 graphically shows, as a function of time, the plasma antibody concentration after co-injection of human IgA and normal mouse CD89-Fc fusion protein.

[FIG. 35] FIG. 35 is a graphical representation of the plasma concentration of soluble human plexin A1 at the 7 hour time point following co-injection of soluble human plexin A1 and anti-plexin A1 antibody into a normal mouse.

Description of Embodiments

The present invention relates to methods that facilitate antigen binding molecule-mediated uptake of antigen into cells. More specifically, the present invention relates to methods for facilitating the uptake of antigen into cells through an antigen binding molecule having human FcRn binding activity in the acidic pH range, where the methods are based on increasing the human FcRn binding activity of the antigen binding molecule in the neutral pH range. The present invention also provides methods for increasing the uptake of antigen into cells by an antigen binding molecule having human FcRn binding activity in the acidic pH range, wherein the methods are based on changing at least one amino acid in the FcRn binding domain of the human antigen binding molecule.

The present invention also relates to methods for facilitating the uptake of antigen into cells by means of an antigen binding molecule having binding activity to human FcRn in the acidic pH range, where the methods are based on the use of a human FcRn binding domain containing an amino acid sequence with the replacement of different amino acids by at least one an amino acid selected from those amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in Fc domain of the parental IgG human FcRn binding domain, including the Fc domain of the parental IgG.

The present invention also provides methods for facilitating antigen binding molecule-mediated uptake of antigen into cells by reducing the antigen binding activity (binding activity) of the above-described antigen binding molecule in the acidic pH range to an antigen binding activity less than in the neutral pH range; and this facilitates the uptake of antigen by cells. The present invention also provides methods for increasing antigen binding molecule-mediated uptake of antigen into cells, where the methods are based on altering at least one amino acid in the antigen binding domain of the antigen binding molecule described above that facilitates uptake of the antigen into cells. The present invention also provides methods for facilitating antigen binding molecule-mediated uptake of antigen into cells, where the methods are based on the replacement of a histidine by at least one amino acid or are based on the insertion of at least one histidine into the antigen binding domain of the antigen binding molecule described above, which increases the uptake of the antigen into cells.

As used herein, “antigen uptake into cells” mediated by an antigen binding molecule means that antigens are taken up by cells through endocytosis. Herein, as used herein, the expression “increases cellular uptake” means that the rate of intracellular uptake of an antigen-binding molecule bound to an antigen in plasma is increased, and/or the amount of recycling of the absorbed antigen in plasma is reduced. This means that the rate of uptake of antigen into cells is increased relative to the rate of uptake of antigen by the antigen binding molecule until the human FcRn binding activity of the antigen binding molecule increases in the neutral pH range, or until the human FcRn binding activity increases and the antigen binding activity (binding activity) of the antigen binding molecule decreases. in the acidic pH range to a value less than its antigen-binding activity in the neutral pH range. Preferably, the rate is increased compared to intact human IgG, and more preferably compared to intact human IgG. Thus, in the present invention, it is possible to evaluate whether the uptake of antigen into cells has been accelerated by the antigen-binding molecule based on the increase in the rate of uptake of antigen into cells. The rate of antigen uptake by cells can be calculated, for example, by monitoring over time the decrease in antigen concentration in a culture medium containing FcRn-expressing cells after adding antigen and antigen-binding molecule to the medium, or by monitoring over time the amount of antigen taken up by human FcRn-expressing cells. By using the methods of the present invention to increase the rate of antigen binding molecule-mediated uptake of antigen into cells, it is possible to increase, for example, the rate of elimination of antigen from plasma by administration of an antigen binding molecule. Thus, it is possible to assess whether an increase in antigen binding molecule-mediated cellular uptake of antigen has occurred, for example, by testing whether the rate of elimination of antigen from plasma is increased or whether the total concentration of antigen in plasma is reduced by administration of an antigen binding molecule.

As used herein, “total plasma antigen concentration” refers to the sum of the concentrations of antigen bound to an antigen binding molecule and unbound antigen, or “free antigen concentration in plasma”, which is the concentration of antigen not bound to an antigen binding molecule. Various methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art, as described hereinafter.

“Intact human IgG” as used herein means unmodified human IgG and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "intact human IgG" provided that it can bind to human FcRn in the acidic pH range. Preferably, the “intact human IgG” may be human IgG1.

The present invention also provides methods for increasing the number of antigens to which a single antigen-binding molecule can bind. More specifically, the present invention relates to methods for increasing the number of antigens to which one antigen binding molecule having human FcRn binding activity in the acidic pH range can bind by increasing the human FcRn binding activity of an antigen binding molecule in the neutral pH range. The present invention also provides methods for increasing the number of antigens to which a single antigen binding molecule having human FcRn binding activity in the acidic pH range can bind by changing at least one amino acid in the human FcRn binding domain of the antigen binding molecule.

The present invention also provides methods for increasing the number of antigens to which a single antigen binding molecule having human FcRn binding activity in the acidic pH range can bind, by using a human FcRn binding domain comprising an amino acid sequence in which at least one amino acid, selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311 , 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in parent Fc- the human IgG FcRn binding domain containing the parent IgG Fc domain is replaced with another amino acid.

"Parental IgG" as used herein means an unmodified IgG that is then modified to produce a variant provided that the modified variant of the parental IgG can bind to human FcRn in the acidic pH range (thus the parental IgG does not require a binding agent). activity with human FcRn under acidic conditions). The parent IgG may be a naturally occurring IgG, or a variant or engineered version of a naturally occurring IgG. Parental IgG may refer to the polypeptide itself, compositions that contain the parental IgG, or an amino acid sequence that encodes the parental IgG. It should be noted that "parental IgG" includes known, commercially available, recombinantly produced IgG as defined above. The original "parental IgG" is not limited and can be obtained from any non-human animal or human organism. Preferably, the organism is selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel and non-human primate. In another embodiment, the “parental IgG” can also be obtained from a cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the “parental IgG” is derived from human IgG1 without limitation to a particular IgG class. This means that human IgG1, IgG2, IgG3 or IgG4 can suitably be used as the “parent IgG”. In a similar manner, any class or subclass of IgG from any organism listed above may preferably be used as the “parent IgG.” An example of a variant or engineered version of a natural IgG is described in Curr Opin Biotechnol. 2009 Dec; 20(6): 685-91, Curr Opin Immunol. 2008 Aug; 20(4): 460-70, Protein Eng Des Sel. 2010 Apr; 23(4): 195-202, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, but not limited to them.

In addition, the present invention provides methods for increasing the number of antigens to which one antigen binding molecule can bind by reducing the antigen binding activity (binding activity) in the acidic pH range of the above-described antigen binding molecule that has an increased number of antigen binding events, less than its antigen-binding activity in the neutral pH range. The present invention also provides methods for increasing the number of antigens that a single antigen binding molecule can bind by changing at least one amino acid in the antigen binding domain of the antigen binding molecule described above that has an increased number of antigen binding events. The present invention also provides methods for increasing the number of antigens to which one antigen molecule can bind by replacing a histidine with at least one amino acid or by inserting at least one histidine into the antigen binding domain of an antigen binding molecule described above that has an increased number of antigen binding events .

As used herein, “the number of antigens to which one antigen-binding molecule can bind” refers to the number of antigens to which one antigen-binding molecule can bind before the molecule is eliminated due to degradation. As used herein, "increasing the number of antigens to which one antigen-binding molecule can bind" refers to the increase in the number of cycles achieved before the antigen-binding molecule is eliminated by degradation, where each cycle consists of binding of an antigen to an antigen-binding molecule in plasma, intracellular uptake of the antigen-binding molecule, associated with the antigen, and dissociation from the antigen in the endosome, followed by the return of the antigen-binding molecule to the plasma. This means that the number of cycles is increased relative to the antigen binding molecule until the human FcRn binding activity of the antigen binding molecule increases in the neutral pH range, or until the human FcRn binding activity increases and the antigen binding activity (binding activity) of the antigen binding molecule decreases in the acidic pH range to less than its antigen-binding activity in the neutral pH range. Thus, it is possible to evaluate whether the number of cycles is increased by testing whether the above-described “whether intracellular absorption is facilitated” or “whether pharmacokinetics are improved”, as described below.

The present invention also provides methods for facilitating intracellular dissociation of an antigen from an antigen-binding molecule that binds to an antigen outside of cells. More specifically, the present invention relates to methods of facilitating the intracellular dissociation of an antigen from an antigen binding molecule that binds to an antigen outside of cells by increasing the human FcRn binding activity in the neutral pH range of an antigen binding molecule that has human FcRn binding activity in the acidic range pH, and a decrease in its antigen-binding activity in the range of acidic pH values to a value less than the range of neutral pH values. The present invention also relates to methods of facilitating the intracellular dissociation of an antigen from an antigen binding molecule that binds to an antigen outside of cells, where the methods are based on changing at least one amino acid in the antigen binding domain of the antigen binding molecule and simultaneously changing at least one amino acid in the FcRn binding domain a human antigen-binding molecule having binding activity to human FcRn in the acidic pH range. The present invention also provides methods for facilitating the intracellular dissociation of an antigen from an antigen binding molecule that binds an antigen outside the cell by replacing a histidine with at least one amino acid or by inserting at least one histidine into the antigen binding domain of the antigen binding molecule and simultaneously replacing at least one one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the parent Fc domain of the human IgG FcRn binding domain, a different amino acid.

In the present invention, antigens can dissociate from the antigen-binding molecule at any location within the cell; however, the preferred site of dissociation is the early endosome. As used herein, “intracellular dissociation of an antigen binding molecule bound antigen from an antigen binding molecule outside the cells” does not necessarily mean that all antigens taken up by cells through binding to an antigen binding molecule are dissociated from the antigen binding molecule within the cell. Thus, it is conceivable that the proportion of antigens dissociated from the antigen-binding molecule within the cell increases compared to the proportion of molecules until the antigen-binding activity of the antigen-binding molecule in the acidic pH range decreases to a value less than in the neutral pH range, and at the same time the activity increases binding to human FcRn in the neutral pH range. Such a method of facilitating the intracellular dissociation of an antigen from an antigen binding molecule bound to an antigen outside a cell is the same as a method which provides the antigen binding molecule with the ability to facilitate the intracellular dissociation of an antigen from an antigen binding molecule by facilitating the uptake of an antigen binding molecule bound by an antigen.

The present invention also provides methods for facilitating the extracellular release of an antigen-free antigen-binding molecule taken up by cells in antigen-bound form. More specifically, the present invention relates to methods of facilitating the extracellular release of an antigen-free antigen-binding molecule taken up by cells in an antigen-bound form by increasing human FcRn binding activity in the neutral pH range of an antigen-binding molecule that has human FcRn binding activity in the pH range acidic pH values, and a decrease in its antigen-binding activity in the range of acidic pH values to a value less than in the range of neutral pH values. The present invention also relates to methods of facilitating the extracellular release of an antigen-free antigen-binding molecule taken up by cells in an antigen-bound form, where the methods are based on changing at least one amino acid in the antigen-binding molecule and simultaneously changing at least one amino acid in the FcRn-binding domain person. The present invention also provides methods for facilitating the extracellular release of an antigen-free antigen-binding molecule taken up by cells in antigen-bound form by replacing a histidine with at least one amino acid, or by inserting at least one histidine into an antigen-binding molecule, and simultaneously replacing at least one amino acid. at least one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308 , 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the parent Fc domain of the human IgG FcRn-binding domain, a different amino acid.

As used herein, “extracellular release of antigen-free antigen-binding molecule taken up by cells in antigen-bound form” does not necessarily mean that each of the cell-taken-up antigen-binding molecules bound to an antigen is released in antigen-free form outside the cell. It is conceivable that the proportion of antigen-binding molecules released in antigen-free form outside cells increases relative to the proportion of molecules until the antigen-binding activity of the antigen-binding molecule in the acidic pH range decreases to less than that in the neutral pH range and the binding activity increases with human FcRn in the neutral pH range. An antigen-binding molecule released outside of cells preferentially retains antigen-binding activity. Such a method of facilitating the extracellular release of an antigen-free antigen-binding molecule taken up by cells in an antigen-free form is the same as a method that provides the antigen-binding molecule with the ability to facilitate the extracellular release of an antigen-free antigen-binding molecule taken up by cells in an antigen-bound form by facilitating the uptake of antigen-binding molecules by cells. molecules associated with antigen in cells.

The present invention also relates to methods of increasing the ability to eliminate plasma antigen by introducing an antigen-binding molecule. In the present invention, “methods for increasing the ability to eliminate plasma antigen” are the same as “methods for enhancing the ability of an antigen-binding molecule to eliminate antigen from plasma.” More specifically, the present invention relates to methods of increasing the plasma antigen elimination capacity of an antigen binding molecule having human FcRn binding activity in the acidic pH range by increasing the human FcRn binding activity of the antigen binding molecule in the neutral pH range. The present invention also relates to methods of increasing the ability to eliminate plasma antigen by an antigen binding molecule having binding activity to human FcRn in the acidic pH range, where the methods are based on changing at least one amino acid in the FcRn binding domain of the human antigen binding molecule.

The present invention also provides methods for increasing the plasma antigen elimination capacity of an antigen binding molecule having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence substituting at least one amino acid selected from amino acids in positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the parental Fc domain of IgG FcRn -human binding domain containing the Fc domain of the parent IgG to another amino acid.

The present invention also provides methods for increasing the plasma antigen elimination ability of an antigen binding molecule by reducing the antigen binding activity in the acidic pH range of the above described antigen binding molecule with increased plasma antigen elimination ability compared to the antigen binding activity in the neutral pH range. The present invention also provides methods for increasing the ability to eliminate plasma antigen by an antigen binding molecule by changing at least one amino acid in the antigen binding domain of the above-described antigen binding molecule with increased ability to eliminate plasma antigen. The present invention also provides methods for increasing the plasma antigen elimination ability by introducing an antigen binding molecule, by replacing a histidine with at least one amino acid, or by inserting at least one histidine into the antigen binding domain of the antigen binding molecule described above with increased plasma antigen elimination ability.

As used herein, “plasma antigen elimination ability” means the ability to eliminate an antigen from plasma upon administration of an antigen binding molecule or secretion in vivo. Thus, “increasing the ability of an antigen-binding molecule to eliminate plasma antigen” as used herein means that the rate of elimination of an antigen from plasma is accelerated upon administration of the antigen-binding molecule compared to the elimination of the antigen before the human FcRn binding activity of the antigen-binding molecule increases in the neutral pH range or up to increasing the binding activity to human FcRn and simultaneously reducing its antigen-binding activity in the acidic pH range to a value less than the neutral pH range. Increased activity of the antigen binding molecule to eliminate antigen from plasma can be assessed, for example, by administering the soluble antigen and the antigen binding molecule in vivo, and measuring the concentration of the soluble antigen in the plasma after administration. If the concentration of a soluble antigen in plasma after administration of a soluble antigen and an antigen-binding molecule is reduced by increasing the binding activity of the antigen-binding molecule to human FcRn in the neutral pH range, or by increasing its binding activity to human FcRn and simultaneously reducing its antigen-binding activity in the acidic pH range to value less than the neutral pH range, the ability of the antigen-binding molecule to eliminate plasma antigen can be considered increased. The form of the soluble antigen may be an antigen-binding molecule bound to an antigen or an antigen-binding molecule not bound to an antigen, the concentration of which can be defined as "the plasma concentration of an antigen-binding molecule bound to an antigen" and "the plasma concentration of an antigen-binding molecule not bound to an antigen" , respectively (the latter is synonymous with “free antigen concentration in plasma”). Since "total antigen concentration in plasma" refers to the sum of the concentrations of antigen-binding molecule bound to the antigen and not bound to the antigen, or "free antigen concentration in plasma" which is the concentration of antigen-binding molecule not bound to the antigen, the concentration of soluble antigen can be defined as “the total concentration of antigen in plasma.” Various methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art, as described hereinafter.

The present invention also relates to methods for improving the pharmacokinetics of antigen-binding molecules. More specifically, the present invention relates to methods for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by increasing the human FcRn binding activity of an antigen binding molecule in the neutral pH range. In addition, the present invention provides methods for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule.

The present invention also provides methods for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence substituting various amino acids with at least one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314 , 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering) in the parent Fc domain of the IgG FcRn-binding human domain containing the Fc domain of IgG.

In addition, the present invention provides methods for improving the pharmacokinetics of an antigen binding molecule by reducing the antigen binding activity in the acidic pH range of the above-described antigen binding molecule with improved pharmacokinetics to a value less than its antigen binding activity in the neutral pH range. The present invention also provides methods for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by altering at least one amino acid in the antigen binding domain of the above described antigen binding molecule with improved pharmacokinetics. The present invention also provides methods for improving pharmacokinetics by replacing a histidine with at least one amino acid or by inserting at least one histidine into the antigen binding domain of an antigen binding molecule with improved pharmacokinetics described above.

As used herein, "improved pharmacokinetics", "improved pharmacokinetics", and "better quality pharmacokinetics" may be otherwise stated as "improved plasma (blood) retention", "improved plasma (blood) retention", "better plasma (blood) retention" (blood). These terms are synonymous.

As used herein, "improvement in pharmacokinetics" means not only an increase in the period until elimination from the plasma (e.g., before the antigen-binding molecule degrades intracellularly or the like and cannot be returned to the plasma) after administration of the antigen-binding molecule to humans or non-human animals such as mice, rats, monkeys, rabbits and dogs, but also by increasing the time the antigen binding molecule is retained in a form that allows antigen binding (eg, an antigen free form of the antigen binding molecule) during the period of administration for elimination due to degradation. Intact human IgG can bind to the FcRn of a non-human animal. For example, a mouse may preferably be used for administration to confirm the properties of the antigen-binding molecule of the invention, since intact human IgG may bind to mouse FcRn more strongly than to human FcRn (Int Immunol. 2001 Dec; 13(12): 1551-9). As another example, a mouse in which its natural FcRn genes are disrupted and which contains a human FcRn gene transgene for expression (Methods Mol Biol. 2010; 602: 93-104) can preferably also be used for administration to confirm antigen-binding properties. molecules of the invention described below herein. Specifically, “improving pharmacokinetics” also includes increasing the period to elimination due to degradation of the non-antigen-bound antigen-binding molecule (the antigen-free form of the antigen-binding molecule). An antigen-binding molecule in plasma does not bind to a new antigen if an antigen-binding molecule is already bound to the antigen. Thus, the longer the period during which an antigen-binding molecule does not bind to an antigen, the longer the period during which it can bind to a new antigen (higher chance of binding to another antigen). This allows the period of time during which the antigen is released from the antigen-binding molecule in vivo to be reduced and the period during which the antigen is bound to the antigen-binding molecule to be increased. The plasma concentration of the antigen-free form of the antigen-binding molecule can be increased, and the period over which the antigen is bound to the antigen-binding molecule can be increased by accelerating the elimination of the antigen from the plasma by administering the antigen-binding molecule. Specifically, as used herein, “improving the pharmacokinetics of an antigen-binding molecule” includes improving the pharmacokinetic parameter of the antigen-free form of the antigen-binding molecule (any increase in plasma half-life, increase in mean plasma retention time, and decrease in plasma clearance), increasing the period over which the antigen binds to the antigen-binding molecule molecule after administration of the antigen-binding molecule, and accelerating the elimination of antigen from plasma mediated by the antigen-binding molecule. Improvement in the pharmacokinetics of an antigen-binding molecule can be assessed by determining any of the parameters: plasma half-life, mean plasma retention time, and plasma clearance of the antigen-binding molecule or its antigen-free form (“Pharmacokinetics: Enshu-niyoru Rikai (Understanding through practice)” Nanzando). For example, the plasma concentration of an antigen-binding molecule or an antigen-free form thereof is determined after administration of the antigen-binding molecule to mice, rats, monkeys, rabbits, dogs or humans. Each parameter is then determined. If the plasma half-life or mean plasma retention time is increased, the pharmacokinetics of the antigen-binding molecule can be considered improved. The parameters can be determined by methods known to those skilled in the art. The parameters can be assessed accordingly, for example, by non-compartmental analysis using WinNonlin pharmacokinetic analysis software (Pharsight) according to the accompanying instructions for use. The plasma concentration of antigen-free antigen-binding molecule can be determined by methods known to those skilled in the art, for example, using the assay method described in Clin Pharmacol. 2008 Apr; 48(4): 406-17.

As used herein, “improving pharmacokinetics” also includes increasing the period during which an antigen binds to an antigen-binding molecule after administration of the antigen-binding molecule. Whether the period during which the antigen binds to the antigen-binding molecule after administration of the antigen-binding molecule is prolonged can be assessed by determining the concentration of free antigen in plasma. The presence of an increase can be judged based on the determined plasma concentration of free antigen or the period of time required for the ratio of free antigen concentration to total antigen concentration to increase.

The plasma concentration of free antigen not bound to an antigen-binding molecule, or the ratio of the concentration of free antigen to the total concentration of antigen, can be determined by methods known to those skilled in the art, for example, the method described in Pharm Res. 2006 Jan; 23 (1): 95-103. Alternatively, if an antigen exhibits a particular function in vivo, whether binding of the antigen to an antigen-binding molecule that neutralizes the function of the antigen (antagonistic molecule) is achieved can be assessed by testing whether the function of the antigen is neutralized. Whether neutralization of antigen function has occurred can be assessed by examining an in vivo marker that reflects antigen function. Whether an antigen binds to an antigen-binding molecule that activates the function of the antigen (agonist molecule) can be assessed by examining an in vivo marker that reflects the function of the antigen.

Determination of the concentration of free antigen in plasma and the ratio of the amount of free antigen in plasma to the amount of total antigen in plasma, in vivo marker analysis, and the like are not particularly limited; however, the assays are preferably performed after a certain period of time has elapsed since administration of the antigen-binding molecule. In the present invention, the period of time after administration of the antigen-binding molecule is not particularly limited; those skilled in the art can determine a suitable period depending on the properties and the like. administered antigen-binding molecule. Such periods include, for example, one day after administration of the antigen-binding molecule, three days after administration of the antigen-binding molecule, seven days after administration of the antigen-binding molecule, 14 days after administration of the antigen-binding molecule, and 28 days after administration of the antigen-binding molecule. As used herein, “plasma antigen concentration” means either “total plasma antigen concentration,” which is the sum of the concentration of antigen bound to an antigen binding molecule and the concentration of unbound antigen, or “plasma free antigen concentration,” which is the concentration of antigen not associated with an antigen-binding molecule.

The total plasma antigen concentration can be reduced by the antigen binding molecule of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or even more compared to administration of a control an antigen binding molecule containing an intact human IgG Fc domain as a human FcRn binding domain or compared to a situation where no antigen binding domain molecule is administered.

The antigen/antigen binding molecule molar ratio can be calculated as follows:

A value: molar concentration of antigen at each time point

B value: molar concentration of antigen-binding molecule at each time point

C-value: molar concentration of antigen per molar concentration of antigen-binding molecule (molar ratio antigen/antigen-binding molecule) at each time point

C=A/B.

A low C value indicates a higher antigen elimination efficiency per antigen binding molecule, while an increased C value indicates a lower antigen elimination efficiency per antigen binding molecule.

The antigen/antigen binding molecule molar ratio can be calculated as described above.

The molar ratio of antigen/antigen binding molecule can be reduced by administering the antigen binding molecule of the present invention by 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times, or even more compared with the administration a control antigen-binding molecule containing an intact human IgG Fc domain as a human FcRn-binding domain.

Herein, intact human IgG1, IgG2, IgG3, or IgG4 is preferably used as intact human IgG for the purpose of using a control intact human IgG compared to antigen binding molecules for their human FcRn binding activity or in vivo activity. Preferably, a control antigen binding molecule comprising the same antigen binding domain as the antigen binding molecule of interest and an intact human IgG Fc domain as the human FcRn binding domain may be suitably used. More preferably, intact human IgG1 is used to control intact human IgG against antigen binding molecules for their human FcRn binding activity or in vivo activity.

The reduction in total plasma antigen concentration or antigen/antibody molar ratio can be assessed as described in Examples 6, 8 and 13. More specifically, using human FcRn transgenic mouse line 32 or human FcRn mouse line 276 (Jackson Laboratories, Methods Mol Biol . 2010; 602: 93-104), they can be assessed either through an antigen-antibody co-injection model or through a steady-state antigen infusion model if the antigen-binding molecule does not cross-react with the corresponding mouse antigen. If the antigen binding molecule cross-reacts with the corresponding mouse antigen, they can be assessed by simply injecting the antigen binding molecule into human FcRn transgenic mouse line 32 or human FcRn line 276 (Jackson Laboratories). In the co-injection model, a mixture of antigen-binding molecule and antigen is injected into mice. In the steady-state antigen infusion model, an infusion pump containing an antigen solution is implanted into mice to achieve a constant plasma antigen concentration, and then the mice are injected with an antigen-binding molecule. The antigen-binding molecule to be tested is administered at the same dose. The total plasma antigen concentration, the plasma free antigen concentration, and the plasma antigen-binding molecule concentration are measured at an appropriate time point using a method known to those skilled in the art.

The total antigen concentration or free antigen concentration in plasma and the antigen/antigen binding molecule molar ratio can be measured 2, 4, 7, 14, 28, 56 or 84 days after administration to assess the duration of effect of the present invention. In other words, to evaluate the properties of the antigen binding molecule of the present invention, the long-term plasma antigen concentration is determined by measuring the total total antigen concentration or the plasma free antigen concentration and the antigen/antigen binding molecule molar ratio in 2, 4, 7, 14, 28, 56 or 84 days after administration of the antigen-binding molecule. Whether a reduction in plasma antigen concentration or antigen/antigen binding molecule molar ratio has been achieved by the antigen binding molecule described in the present invention can be determined by assessing the reduction at each of the time points or multiple time points described above.

The total antigen concentration or free antigen concentration in plasma and the antigen/antigen binding molecule molar ratio can be measured at 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration to evaluate the short-term effect of the present invention. In other words, the short-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration in the plasma and the antigen/antigen-binding molecule molar ratio at 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration of the antigen-binding molecule to evaluate properties antigen binding molecule of the present invention.

The route of administration of the antigen binding molecule of the present invention can be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral and intramuscular injection.

In the present invention, improvement of pharmacokinetics in humans is preferred.

If the retention time in humans is difficult to determine, it can be calculated based on the retention time in mice (eg, normal mice, transgenic mice expressing human antigen, transgenic mice expressing human FcRn) or monkeys (eg, cynomolgus monkeys).

As used herein, the acidic pH range generally refers to pH values between 4.0 and 6.5. The acidic pH range is preferably the range indicated by any pH value within pH 5.5 to 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6 .0, 6.1, 6.2, 6.3, 6.4 and 6.5, more preferably a pH of 5.8 to 6.0, which are values close to the pH value in the endosome in vivo. Meanwhile, as used herein, the neutral pH range generally refers to a pH value of 6.7 to 10.0. The neutral pH range is preferably a range indicated by any pH value within the range of pH 7.0 to 8.0, preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4 , 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, more preferably pH 7.4, which are values close to the pH value in plasma (blood) in vivo. pH 7.0 can be used as an alternative to pH 7.4 if it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to the latter's low affinity at pH 7.4. Since temperature is used in the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature between 10 degrees C and 50 degrees C. Preferably, a temperature between 15 degrees C and 40 degrees C is used to determine the binding affinity between human FcRn-binding domain and human FcRn. More preferably, any temperature between 20 degrees C and 35 degrees C, as well as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 degrees C is also used to determine the binding affinity between the human FcRn binding domain and human FcRn. The temperature of 25 degrees C described in Example 5 is one example for an embodiment of the present invention.

Thus, as used herein, “a decrease in the antigen-binding activity of an antigen-binding molecule in the acidic pH range to a value less than in the neutral pH range” means that the antigen-binding activity of the antigen-binding molecule at pH 4.0 to pH 6.5 is worse compared with its antigen-binding activity at pH from 6.7 to 10.0. Preferably, the expression above means that the antigen binding activity of the antigen binding molecule at a pH of 5.5 to 6.5 is inferior compared to the antigen binding activity at a pH of 7.0 to 8.0, more preferably means that its antigen binding activity at pH values close to the pH value in the early endosome, is worse compared to its antigen-binding activity at plasma pH in vivo. Specifically, the antigen binding activity of an antigen binding molecule at pH 5.8 to 6.0 is inferior compared to the antigen binding activity of an antigen binding molecule at pH 7.4.

Meanwhile, herein, the expression “a decrease in the antigen-binding activity of an antigen-binding molecule in the acidic pH range to a value less than in the neutral pH range” is also denoted as “an increase in the antigen-binding activity of the antigen-binding molecule in the neutral pH range to a value greater than in range of acidic pH values.” Specifically, in the present invention, it is possible to increase the ratio of antigen-binding activity of an antigen-binding molecule between acidic and neutral pH ranges. For example, the value of KD (pH 5.8)/KD (pH 7.4) is increased in the embodiment described below. The ratio of antigen-binding activity of an antigen-binding molecule between acidic and neutral pH ranges can be increased, for example, by decreasing its antigen-binding activity in the acidic pH range, increasing its antigen-binding activity in the neutral pH range, or both.

Herein, the expression “deterioration of antigen-binding activity in the acidic pH range compared to antigen-binding activity in the neutral pH range” is sometimes used instead of the expression “decreased antigen-binding activity in the acidic pH range to a value less than the neutral pH range.”

As used herein, human FcRn binding activity in the acidic pH range means human FcRn binding activity at pH 4.0 to 6.5, preferably human FcRn binding activity at pH 5.5 to 6.5, and more preferably binding activity to human FcRn at pH 5.8 to pH 6.0, which is comparable to the pH of the early endosome in vivo. Meanwhile, herein, human FcRn binding activity in the neutral pH range means human FcRn binding activity at pH 6.7 to 10.0, preferably human FcRn binding activity at pH 7.0 to 8.0, and more preferably, binding activity to human FcRn occurs at pH 7.4, which is comparable to the in vivo plasma pH.

The antigen binding molecules of the present invention have a human FcRn binding domain. The human FcRn binding domain is not particularly limited so long as the antigen binding molecules exhibit binding activity to human FcRn in the acidic and neutral pH range. Alternatively, the domain may have direct or indirect human FcRn binding activity. Such domains include, for example, IgG immunoglobulin Fc domain, albumin, albumin domain 3, anti-human FcRn antibodies, anti-human FcRn peptides and anti-human FcRn scaffold molecules, each of which has direct binding activity to human FcRn; and molecules that bind to IgG or albumin, which have indirect binding activity to human FcRn. Such preferred domains of the present invention have human FcRn binding activity in the acidic and neutral pH ranges. It is possible to use the domains without any modification, provided that they already have binding activity to human FcRn in the acidic and neutral pH range. If the domains have only weak human FcRn binding activity or no human FcRn binding activity in the acidic and/or neutral pH range, human FcRn binding activity can be provided by altering the amino acids in the antigen binding molecules. However, it is preferred that human FcRn binding activity in the acidic and/or neutral pH range is provided by altering the amino acids in the human FcRn binding domain. Alternatively, amino acids in domains that already have human FcRn binding activity in the acidic and/or neutral pH range can be modified to increase human FcRn binding activity. Desired amino acid changes in the human FcRn binding domain can be selected by comparing binding activity to human FcRn over a range of acidic and/or neutral pH values before and after the amino acid change.

The preferred human FcRn binding domain is a region that directly binds to human FcRn. Such preferred FcRn binding regions include, for example, the Fc domains of an antibody. Meanwhile, regions capable of binding to a polypeptide such as albumin or IgG that have human FcRn binding activity can indirectly bind to human FcRn through albumin, IgG or the like. Thus, such a human FcRn-binding region of the present invention may be a region that binds to a polypeptide having human FcRn binding activity.

The antigen binding molecules of the present invention are not particularly limited as long as they include an antigen binding domain having binding activity specific for the target antigen. Such preferred antigen binding domains include, for example, domains having the antigen binding region of an antibody. The antigen binding region of an antibody contains, for example, CDRs and variable regions. If the antigen binding region of an antibody is a CDR, it may contain all six CDRs of the entire antibody, or one, two, or more CDRs. When CDRs are included as an antibody binding region, they may include deletions, substitutions, additions and/or insertions of amino acids, or may be part of the CDR.

On the other hand, antigen-binding molecules that are used in the methods of the present invention contain antigen-binding molecules that have antagonistic activity (antagonistic antigen-binding molecules), antigen-binding molecules that have agonistic activity (agonistic antigen-binding molecule), and molecules that have cytotoxicity. In a preferred embodiment, the antigen binding molecules comprise antagonistic antigen binding molecules, in particular antagonistic antigen binding molecules that recognize an antigen, such as a receptor or cytokine.

In the present invention, the antigen binding molecule of interest is not particularly limited and may be any antigen binding molecule. The antigen binding molecule of the present invention preferably includes both antigen binding activity (antigen binding domain) and a human FcRn binding domain. In particular, a preferred antigen-binding molecule of the present invention relates to a domain that binds to human FcRn. An antigen binding molecule containing both an antigen binding domain and a human FcRn binding domain includes, for example, antibodies. Preferred antibodies in the context of the present invention include, for example, IgG antibodies. If the antibody used is an IgG antibody, the type of IgG is not limited; IgG belonging to any isotype (subclass) such as IgG1, IgG2, IgG3 or IgG4 can be used. In addition, the antigen binding molecules of the present invention may include an antibody constant region, and amino acid mutations may be introduced into the constant region. Amino acid mutations introduced include, for example, those that enable or disrupt binding to the Fcgamma receptor (Proc Natl Acad Sci USA. 2006 Mar 14; 103(11): 4005-10), but are not limited to these examples. Alternatively, it is also possible to change the pH-dependent binding by selecting an appropriate constant region, such as from IgG2.

If the antigen binding molecule of interest of the present invention is an antibody, it may be an antibody obtained from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. In addition, the antibody may be an altered antibody, such as a chimeric antibody, and in particular, an altered antibody including an amino acid substitution in the humanized antibody sequence, etc. Antibodies also include bispecific antibodies, antibody modification products, molecule-linked products, and polypeptides comprising antibody fragments.

"Chimeric antibodies" are antibodies produced by combining sequences obtained from different animals. Specifically, the chimeric antibody includes, for example, antibodies having heavy and light chain variable regions (V) from a mouse antibody and heavy and light chain constant regions (C) from a human antibody.

“Humanized antibodies,” also referred to as reconstituted human antibodies, are antibodies in which the complementarity determining region (CDR) of an antibody obtained from a non-human mammal, such as a mouse, is transplanted into the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). Basic genetic recombination technologies suitable for this purpose are also well known (see European patent application EP 125023; and WO 96/02576).

A bispecific antibody refers to an antibody that has, in the case of some antibody molecules, variable regions that recognize different epitopes. A bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

In addition, polypeptides comprising antibody fragments include, for example, Fab fragments, F(ab') ₂ fragments, scFvs (Nat Biotechnol. 2005 Sep; 23(9): 1126-36), antibody domains (dAbs) (WO 2004 /058821, WO 2003/002609), scFv-Fc (WO 2005/037989), dAb-Fc, and Fc fusion proteins. The Fc domain can be used as a human FcRn binding domain if the molecule includes an Fc domain. Alternatively, the FcRn binding domain can be fused to these molecules.

Additionally, antigen binding molecules that are applicable to the present invention may be antibody-like molecules. Antibody-like molecules (scaffold molecule, peptide molecule) are a molecule that can exert its functions by binding to a target molecule (Current Opinion in Biotechnology (2006) 17: 653-658; Current Opinion in Biotechnology (2007) 18: 1-10 ; Current Opinion in Structural Biology (1997) 7: 463-469; Protein Science (2006) 15: 14-27), and includes, for example, DARPin (WO 2002/020565), affibody (WO 1995/001937), Avimer ( WO 2004/044011; WO 2005/040229) and Adnectin (WO 2002/032925). If these antibody-like molecules can bind to molecules in a pH-dependent manner and/or have binding activity to human FcRn in the neutral pH range, then it is possible to facilitate the uptake of antigen into cells through antigen-binding molecules, helping to reduce the concentration of antigen in plasma through the administration of antigen-binding molecules, and improving the pharmacokinetics of antigen-binding molecules, and increasing the number of antigens to which one antigen-binding molecule can bind.

In addition, the antigen binding molecule may be a protein resulting from a fusion between a human FcRn binding domain and a receptor protein that binds to a target including a ligand and includes, for example, TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc proteins and CTLA4-Fc fusion proteins (Nat Med. 2003, Jan; 9(1): 47-52; BioDrugs. (2006) 20(3): 151-60). If such human receptor-FcRn-binding domain fusion proteins bind in a pH-dependent manner to a target ligand-containing molecule and/or have human FcRn binding activity in the neutral pH range, it is possible to facilitate antigen uptake into cells via antigen-binding molecules, promoting a decrease in concentration antigen in plasma by administering antigen-binding molecules, and improve the pharmacokinetics of antigen-binding molecules, and increase the number of antigens to which one antigen-binding molecule can bind. The receptor protein is suitably designed and modified to include a receptor protein binding domain for a target including a ligand. As set forth in the example above, including TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion proteins and CTLA4-Fc fusion proteins, a soluble receptor molecule containing the extracellular domain of such a receptor protein that is required for binding to such targets, including ligands are advantageously used in the present invention. Such designed and modified receptor molecules are referred to in the present invention as an artificial receptor. The method used to design and modify a receptor molecule to construct an artificial receptor molecule is known in the art.

In addition, the antigen binding molecule may be a fusion protein in which an artificial ligand protein that binds to a target and has a neutralizing effect is fused to a human FcRn binding domain, and the artificial ligand protein includes, for example, a mutant IL-6 ( EMBO J 1994 Dec 15;13(24):5863-70). If such artificial ligand fusion proteins can bind to target molecules in a pH-dependent manner and/or have binding activity to human FcRn in the neutral pH range, it is possible to facilitate the uptake of antigen into cells through antigen-binding molecules, facilitate the reduction of plasma antigen concentration through the administration of antigen-binding molecules and improve the pharmacokinetics of antigen-binding molecules, and increase the number of antigens to which one antigen-binding molecule can bind.

In addition, the antibodies of the present invention may include modified sugar chains. Antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (WO 99/54342), antibodies that are defective in fucose added to the sugar chain (WO 00/61739; WO 02/31140; WO 2006/067847; WO2 006/067913), and antibodies having sugar chains separated by GlcNAc (WO 02/79255).

Conditions used in the assay of antigen binding activity or human FcRn binding activity other than pH may be appropriately selected by those skilled in the art, and the conditions are not particularly limited. For example, conditions using MES buffer at 37 degrees C, as described in WO 2009/125825, can be used to determine activity. In another embodiment, Na-phosphate buffer at 25 degrees C, as described in Examples 4 or 5, can be used to determine activity. Meanwhile, the antigen binding activity and human FcRn binding activity of an antigen binding molecule can be determined by methods known to those skilled in the art, for example, using Biacore (GE Healthcare) or the like. If the antigen is a soluble antigen, the activity of the antigen binding molecule to bind to the soluble antigen can be determined by placing the antigen as an analyte on a chip with an immobilized antigen binding molecule. Alternatively, if the antigen is a membrane-type antigen, the activity of the antigen-binding molecule to bind to the membrane-type antigen can be determined by placing the antigen-binding molecule as an analyte on an antigen-immobilized chip. The human FcRn binding activity of an antigen binding molecule can be determined by placing the human FcRn or antigen binding molecule as an analyte on a chip immobilized with the antigen binding molecule or human FcRn, respectively.

In the present invention, the relationship between the antigen binding activity in the acidic pH range and the antigen binding activity in the neutral pH range is not particularly limited as long as the antigen binding activity in the acidic pH range is lower than that in the neutral pH range. However, the value of KD (pH 5.8)/KD (pH 7.4), which is the ratio of the dissociation constant (KD) with respect to the antigen at pH 5.8 and pH 7.4, is preferably 2 or higher, more preferably 10 or higher, and even more preferably 40 or higher. The upper limit of the value of KD(pH 5.8)/KD(pH 7.4) is not particularly limited, and may be any value, for example, 400, 1000, or 10000, provided that production is possible using techniques known to those skilled in the art. in this area.

If the antigen is a soluble antigen, the antigen binding activity value can be expressed in terms of the dissociation constant (KD). On the other hand, if the antigen is a membrane type antigen, the activity can be expressed in terms of the apparent dissociation constant (apparent KD). Dissociation constant (KD) and apparent dissociation constant (apparent KD) can be determined by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scatchard plot, flow cytometer, or the like.

In the present invention, other parameters that are characteristic of the relationship of antigen binding activity between values in the acidic and neutral pH range include, for example, the dissociation rate constant k _d . If the dissociation rate constant (k _d ) is used instead of the dissociation constant (KD) as a parameter characteristic of the binding activity ratio, the value of k _d (in the acidic pH range)/k _d (in the neutral pH range) which represents the ratio k _d (dissociation rate constant) for the antigen in the range of acidic pH values and neutral pH values is preferably 2 or higher, more preferably 5 or higher, even more preferably 10 or higher and even more preferably 30 or higher. The upper limit of the value of k _d (in the acidic pH range)/k _d (in the neutral pH range) is not particularly limited and may be any value, for example, 50, 100 or 200, provided that production is possible using technologies known to those skilled in the art.

If the antigen is a soluble antigen, the antigen binding activity value can be expressed using the dissociation rate constant (k _d ). Alternatively, if the antigen is a membrane type antigen, the value may be expressed in terms of apparent k _d (apparent dissociation rate constant). The dissociation rate constant (k _d ) and the apparent dissociation rate constant (apparent k _d ) can be determined by methods known to those skilled in the art, for example, using a Biacore (GE Healthcare), a flow cytometer, or the like.

In the present invention, if the antigen binding activity of an antigen binding molecule is determined at different pH values, it is preferable that the measurement conditions are constant except for pH.

Methods for reducing (impairing) the antigen-binding activity of an antigen-binding molecule in the acidic pH range to a value less than the neutral pH range (methods for imparting pH-dependent binding activity) are not particularly limited and can be achieved by any means. Specifically, as described in WO 2009/125825, methods include, for example, methods of reducing (impairing) antigen binding activity in the acidic pH range to a value less than in the neutral pH range by replacing histidine with an amino acid in the antigen binding molecule or by incorporating histidine into an antigen-binding molecule. It is already known that an antibody can be imparted with pH-dependent antigen-binding activity by replacing histidine with an amino acid in the antibody (FEBS Letter (1992) 309(1): 85-88). Such histidine mutation (substitution) or insertion sites are not particularly limited; and histidine can be replaced by an amino acid at any site or inserted into any site. Preferred sites for histidine mutation (substitution) or insertion include, for example, regions where the mutation or insertion has an effect on the antigen binding activity of the antigen binding molecule. Such regions include regions where a mutation or insertion reduces (impairs) antigen binding activity in the acidic pH range to less than that in the neutral pH range (KD (acidic pH range)/KD (neutral pH range) value) increases) compared to the value before the introduction of the mutation or insertion. If the antigen binding molecule is an antibody, such regions include, for example, antibody variable regions and CDRs. The number of histidine mutations or insertions to be introduced (achieved) can be determined accordingly by one skilled in the art. The histidine substitution can be introduced in only one site or in two or more. Alternatively, the histidine may be inserted at only one site or at two or more sites. In addition, mutations other than histidine mutation (mutation (deletion, addition, insertion and/or substitution) with an amino acid other than histidine) can be introduced in addition to the histidine mutation. Alternatively, a histidine mutation can be combined with a histidine insertion. Such a histidine substitution or insertion can be carried out by a probabilistic method such as a histidine scan, which is carried out using histidine instead of alanine in an alanine scan known to those skilled in the art. Further, antigen binding molecules having a higher KD (in the acidic pH range)/KD (in the neutral pH range) than before the mutation was introduced can be selected from a library of antigen binding molecules with random histidine mutations or insertions introduced.

If a histidine is replaced with an amino acid in an antigen-binding molecule or a histidine is inserted into an antigen-binding molecule, the antigen-binding activity of the antigen-binding molecule in the neutral pH range after the histidine substitution or insertion is preferably equivalent to, but not specifically, equivalent to the antigen-binding activity of the antigen-binding molecule in the neutral pH range before the histidine substitution or insertion. limited. As used herein, "the antigen binding activity of an antigen binding molecule in the neutral pH range after a histidine substitution or insertion is equivalent to the antigen binding activity of an antigen binding molecule in the neutral pH range before the histidine substitution or insertion" means that the antigen binding molecule after the histidine substitution or insertion retains 10% or more , preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the antigen binding activity of the antigen binding molecule prior to the histidine substitution or insertion. If the antigen binding activity of an antigen binding molecule is reduced due to a histidine substitution or insertion, the antigen binding activity can be adjusted by substitution, deletion, addition and/or insertion of one or more amino acids into the antigen binding molecule such that the antigen binding activity becomes equivalent to the antigen binding activity before the histidine substitution or insertion. The present invention also includes such antigen binding molecules whose binding activity is made equivalent by substitution, deletion, addition and/or insertion of one or more amino acids into the antigen binding molecule following a histidine substitution or insertion.

Other methods of reducing (impairing) the antigen-binding activity of an antigen-binding molecule in the acidic pH range to less than in the neutral pH range include methods of replacing amino acids with unnatural amino acids in the antigen-binding molecule or inserting unnatural amino acids into the antigen-binding molecule. It is known that pKa can be artificially adjusted using unnatural amino acids (Angew. Chem. Int. Ed. 2005, 44, 34; Chem Soc Rev. 2004 Sep 10, 33 (7): 422-30; Amino Acids. (1999) 16 (3-4): 345-79). Thus, in the present invention, it is possible to use non-natural amino acids instead of the histidine described above. The sites where unnatural amino acids are introduced are not particularly limited, and unnatural amino acids can be replaced or inserted at any site. Preferred sites of unnatural amino acid substitution or insertion include, for example, regions where the substitution or insertion has an effect on the antigen binding activity of the antigen binding molecule. For example, if the antigen binding molecule is an antibody, such regions include antibody variable regions and complementarity determining regions (CDRs). Meanwhile, the amount of non-natural amino acids to be introduced is not particularly limited; and it is possible to replace unnatural amino acids in only one region or two or more regions. Alternatively, unnatural amino acids may be incorporated at only one site or at two or more sites. In addition, other amino acids can be deleted, added, inserted and/or replaced in addition to the substitution or insertion of unnatural amino acids. In addition, unnatural amino acids can be substituted and/or inserted in combination with the histidine substitutions and/or insertions described above. Any non-natural amino acid can be used in the present invention. It is possible to use non-natural amino acids known to those skilled in the art.

In the present invention, if the antigen binding molecule is an antibody, possible substitution sites for histidine or unnatural amino acids include, for example, CDR sequences and sequences responsible for the CDR structure of the antibody, including, for example, regions described in WO 2009/125825. Amino acid positions are indicated according to Kabat numbering (Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, NIH).

The Kabat numbering system is primarily used when referring to residues in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (eg, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service , National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is primarily used when referring to residues in the immunoglobulin heavy chain constant region (eg, EU index published in Kabat et al., supra). "EU index as in Kabat" refers to the EU numbering system for human IgG ₁ antibody residues. Unless otherwise indicated herein, reference to residue numbers in the antibody variable domain refers to the numbering of residues using the Kabat numbering system. Unless otherwise indicated herein, reference to residue numbers in the antibody constant domain refers to residue numbering by the EU numbering system (see, for example, WO 2006073941).

Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b and H102.

Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92 and L94.

Of these alteration sites, H32, H61, L53, L90, and L94 are predicted to be highly abundant alteration sites.

If the antigen is an IL-6 receptor (eg, human IL-6 receptor), preferred sites of change include the following. However, the areas of change are not specifically limited to them.

Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b and H102.

Light chain: L24, L27, L28, L32, L53, L56, L90, L92 and L94.

Specifically, preferred combinations of histidine or unnatural amino acid substitution sites include, for example, the combination of H27, H31 and H35; a combination of H27, H31, H32, H35, H58, H62 and H102; combination of L32 and L53; and a combination of L28, L32 and L53. In addition, preferred combinations of substitution sites in the heavy and light chains include, for example, a combination of H27, H31, L32 and L53.

Of these sites, histidine or unnatural amino acids are present in only one site or in many sites.

Meanwhile, if the antigen binding molecule is an agent having an antibody constant region, methods of reducing (impairing) the antigen binding activity of the antigen binding molecule in the acidic pH range to a value less than in the neutral pH range include, for example, methods of changing amino acids in the constant region antibodies. Specifically, such methods include, for example, the constant region replacement methods described in WO 2009/125825 (SEQ ID NOs: 11, 12, 13 and 14). Meanwhile, methods for changing the constant region of an antibody include, for example, methods for evaluating the constant regions of different isotypes (IgG1, IgG2, IgG3 and IgG4) and selecting isotypes that reduce antigen binding activity in the acidic pH range (increase the dissociation rate in the acidic pH range) . Such methods also include methods of reducing antigen-binding activity in the acidic pH range (increasing the dissociation rate in the acidic pH range) by introducing amino acid substitutions into wild-type isotype amino acid sequences (wild-type IgG1, IgG2, IgG3, or IgG4 amino acid sequences). The hinge region sequence in the antibody constant region varies significantly among isotypes (IgG1, IgG2, IgG3 and IgG4), and differences in the amino acid sequence of the hinge region have a major impact on antigen-binding activity. Thus, it is possible to select a suitable isotype to reduce antigen-binding activity in the acidic pH range (increasing dissociation rate in the acidic pH range) depending on the type of antigen or epitope. In addition, since differences in the amino acid sequence of the hinge region have a large impact on antigen-binding activity, preferred sites of amino acid substitutions in the amino acid sequences of wild-type isotypes are allowed within the hinge region.

If the antigen-binding activity of an antigen-binding molecule in the acidic pH range is reduced (decreased) to a value less than that in the neutral pH range (when the KD (acidic pH range)/KD (neutral pH range) is increased) by the above-described methods etc., it is generally important that the KD (in the acidic pH range)/KD (in the neutral pH range) value is two times or higher, preferably five times or higher, and more preferably ten times or greater than the original antibody, but is not particularly limited to these values.

The methods described above can be used to produce antigen-binding molecules whose antigen-binding activity in the acidic pH range is reduced (reduced) to less than in the neutral pH range (antigen-binding molecules that bind in a pH-dependent manner) by amino acid substitution or insertion from antigen-binding molecules that do not have such properties. Other methods include methods for directly producing antigen-binding molecules having the properties described above. For example, antibodies having a desired property of interest can be directly selected by screening using pH-dependent antigen binding as an indicator from antibodies obtained by immunizing animals (mice, rats, hamsters, rabbits, human immunoglobulin transgenic mice, transgenic rats) with the antigen with human immunoglobulin, transgenic rabbits with human immunoglobulin, llamas, camels, etc.). Antibodies can be obtained through hybridoma technology or B-cell cloning technology (Proc Natl Acad Sci USA. 1996 Jul 23; 93(15): 7843-8; J Immunol Methods. 2006 Oct 20; 316(1-2): 133-43 ; Journal of the Association for Laboratory Automation; WO 2004/106377; WO 2008/045140; and WO 2009/113742), which are methods known to those skilled in the art, but are not limited to these methods. Alternatively, antibodies that have properties of interest can be directly selected by screening using pH-dependent antigen binding as an indicator from a library providing the antigen binding domain in vitro. Such a library includes a natural human library, a library of immunized non-human animals and humans, a semi-synthetic library and a synthetic library, which are libraries well known to those skilled in the art (Methods Mol Biol. 2002; 178: 87-100; J Immunol Methods. 2004 Jun;289(1-2):65-80; and Expert Opin Biol Ther. 2007 May;7(5):763-79), but are not limited to them. However, the methods are not particularly limited to these examples.

The present invention exploits the difference in pH as the difference in environmental conditions between the plasma and the endosome in relation to the different binding affinities of the antigen binding molecule to the antigen in the plasma and the endosome (strong binding in the plasma and weak binding in the endosome). Since differences in environmental conditions between plasma and endosome are not limited to differences in pH, the pH-dependent binding property of an antigen-binding molecule to antigen can be replaced by using other factors whose concentration is different within the plasma and endosome. Such a factor can also be used to produce an antibody that binds to an antigen within the plasma but dissociates from the antigen within the endosome. Thus, the present invention also includes an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has binding activity to human FcRn in the acidic and neutral pH range, and lower antigen binding activity in the endosome than in plasma, where binding activity with human FcRn in plasma is higher than the binding activity of intact human IgG.

Methods for increasing the human FcRn binding activity of the human FcRn binding domain in the antigen binding molecule of the present invention in the neutral pH range are not particularly limited, and the binding activity can be increased by any methods. Specifically, if the Fc domain of an IgG immunoglobulin is used as a human FcRn binding domain, the binding activity of human FcRn in the neutral pH range can be increased by changing its amino acids. Such preferred IgG immunoglobulin Fc domain to be modified includes, for example, the Fc domain of parental human IgG (IgG1, IgG2, IgG3 or IgG4 and engineered variants thereof). Amino acids at any sites can be replaced by other amino acids, provided they impart or increase human FcRn binding activity in the neutral pH range. If the antigen binding IgG1 molecule has a human Fc domain as the human FcRn binding domain, it is preferred that the molecule has substitutions that allow binding to human FcRn in a neutral pH range compared to the parent human IgG1. Amino acids for which such changes can be made include, for example, amino acids at positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, from 291 to 312, from 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362, 370, from 375 to 378, 380, 382, from 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440 and 442 (EU numbering). More specifically, such amino acid changes include, for example, those listed in Table 1. Binding of the Fc domain of an IgG immunoglobulin to human FcRn in the neutral pH range can be made possible by using the changes described above.

In addition, changes that allow binding to human FcRn in the acidic pH range, compared to parental human IgG, are presented as an example in Table 2. If suitable changes that also allow binding to human FcRn in the neutral pH range , selected from the changes listed above, they can be used in the present invention. Meanwhile, combinations of changes that allow Fv4-IgG1 to bind to human FcRn under acidic conditions are presented in Tables 6-1 and 6-2. Particularly preferred amino acids to be changed in the human Fc domain of the parent IgG include, for example, amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289 , 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 4 24 , 428, 433, 434 and 436 (EU numbering). The binding activity of an antigen binding molecule to human FcRn can be increased in the neutral pH range by replacing various amino acids with at least one amino acid selected from the amino acids listed above.

Particularly preferred changes include, for example

replacement of the amino acid Met with Gly at position 237;

replacement of amino acid Ala with Pro at position 238;

replacement of amino acid Lys with Ser at position 239;

replacement of the amino acid Ile with Lys at position 248;

replacing the amino acid Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr with Thr at position 250;

replacing the amino acid Phe, Trp or Tyr with Met at position 252;

replacing the amino acid Thr with Ser at position 254;

replacement of the amino acid Glu with Arg at position 255;

replacement of amino acids Asp, Glu or Gln with Thr at position 256;

replacing the amino acids Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val with Pro at position 257;

replacement of the amino acid His with Glu at position 258;

replacement of the amino acid Ala with Asp at position 265;

replacing the amino acid Phe with Asp at position 270;

replacement of amino acids Ala or Glu with Asn at position 286;

replacement of the amino acid His with Thr at position 289;

replacement of the amino acid Ala with Asn at position 297;

replacement of the amino acid Gly with Ser at position 298;

replacing the amino acid Ala with Val at position 303;

replacement of amino acids Ala with Val at position 305;

replacing the amino acids Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr with Thr at position 307;

replacing the amino acids Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr with Val at position 308;

replacing the amino acids Ala, Asp, Glu, Pro or Arg with Leu or Val at position 309;

replacement of amino acids Ala, His or Ile with Gln at position 311;

replacement of amino acids Ala or His with Asp at position 312;

replacement of amino acids Lys or Arg with Leu at position 314;

replacing the amino acid Ala or His with Asn at position 315;

replacement of amino acid Ala with Lys at position 317;

replacement of the amino acid Gly with Asn at position 325;

replacing the amino acid Val with Ile at position 332;

replacement of the amino acid Leu with Lys at position 334;

replacement of the amino acid His with Lys at position 360;

replacement of the amino acid Ala with Asp at position 376;

replacing the amino acid Ala with Glu at position 380;

replacement of the amino acid Ala with Glu at position 382;

replacing the amino acid Ala with Asn or Ser at position 384;

replacement of amino acids Asp or His with Gly at position 385;

replacement of amino acid Pro with Gln at position 386;

replacement of the amino acid Glu with Pro at position 387;

replacement of amino acids Ala or Ser with Asn at position 389;

replacement of the amino acid Ala with Ser at position 424;

replacing the amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr with Met at position 428;

replacement of the amino acid Lys with His at position 433;

replacement of amino acids Ala, Phe, His, Ser, Trp or Tyr with Asn at position 434;

and replacement of the amino acids His or Phe with Tyr at position 436 (EU numbering) in the Fc domain of the parental IgG. Meanwhile, the number of amino acids to be changed is not particularly limited; and it is possible to change amino acids in only one region or two or more regions. Combinations of two or more amino acid changes include, for example, those presented in Table 3. Meanwhile, combinations of changes that allow binding to human FcRn in the acidic pH range, compared to parental human IgG, are presented in Tables 4-1 up to 4-5. If suitable combinations of changes that can enable binding to human FcRn in the neutral pH range are selected from the changes described above, they can be used in the present invention. In addition, combinations of changes that allow Fv4-IgG1 to bind to human FcRn under neutral conditions are presented in Tables 6-1 and 6-2.

The symbol "^" in the tables indicates the insertion of an amino acid after the indicated number in the EU numbering. For example, ^{^} 281S indicates that S is inserted between positions 281 and 282 in EU numbering.

Table 1 POSITION CHANGE IN AMINO ACID 256 P 280 K 339 T 385 H 428 L 434 W, Y, F, A, H

Table 4-2 is a continuation of Table 4-1.

Table 4-3 is a continuation of Table 4-2.

Table 4-4 is a continuation of Table 4-3.

Table 4-5 is a continuation of Table 4-4.

Such amino acid changes can suitably be introduced using known methods. For example, changes in the Fc domain of intact human IgG1 are described in Drug Metab Dispos. 2007 Jan 35(1): 86-94; Int Immunol. 2006 Dec 18, (12): 1759-69; J Biol Chem. 2001 Mar. 2, 276(9): 6591-604; J Biol Chem. (2007) 282(3): 1709-17; J Immunol. (2002) 169(9): 5171-80; J Immunol. (2009) 182(12): 7663-71; Molecular Cell, Vol. 7, 867-877, April, 2001; Nat Biotechnol. 1997 July 15, (7): 637-40; Nat Biotechnol. 2005 Oct. 23, (10): 1283-8; Proc Natl Acad Sci USA. 2006 Dec. 5, 103(49): 18709-14; EP 2154157; US 20070141052; WO 2000/042072; WO 2002/060919; WO 2006/020114; WO 2006/031370; WO 2010/033279; WO 2006/053301; and WO 2009/086320.

According to the Journal of Immunology (2009) 182: 7663-7671, the human FcRn binding activity of intact human IgG1 in the acidic pH range (pH 6.0) has a KD of 1.7 micromoles (µM), while in the acid pH range At neutral pH values, activity is almost undetectable. Thus, in a preferred embodiment, the antigen binding molecules to be used in the methods of the present invention are those whose binding activity to human FcRn in the acidic pH range has a KD of 20 micromolar or greater, and is identical or greater in the acidic pH range. neutral pH values than that of intact human IgG. In a more preferred embodiment, the antigen binding molecule includes antigen binding molecules whose binding activity to human FcRn has a KD value of 2.0 micromolar or greater in the acidic pH range and a KD of 40 micromolar or greater in the neutral pH range. In an even more preferred embodiment, the antigen binding molecule includes antigen binding molecules whose binding activity to human FcRn has a KD value of 0.5 micromolar or greater in the acidic pH range and a KD of 15 micromolar or greater in the neutral pH range. The KDs described above are determined in the manner described in Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen-binding molecule on a chip and applying human FcRn as the analyte).

The dissociation constant (KD) can be used as a value for binding activity to human FcRn. However, the human FcRn binding activity of intact human IgG has little human FcRn binding activity in the neutral pH range (pH 7.4), and thus it is difficult to calculate the activity as a KD. Methods for assessing whether human FcRn binding activity is greater than that of intact human IgG at pH 7.4 include methods for assessing by comparing the intensity of the response in Biacore after application of the same concentration of analyte. Specifically, if the response following application of a human FcRn chip immobilized with an antigen-binding molecule at pH 7.4 is greater than the response following application of human FcRn to a chip immobilized with intact human IgG at pH 7.4, human FcRn binding activity of antigen-binding molecules is estimated to be higher than that of intact human IgG at pH 7.4.

pH 7.0 can also be used as a pH neutral range. Using pH 7.0 as a neutral pH may promote weak interaction between human FcRn and the FcRn-binding domain. Since temperature is used in the assay conditions, binding affinity can be assessed at any temperature from 10 degrees C to 50 degrees C. Preferably, a temperature from 15 degrees C to 40 degrees C is used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably, any temperature between 20 degrees C and 35 degrees C, as well as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, is also used to determine the binding affinity between the human FcRn binding domain and human FcRn , 29, 30, 31, 32, 33, 34 and 35 degrees C. The temperature of 25 degrees C described in Example 5 is one example of an embodiment of the present invention. In a preferred embodiment, the interaction between human FcRn and the FcRn binding domain can be measured at pH 7.0 and 25 degrees C as described in Example 5. The binding affinity of the antigen binding molecule to human FcRn can be measured by Biacore as described in Example 5.

In a more preferred embodiment, the antigen binding molecules of the present invention have a human FcRn binding activity at pH 7.0 and 25 degrees C that is higher than that of intact human IgG. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 degrees C is 28 times greater than that of intact human IgG, or greater than the KD of 3.2 micromoles. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 degrees C is 38 times greater than that of intact human IgG, or greater than the KD of 2.3 micromoles.

Intact human IgG1, IgG2, IgG3 or IgG4 is preferably used as intact human IgG for the purpose of control intact human IgG to be compared with antigen binding molecules for their human FcRn binding activity or in vivo activity. Preferably, a control antigen binding molecule comprising the same antigen binding domain as the antigen binding molecule of interest and an intact human IgG Fc domain as the human FcRn binding domain may be suitably used. More preferably, intact human IgG1 is used to control intact human IgG to be compared with antigen binding molecules for their human FcRn binding activity or in vivo activity.

More specifically, the antigen-binding molecules with long-lasting plasma antigen elimination activity described in the present invention have binding activity to human FcRn at pH 7.0 and at 25 degrees C within a range of 28-fold to 440-fold greater than for intact human IgG1, or KD within the range of 3.0 to 0.2 micromoles. Long-term plasma antigen concentration is determined by measuring the plasma concentration of total or free antigen and the antigen/antigen-binding molecule molar ratio at 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to assess the effect of the long-term effect of the antigen-binding molecule on the present invention on antigen elimination activity in plasma. Whether a reduction in plasma antigen concentration or antigen/antigen binding molecule molar ratio is achieved by the antigen binding molecule described in the present invention can be determined by assessing the reduction at any one or more of the time points described above.

Even more specifically, the antigen-binding molecules with a short-term effect on plasma antigen elimination activity described in the present invention have binding activity to human FcRn at pH 7.0 and at 25 degrees C that is 440 times greater than that of intact human IgG , or KD, greater than 0.2 micromoles. Short-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio at 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration of the antigen-binding molecule to assess the short-term effect of the antigen-binding molecule of the present invention on antigen elimination activity in plasma.

The methods of the present invention are applicable to any antigen-binding molecule, regardless of the type of antigen being targeted.

Antigens that are recognized by antigen-binding molecules, such as antibodies of interest, are not particularly limited in the methods of the present invention. Such antibodies of interest can recognize any antigen. Antibodies whose pharmacokinetics are enhanced by the methods of the present invention include, for example, receptor proteins (membrane-bound receptors and soluble receptors), antibodies that recognize membrane antigen, such as cell surface markers, and antibodies that recognize soluble antigen, such as cytokines. Specific examples of an antigen that is recognized by an antibody whose pharmacokinetics are improved by the methods of the present invention include, for example: 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adressin, adiponectin, ADP-ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin, alpha1-antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, amine, amylin, amyloid beta, amyloid immunoglobulin variable heavy chain, amyloid immunoglobulin variable light chain, Androgen, ANG, angiotensinogen, angiopoietin-2 ligand, anti -Id, antithrombin III, anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A Apo-SAA, APP, APRIL, AR, ARC, ART, artemin, aspartic acid, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptides A, atrial natriuretic peptides B, atrial natriuretic peptides C, integrin av/b3, Ax1, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF -R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK , B-lymphocyte stimulator (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP -7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMP, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecules, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a ), CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cardiotrophin-1, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1/I-309, CCLll/eotaxin, CCL12/MCP-5, CCL13/ MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL20/MIP-3-alpha, CCL21/ SLC, CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-alpha, CCL3L1/LD-78- beta, CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/10/MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95 , CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN -1, complement factor 3 (C3), complement factor D, corticosteroid binding globulin, colony stimulating factor receptor-1, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 /fractalkine, CX3CR1, CXCL, CXCLl/Gro-alpha, CXCL10, CXCL11/I-TAC, CXCL12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine, CXCL16, CXCL16, CXCL2 /Gro-beta CXCL3/Gro-gamma, CXCL3, CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCLIO/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, tumor-associated antigen cytokeratin, DAN, DCC, DcR3, DC-SIGN, complement-dependent stimulator of hemolysis, delta-like ligand protein 4, des(1-3)-IGF -1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidase IV, DK1, DNAM-1, DNase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA- A2, EDAR, EGF, EGFR (ErbB-1), EGF domain-like protein 7, Elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, endosialin, endothelin receptor, endotoxin, enkephalinase, eNOS, Eot, Eotaxin, Eotaxin -2, eotaxins, EpCAM, ephrin B2/EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus -2, F protein RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, Factor VIII, Factor VIIIc, Fas, FalphaR, FcepsilonRI, FcgammaIIb, FcgammaRI, FcgammaRIIa, FcgammaRIIIa , FcgammaRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, acidic FGF, basic FGF, FGFR, FGFR-3, fibrin, activation protein fibroblast growth factor (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14/CDMP-1), GDF-6 (BMP-13/CDMP-2), GDF-7 (BMP- 12/CDMP-3), GDF-8 (myostatin), GDF-9, GDNF, gelsolin, GFAP, GF-CSF, GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-beta1, gH envelope glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide receptor 1, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptors, glycoprotein IIb/IIIa (GP IIb/IIIa), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, growth hormone releasing factor, GRO-beta, GRO-gamma, H. pylori, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1 , HCMV gB envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, liver growth factor, Bacillus anthracis protective antigen, hepatitis C virus glycoprotein E2, hepatitis E, hepcidin, Her1, Her2/ neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope proteins, such as GP120, MIB gp 120 with HIV V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV) , human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta , IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptors , IL-11, IL-11 receptors, IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL-16, IL-16 receptors, IL-17, receptors IL-17, IL-18 (IGIF), receptors IL-18, IL-1alpha, IL-1beta, receptors IL-1, IL-2, receptors IL-2, IL-20, receptors IL-20, IL- 21, IL-21 receptors, IL-23, IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3 receptors, IL-4, IL receptors -4, IL-5, IL-5 receptors, IL-6, IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9 receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha, INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gamma receptors, IFN type I, IFN type I receptor, influenza, inhibin, inhibin alpha, inhibin beta, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding proteins, integrin, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha- V/beta-3, integrin alpha-V/beta-6, integrin alpha4/alpha7, integrin alpha5/alpha1, integrin alpha5/beta3, integrin alpha5/beta6, integrin alpha-delta (alphaV), integrin alpha-theta, integrin beta1 , integrin beta2, integrin beta3(GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2 , kallikrein L3, kallikrein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor-2 (KGF-2), KGF, immunoglobulin-like killer cell receptor, kit ligand (KL), tyrosine kinase kit, laminin 5, LAMP, LAPP (amylin islet amyloid polypeptide), LAP (TGF-1), latent associated peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (LH), Lewis-Y antigen, Lewis-Y associated antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a , LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, lymphotactin, lymphotoxin beta receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.k.), MDC ( 69 a.k.), megsin, Mer, MET tyrosine kinase family receptor, metalloproteases, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP -15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein, monocyte colony inhibitory factor, murine gonadotropin-associated peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, Mucin (Mud), Müllerian inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid precursor inhibitory factor-1 (MPIF-1), NAIP, nanobody, NAP, NAP-2, NCA 90, NCAD , N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, neuroserpin, neuronal growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, neuroturin, NGF-beta, NGFR, NKG20, N- methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, nonstructural protein type 3 (NS3) from hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductive factors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P -Cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PEC AM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2 , PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet-derived growth factor, plgR, PLP, polyglycol chains of various sizes (e.g. PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, programmed cell death protein 1, proinsulin, prolactin, PC9 proprotein convertase, prorelaxin, prostate specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z , PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, glycoprotein ligand of P-selectin 1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A-chain, relaxin B-chain, renin, respiratory- syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, sclerostin, SDF-1, SDF1 alpha, SDF1 beta, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine-1-phosphate receptor 1, Staphylococcal Lipoteichoic Acid, Stat, STEAP, STEAP-II, Stem Cell Factor (SCF), Streptokinase, Superoxide Dismutase, Syndecan-1, TACE, TACI, TAG-72 (Tumor Associated Glycoprotein 72), TARC, TB, TCA- 3, alpha/beta T cell receptor, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, pan-specific TGF-beta , TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta R1 (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid-stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, tissue factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2/DR4), TNFRSF10B (TRAIL R2 DR5/KILLER /TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3 DcR1/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODF R/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 ( TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR/HveA/LIGHT R/TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 ( TROY TAJ/TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 /LARD/TR-3/TRAMP/WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII/TNFC R), TNFRSF4 (OX40 ACT35/TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1 /APT1/CD95), TNFRSF6B (DcR3 M68/TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 ligand/ TL2), TNFSF11 (TRANCE/RANK ligand ODF/OPG ligand), TNFSF12 (TWEAK Apo-3 ligand/DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEM ligand /LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand/TL6), TNFSF1A (TNF-a connectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3 (LTb TNFC/p33) , TNFSF4 (OX40 ligand gp34/TXGP1), TNFSF5 (CD40 ligand CD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas ligand Apo-1 ligand/APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153 ), TNFSF9 (4-1 BB ligand CD137 ligand), TNF-alpha, TNF-beta, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2 , TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF) , tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD , VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VitB12 receptor, receptor vitronectin, VLA, VLA-1, VLA-4, integrin VNR, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1beta, XCL1/lymphotactin, XCR1, XEDAR, XIAP and XPD.

The antigen-binding molecules described in the present invention are capable of reducing the total concentration of the above antigens in plasma. The antigen-binding molecules described in the present invention are also capable of eliminating virus, bacteria and fungi from plasma by binding to structural components of the virus, bacteria and fungi. Specifically, RSV F protein, Staphylococcus lipoteichoic acid, Clostridium difficile toxin, Shiga-like toxin II, Bacillus anthracis protective antigen and Hepatitis C virus E2 glycoprotein can be used as structural components of virus, bacteria and fungi.

Although the methods of the present invention are not limited to any particular theory, the relationship between a decrease (deterioration) in the antigen-binding ability of an antigen-binding molecule in the acidic pH range to a value less than in the neutral pH range and/or an increase (increase) in binding activity to Human FcRn in the neutral pH range and an increase in the number of antigens to which one antigen-binding molecule can bind, due to easier uptake of antigen-binding molecules into cells, and increased elimination of antigen from plasma can be explained as follows.

For example, if the antigen-binding molecule is an antibody that binds to a membrane antigen, the antibody introduced into the body bound to the antigen is then taken up by internalization within endosomes into cells along with the antigen as the antibody remains bound to the antigen. The antibody then moves to the lysosomes as the antibody is bound to the antigen, and the antibody is degraded by the lysosome along with the antigen. Internalization-mediated elimination from plasma is called antigen-dependent elimination, and this type of elimination has already been described for a number of antibody molecules (Drug Discov Today. 2006 Jan; 11(1-2): 81-8). If one IgG antibody molecule binds to antigens in a bivalent manner, one antibody molecule is internalized because the antibody is bound to two antigen molecules and is degraded in the lysosome. Thus, in the case of typical antibodies, one IgG antibody molecule cannot bind three or more antigen molecules. For example, one IgG antibody molecule that has neutralizing activity cannot neutralize three or more antigen molecules.

The relatively longer retention time (slow elimination) of IgG molecules in plasma is a consequence of the function of human FcRn, which is known as the salvage receptor for IgG molecules. When taken up into endosomes via pinocytosis, IgG molecules bind to human FcRn expressed in endosomes under the acidic conditions of the endosome. Because IgG molecules that do not bind to human FcRn move to lysosomes where they are degraded, IgG molecules that bind to human FcRn move to the cell surface and return to the plasma through dissociation from human FcRn under neutral conditions in the plasma.

Alternatively, if the antigen-binding molecule is an antibody that binds to a soluble antigen, the administered antibody binds to the antigen and is then taken up by cells because the antibody is bound to the antigen. Many antibodies taken up by cells are released outside the cell via FcRn. However, as antibodies are released outside the cells, antigens remain bound to the antibodies, and the antibodies cannot bind to the antigens again. Thus, like antibodies that bind to membrane antigens, in the case of typical antibodies, one IgG antibody molecule cannot bind to three or more antigen molecules.

pH-dependent antigen-binding antibodies that are strongly bound to antigen under neutral conditions in the plasma but dissociate from the antigen under acidic conditions in the endosome (antibodies that bind under neutral conditions but dissociate under acidic conditions) can dissociate from the antigen in the endosome. Such pH-dependent antigen-binding antibodies can bind to antigens again if they are recycled into the plasma via FcRn after antigen dissociation; thus, each antibody can rebind to a number of antigens. In addition, the antigen bound to the antigen-binding molecule dissociates in the endosome and does not return to the plasma. This facilitates antigen binding molecule-mediated uptake of antigen into cells. Thus, administration of an antigen-binding molecule can enhance antigen elimination and thereby reduce the concentration of antigens in plasma.

Antigen binding molecule-mediated cellular uptake of antigen can be further enhanced by providing human FcRn binding activity under neutral conditions (pH 7.4) to an antibody that binds antigen in a pH-dependent manner (binds under neutral conditions but dissociates under acidic conditions). Thus, administration of an antigen-binding molecule can enhance the elimination of antigen and thereby reduce the concentration of antigen in plasma. Typically, both the antibody and the antigen-antibody complex are taken up by cells through nonspecific endocytosis and then transported to the cell surface through binding to FcRn under acidic conditions in the endosome. The antibody and antigen-antibody complex are recycled into the plasma by dissociation from FcRn under neutral conditions to the cell surface. Thus, if an antibody that exhibits proper pH dependence in antigen binding (binds under neutral conditions but dissociates under acidic conditions) binds to an antigen in the plasma and then dissociates from the bound antigen in the endosome, the rate of antigen elimination is estimated to be equal to the rate of uptake antigen by cells through nonspecific endocytosis. On the other hand, if the pH dependence is inappropriate, antigen that does not dissociate in the endosome is also recycled into the plasma. Meanwhile, if the pH dependence is pH proper, the rate-determining step in antigen elimination is cellular uptake through nonspecific endocytosis. Some of the FcRns are considered cell surface localized because FcRns transport antibodies from the endosome to the cell surface.

The present inventors have proposed that immunoglobulins of the IgG type, which are one of the antigen-binding molecules, generally have little FcRn-binding activity in the neutral pH range, but those that exhibit FcRn-binding activity in the neutral pH range can bind to FcRn on the cell surface and thus be taken up by cells in an FcRn-dependent manner through the binding of FcRn presented on the cell surface. The rate of FcRn-mediated cellular uptake is higher than the rate of cellular uptake through nonspecific endocytosis. Thus, the rate of antigen elimination can be further increased by ensuring the presence of FcRn binding activity in the neutral pH range. Specifically, an antigen-binding molecule with FcRn-binding activity in the neutral pH range moves antigen into cells more quickly than typical IgG (intact human) immunoglobulin, and then the antigen-binding molecule dissociates from the antigen in the endosome. The antigen-binding molecule is recycled to the cell surface or plasma and again binds to another antigen or is taken up by cells via FcRn. The rate of this cyclic process can be increased by increasing FcRn binding activity in the neutral pH range, thereby increasing the rate of antigen elimination from plasma. In addition, the effectiveness can be further increased by reducing the antigen binding activity of the antigen binding molecule in the acidic pH range to a value less than the neutral pH range. In addition, the number of antigens to which one antigen-binding molecule can bind increases as the number of cycles completed by one antigen-binding molecule increases. The antigen binding molecule of the present invention contains an antigen binding domain and an FcRn binding domain. Since the FcRn binding domain has no effect on antigen binding, or in terms of the mechanism described above, an increase in antigen binding molecule-mediated cellular uptake of antigen can be expected regardless of the type of antigen, and as a result, an increase in the rate of antigen elimination by reducing the antigen binding activity of antigen binding molecules in the acidic pH range (binding activity) to a value less than the neutral pH range, and/or increase its binding activity to FcRn at plasma pH.

<Agents that serve as an antigen-binding molecule>

In addition, the present invention relates to antigen binding molecules that have binding activity to human FcRn in the acidic and neutral pH ranges and those whose antigen binding activity in the acidic pH range is lower than in the neutral pH range. Specific examples of antigen-binding molecules include those that have binding activity to human FcRn at pH 5.8 to pH 6.0 and pH 7.4, which are believed to be the in vivo pH values of early endosome and plasma, respectively, and those whose antigen-binding activity is lower at pH 5.8 than at pH 7.4. An antigen binding molecule whose antigen binding activity is lower at pH 5.8 than at pH 7.4 may also be referred to as an antigen binding molecule whose antigen binding activity is stronger at pH 7.4 than at pH 5.8.

The antigen binding molecules of the present invention having binding activity to human FcRn in the acidic and neutral pH range are preferably antigen binding molecules that also have binding activity to human FcRn in the acidic pH range and stronger binding activity to human FcRn than for intact human IgG, in the neutral pH range. The ratio of binding activity is not limited, provided that their binding activity to human FcRn is even slightly stronger at pH 7.4.

According to Journal of Immunology (2009) 182: 7663-7671, the binding activity to human FcRn and intact human IgG1 has a KD of 1.7 micromolar in the acidic pH range (pH 6.0), while the activity is almost undetectable in the neutral range. pH. Thus, in a preferred embodiment, antigen binding molecules of the present invention with human FcRn binding activity in the acidic and neutral pH range include antigen binding molecules that have human FcRn binding activity with a KD of 20 micromoles or higher in the acidic pH range , which is equal to or stronger than that in the neutral pH range of intact human IgG. In a more preferred embodiment, the antigen binding molecules of the present invention include antigen binding molecules whose binding activity to human FcRn has a KD of 2.0 micromolar or greater in the acidic pH range and a KD of 40 micromolar or greater in the neutral pH range. pH values. In an even more preferred embodiment, the antigen binding molecules of the present invention include antigen binding molecules whose binding activity to human FcRn is a KD of 0.5 micromolar or greater in the acidic pH range and a KD of 15 micromolar or greater in range of neutral pH values. The above KD values are determined in the manner described in Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen-binding molecule on a chip and applying human FcRn as an analyte).

The present invention relates to an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen binding molecule has binding activity to human FcRn in the acidic and neutral pH range, in which human FcRn and lower antigen binding activity in the acidic pH range than in the neutral pH range, is a higher value than the KD of 3.2 micromoles. The present invention also provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is a value 28 times greater higher than that for intact human IgG. The antigen-binding molecules of the present invention have binding activity to human FcRn at pH 7.0 and 25 degrees C, which is higher than that of intact human IgG. In a more preferred embodiment, the binding activity to human FcRn at pH 7.0 and 25 degrees C is a value 28 times higher than for intact human IgG, or with a KD of greater than 3.2 micromoles.

The present invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen binding molecule has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is a KD of greater than 2.3 micromoles. The present invention also provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen binding molecule has binding activity to human FcRn in the neutral pH range, wherein binding activity to human FcRn in the neutral pH range is 38 times higher than that for intact human IgG.

As used herein, the acidic pH range generally refers to a pH value between pH 4.0 and pH 6.5. The acidic pH range is preferably the range indicated by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5, more preferably pH 5.8 to pH 6.0, which are values close to the pH value in the early endosome in vivo. Meanwhile, herein, the neutral pH range generally refers to pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value within pH 7.0 to pH 8.0, preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4 , 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, more preferably pH 7.4, which is a value close to the pH value in plasma (blood) in vivo. A pH value of 7.0 may be used as an alternative to a pH value of 7.4 if it is difficult to determine the binding affinity between the human FcRn binding domain and human FcRn due to the latter's low binding affinity at pH 7.4. Since temperature is used in the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature between 10 degrees C and 50 degrees C. Preferably, a temperature between 15 degrees C and 40 degrees C is used to determine the binding affinity between human FcRn-binding domain and human FcRn. More preferably, any temperature between 20 degrees C and 35 degrees C, as well as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 degrees C is also used to determine the binding affinity between the human FcRn binding domain and human FcRn. The temperature of 25 degrees C described in Example 5 is one example for an embodiment of the present invention.

In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 degrees C is 38 times greater than that of intact human IgG or greater than the KD of 2.3 micromoles. Intact human IgG1, IgG2, IgG3 or IgG4 is used as intact human IgG for the purpose of control intact human IgG compared with antigen binding molecules for their binding activity to human FcRn. More preferably, intact human IgG1 is used to control intact human IgG against antigen binding molecules for their binding activity to human FcRn.

The present invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the total plasma antigen concentration after administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration after administration of a control antigen binding molecule to a non-human animal. animal.

The present invention also provides an antigen-binding molecule for which the plasma antigen concentration after administration of the antigen-binding molecule to a non-human animal is lower than the total plasma antigen concentration obtained for a non-human animal that has not been administered the antigen-binding molecule.

The total antigen concentration in plasma can be reduced by administering the antigen binding molecule of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, and 1000-fold or even more compared to administration of a control antigen-binding molecule. molecule containing the Fc domain of an intact human IgG as the human FcRn binding domain, or compared with the case where the antigen binding domain molecule of the present invention is not introduced.

In another embodiment, the present invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, for which the antigen/antigen binding molecule molar ratio (C) of the antigen binding molecule is calculated by the following method:

C=A/B,

is lower than the antigen/antigen binding molecule (C') molar ratio of an antigen binding molecule containing the same antigen binding domain and an intact human IgG Fc domain as the human FcRn binding domain, calculated by the formula:

C'=A'/B',

Where:

A is the total concentration of antigen in plasma after administration of the antigen-binding molecule to a non-human animal,

B is the plasma concentration of the antigen binding molecule following administration of the antigen binding molecule to a non-human animal,

A' is the total antigen concentration in plasma following administration of a control antigen-binding molecule to a non-human animal,

B' is the plasma concentration of antigen-binding molecule following administration of a control antigen-binding molecule to a non-human animal.

The molar ratio of antigen/antigen binding molecule can be reduced by administering the antigen binding molecule of the present invention by 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times and 1000 times or even more compared with the administration of the antigen binding molecule a molecule containing an intact human IgG Fc domain as a human FcRn binding domain.

The reduction in total plasma antigen concentration or antigen/antibody molar ratio can be assessed as described in Examples 6, 8 and 13. More specifically, using transgenic line 32 or line 276 of human FcRn mice (Jackson Laboratories, Methods Mol Biol. (2010) 602: 93-104), they can be assessed either through an antigen-antibody co-injection model or a steady-state antigen infusion model if the antigen-binding molecule does not cross-react with the corresponding mouse antigen. If the antigen binding molecule cross-reacts with the corresponding mouse antigen, they can be assessed by simply injecting the antigen binding molecule into human FcRn 32 or 276 transgenic mice (Jackson Laboratories). In the co-injection model, a mixture of antigen-binding molecule and antigen is injected into mice. In the steady-state antigen infusion model, an infusion pump containing an antigen solution is implanted into mice to achieve a constant plasma antigen concentration, and then the mice are injected with an antigen-binding molecule. The antigen-binding molecule to be tested is administered at the same dose. Total plasma antigen concentration, plasma free antigen concentration, and plasma antigen-binding molecule concentration are measured at the appropriate time point using methods known to those skilled in the art.

The route of administration of the antigen binding molecule of the present invention can be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral and intramuscular injection.

More specifically, the antigen-binding molecules with a long-term effect on plasma antigen elimination activity described in the present invention have binding activity to human FcRn at pH 7.0 and 25 degrees C within a range of 28 times to 440 times higher than for intact human IgG1, or with a KD within the range of 3.0 micromolar to 0.2 micromolar. Short-term plasma antigen concentration is determined by measuring the total antigen concentration or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio at 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to assess the long-term effect of the antigen-binding molecule of the present invention on antigen elimination activity in plasma. Whether a reduction in plasma antigen concentration or antigen/antigen binding molecule molar ratio is achieved by the antigen binding molecule described in the present invention can be determined by assessing the reduction at any one time point or multiple time points described above.

Even more specifically, the antigen-binding molecules with a transient effect on antigen elimination in plasma described in the present invention have binding activity to human FcRn at pH 7.0 and at 25 degrees C, 440 times higher than intact human IgG, or to KD higher than 0.2 micromolar. Short-term plasma antigen concentration is determined by measuring total antigen concentration or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio at 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration of antigen-binding molecules to assess the short-term effect of antigen-binding molecules. molecules of the present invention on antigen elimination activity in plasma.

Moreover, in the antigen binding molecule of the present invention, which has lower antigen binding activity in the acidic pH range than in the neutral pH range, the ratio of binding activity is not limited, provided that the antigen binding activity is lower in the acidic pH range, than in the neutral pH range. Provided that the antigen binding activity in the acidic pH range is even slightly lower, the antigen binding molecule is suitable. In a preferred embodiment, the antigen binding molecules of the present invention include antigen binding molecules whose antigen binding activity at pH 7.4 is double or greater than that at pH 5.8. In a more preferred embodiment, the antigen binding molecules of the present invention include antigen binding molecules whose antigen binding activity at pH 7.4 is 10 times or greater than that at pH 5.8. In an even more preferred embodiment, the antigen binding molecules of the present invention include antigen binding molecules whose antigen binding activity at pH 7.4 is 40 times or greater than that at pH 5.8.

Specifically, the antigen binding molecules of the present invention include, for example, the embodiments described in WO 2009/125825. More specifically, in a preferred embodiment, the antigen binding molecule of the present invention has an antigen binding activity at pH 5.8 that is lower than that at pH 7.4, where the value of KD (pH 5.8)/KD (pH 7. 4), which is the ratio of the KD for the antigen at pH 5.8 and the KD at pH 7.4, is preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD (pH 5.8)/KD (pH7.4) is not particularly limited and may be any value, for example, 400, 1000 or 10000, provided that the preparation is possible using technologies known to those skilled in the art. areas.

In another preferred embodiment, an antigen binding molecule of the present invention whose antigen binding activity at pH 5.8 is lower than that at pH 7.4 has a value of k _d (pH 5.8)/k _d (pH 7.4) , which is the ratio of k _d for an antigen at pH 5.8 and k _d for an antigen at pH 7.4, which is 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of the value of k _d (pH 5.8)/k _d (pH 7.4) is not particularly limited and may be any value, for example, 50, 100 or 200, provided that it is possible to obtain using technologies known in the art. specialists in this field.

Conditions other than pH at which antigen binding activity and human FcRn binding activity are measured can be appropriately selected by those skilled in the art, and the conditions are not particularly limited; however, measurements can be carried out, for example, under conditions with MES buffer and at 37 degrees C, as described in the examples. In addition, the antigen binding activity of an antigen binding molecule can be determined by methods known to those skilled in the art, for example, using Biacore T100 (GE Healthcare) or the like, as described in the examples.

The antigen-binding molecules of the present invention enhance the uptake of antigen by cells. The molecules readily dissociate from the antigen in the endosome and are then released outside the cell through binding to human FcRn. The antigen-binding molecules of the present invention are believed to readily bind to antigen in plasma again. Thus, for example, if the antigen binding molecule of the present invention is a neutralizing antigen binding molecule, the reduction in plasma antigen concentration can be enhanced by the administered molecules. Thus, an antigen binding molecule that has binding activity to human FcRn in the acidic pH range has lower antigen binding activity in the acidic pH range than in the neutral pH range; and an antigen binding molecule that has human FcRn binding activity in the neutral pH range is likely to be an antigen binding molecule that has better pharmacokinetics and can bind more antigens per molecule.

In a preferred embodiment, such antigen binding molecules having human FcRn binding activity in the acidic and neutral pH range include those comprising a human FcRn binding domain having the ability to bind directly or indirectly to human FcRn. If a domain already has human FcRn binding activity in the acidic to neutral pH range, it can be used in its low form. Alternatively, even if a domain has human FcRn binding activity in the acidic pH range but exhibits only weak or no human FcRn binding activity in the neutral pH range, it can be used after changing the amino acids in the domain to impart human FcRn binding activity in the pH range. range of neutral pH values. Alternatively, it is possible to increase human FcRn binding activity by changing the amino acids in a domain that already has human FcRn binding activity in the acidic and neutral pH ranges. Such antigen-binding molecules include, for example, those that have an IgG Fc domain amino acid sequence that contains a change in at least one amino acid. The change in amino acids is not particularly limited; and the change can be made at any site as long as the human FcRn binding activity in the neutral pH range is higher than before the change.

Specifically, amino acid changes that provide human FcRn binding activity in the acidic to neutral pH range include, for example, amino acid changes at positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252 , from 254 to 260, from 262 to 272, 274, 276, from 278 to 289, from 291 to 312, from 315 to 320, 324, 325, from 327 to 339, 341, 343, 345, 360, 362, 370 , 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442 (EU numbering) in the parental IgG Fc domain described above. More specifically, amino acid changes include changes in the regions (in EU numbering) presented in Tables 1, 2, 6-1, 6-2 and 9. Preferred antigen-binding molecules include antigen-binding molecules containing an amino acid sequence that results from a change in at least at least one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308 , 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 in EU numbering. In a preferred embodiment, such amino acid changes include:

replacement of the amino acid Met with Gly at position 237;

replacement of amino acid Ala with Pro at position 238;

replacement of amino acid Lys with Ser at position 239;

replacement of the amino acid Ile with Lys at position 248;

replacing the amino acid Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr with Thr at position 250;

replacing the amino acid Phe, Trp or Tyr with Met at position 252;

replacing the amino acid Thr with Ser at position 254;

replacement of the amino acid Glu with Arg at position 255;

replacement of amino acids Asp, Glu or Gln with Thr at position 256;

replacing the amino acids Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val with Pro at position 257;

replacement of the amino acid His with Glu at position 258;

replacement of the amino acid Ala with Asp at position 265;

replacing the amino acid Phe with Asp at position 270;

replacement of amino acids Ala or Glu with Asn at position 286;

replacement of the amino acid His with Thr at position 289;

replacement of the amino acid Ala with Asn at position 297;

replacement of the amino acid Gly with Ser at position 298;

replacing the amino acid Ala with Val at position 303;

replacement of amino acids Ala with Val at position 305;

replacing the amino acids Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr with Thr at position 307;

replacing the amino acids Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr with Val at position 308;

replacing the amino acids Ala, Asp, Glu, Pro or Arg with Leu or Val at position 309;

replacement of amino acids Ala, His or Ile with Gln at position 311;

replacement of amino acids Ala or His with Asp at position 312;

replacement of amino acids Lys or Arg with Leu at position 314;

replacing the amino acid Ala or His with Asn at position 315;

replacement of amino acid Ala with Lys at position 317;

replacement of the amino acid Gly with Asn at position 325;

replacing the amino acid Val with Ile at position 332;

replacement of the amino acid Leu with Lys at position 334;

replacement of the amino acid His with Lys at position 360;

replacement of the amino acid Ala with Asp at position 376;

replacing the amino acid Ala with Glu at position 380;

replacement of the amino acid Ala with Glu at position 382;

replacing the amino acid Ala with Asn or Ser at position 384;

replacement of amino acids Asp or His with Gly at position 385;

replacement of amino acid Pro with Gln at position 386;

replacement of the amino acid Glu with Pro at position 387;

replacement of amino acids Ala or Ser with Asn at position 389;

replacement of the amino acid Ala with Ser at position 424;

replacing the amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr with Met at position 428;

replacement of the amino acid Lys with His at position 433;

replacement of amino acids Ala, Phe, His, Ser, Trp or Tyr with Asn at position 434;

and replacing the amino acids His or Phe with Tyr at position 436 (EU numbering).

The number of amino acids to be changed is not particularly limited; it is possible to change an amino acid in only one site or two or more sites. Combinations of two or more amino acid changes include, for example, those presented in Tables 3, 4-1 to 4-5, 6-1, 6-2 and 9.

Meanwhile, domains that already have human FcRn binding activity in the acidic and neutral pH ranges include, for example, human FcRn binding domains containing at least one amino acid selected from:

Met at amino acid position 237;

Ala at amino acid position 238;

Lys in at amino acid position 239;

Ile at amino acid position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr at amino acid position 250;

Phe, Trp or Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

Asp, Glu, or Gln at amino acid position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Phe at amino acid position 270;

Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Gly at amino acid position 298;

Ala at amino acid position 303;

Ala at amino acid position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr at amino acid position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr at amino acid position 308;

Ala, Asp, Glu, Pro or Arg at amino acid position 309;

Ala, His or Ile at amino acid position 311;

Ala or His at amino acid position 312;

Lys or Arg at amino acid position 314;

Ala or His at amino acid position 315;

Ala at amino acid position 317;

Gly at amino acid position 325;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr at amino acid position 428;

Lys at amino acid position 433;

Ala, Phe, His, Ser, Trp or Tyr at amino acid position 434;

and His or Phe at amino acid position 436 (EU numbering) in the parental IgG Fc domain.

An amino acid in a region or amino acids in two or more regions may have these amino acids. Combinations of amino acids at two or more positions include, for example, those presented in Table 3, 4-1 to 4-5, 6-1, 6-2 and 9.

Alternatively, in a preferred embodiment, the antigen binding molecule, whose antigen binding activity in the acidic pH range is lower than that in the neutral pH range, includes antigen binding molecules in which at least one amino acid in the antigen binding molecule is replaced by histidine or a non-natural amino acid, or in which at least one histidine or unnatural amino acid is already incorporated. The site at which the histidine or non-natural amino acid mutation is introduced is not particularly limited and may be any region as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range, and the KD value (in the range acidic pH values)/KD (in the neutral pH range) is large, or k _d (in the acidic pH range)/k _d (in the neutral pH range) is large) compared to that before replacement. Examples include antibody variable regions and CDRs in the case where the antigen binding molecule is an antibody. The number of amino acids to be replaced by histidine or non-natural amino acid and the number of inserted amino acids to be inserted can be determined accordingly by those skilled in the art. One amino acid may be replaced by histidine or an unnatural amino acid, or one amino acid may be inserted, or two or more amino acids may be replaced by histidine or unnatural amino acids, or two or more amino acids may be inserted. In addition, with the exception of substitutions with histidine or a non-natural amino acid or insertion of a histidine or non-natural amino acid, deletion, addition, insertion, and/or substitution of other amino acids can also be carried out simultaneously. Substitutions of histidine or unnatural amino acid or insertion of histidine or unnatural amino acid can be carried out randomly using a method such as histidine scanning, which uses histidine instead of alanine in alanine scanning, which is known to those skilled in the art. Antigen binding molecules whose KD (pH 5.8)/KD (pH 7.4) or k _d (pH 5.8)/k _d (pH 7.4) is increased compared to that before the mutation can be selected from antigen binding molecules , in which a mutation with a histidine or an unnatural amino acid is introduced randomly.

Preferred antigen binding molecules with a mutation to histidine or to a non-natural amino acid, and whose antigen binding activity in the acidic pH range is lower than that in the neutral pH range, include, for example, antigen binding molecules whose antigen binding activity at pH 7.4 after mutation to histidine or to an unnatural amino acid is equivalent to antigen binding activity at pH 7.4 before mutation to histidine or to an unnatural amino acid. In the present invention, "an antigen binding molecule after mutation to histidine or an unnatural amino acid has an antigen binding activity that is equivalent to the antigen binding activity of the antigen binding molecule before mutation to histidine or an unnatural amino acid" means that if the antigen binding activity of the antigen binding molecule before mutation to histidine or an unnatural amino acid is determined to be 100 %, the antigen binding activity of the antigen binding molecule after mutation to histidine or a non-natural amino acid is at least 10% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. Antigen binding activity at pH 7.4 after mutation to histidine or unnatural amino acid may be higher than antigen binding activity at pH 7.4 before mutation to histidine or unnatural amino acid. If the antigen-binding activity of an antigen-binding molecule is reduced due to the substitution or insertion of a histidine or unnatural amino acid, the antigen-binding activity can be adjusted by introducing the substitution, deletion, addition and/or insertion of such one or more amino acids into the antigen-binding molecule such that the antigen-binding activity becomes equivalent to the antigen-binding activity of histidine. replacement or insertion. The present invention also relates to such antigen-binding molecules whose binding activity is made equivalent by substitution, deletion, addition and/or insertion of one or more amino acids following the histidine substitution or insertion.

Additionally, if the antigen binding molecule is an agent comprising an antibody constant region, in another preferred embodiment, antigen binding molecules whose antigen binding activity at pH 5.8 is lower than the antigen binding activity at pH 7.4, the present invention relates to methods for altering constant regions of antibodies contained in antigen-binding molecules. Specific examples of antibody constant regions after modification include those described in the examples in WO 2009/125825 (SEQ ID NOs: 11, 12, 13 and 14).

If the antigen binding activity of an antigen binding agent at pH 5.8 is reduced compared to the antigen binding activity at pH 7.4 (if the KD (pH 5.8)/KD (pH7.4) value is increased) by the methods described above, then in Generally, a KD (pH 5.8)/KD (pH 7.4) value that is two times or more, more preferably five times or more, and even more preferably ten times or more is preferred. compared to that of the parent antibody, but is not specifically limited to these values.

In addition, the present invention relates to antigen-binding molecules with the replacement of histidine or a non-natural amino acid by at least one amino acid at one of the sites described above. Amino acid positions are presented according to 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 and H102.

Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92 and L94.

Of all the change sites, H32, H61, L53, L90 and L94 are hypothesized to be the most important sites for change.

Specifically, preferred combinations of sites for substitution with histidine or a non-natural amino acid include, for example, a combination of H27, H31 and H35; a combination of H27, H31, H32, H35, H58, H62 and H102; combination of L32 and L53; and a combination of L28, L32 and L53. In addition, preferred combinations of heavy and light chain substitution sites include, for example, a combination of H27, H31, L32 and L53.

The antigen binding molecule of the present invention may have other properties, and for example, may be an agonistic or antagonistic antigen binding molecule, provided that its antigen binding activity is lower in the acidic pH range than in the neutral pH range, and it has binding activity with human FcRn in the range of acidic and neutral pH values. Preferred antigen binding molecules of the present invention include, for example, antagonistic antigen binding molecules. Such an antagonistic antigen binding molecule is typically an antigen binding molecule that inhibits a receptor-mediated intracellular signal transduction pathway by blocking the binding between a ligand (agonist) and a receptor.

Meanwhile, the antigen binding molecule of the present invention can recognize any antigen. Specifically, antigens recognized by the antigen binding molecule of the present invention include, for example, the receptor proteins (membrane-bound receptors and soluble receptors) described above, membrane antigens such as cell surface markers, and soluble antigens such as cytokines. Such antigens include, for example, those described above.

In a preferred embodiment, the antigen binding molecules of the present invention include IgG type immunoglobulins (IgG antibodies) with an antigen binding domain and a human FcRn binding domain. If an IgG antibody is used as an antigen-binding molecule, the type is not limited; and it is possible to use IgG1, IgG2, IgG3, IgG4 and the like.

The origin of the antigen binding molecules of the present invention is not particularly limited, and they can be of any origin. It is possible to use, for example, mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies and others. In addition, the antibodies may, for example, be chimeric antibodies as described above, and in particular, altered antibodies with amino acid sequence substitutions, such as humanized antibodies. Antibodies may be bispecific antibodies described above, modification products of antibodies to which various molecules are linked, polypeptides including antibody fragments, and antibodies with modified sugar chains.

A bispecific antibody refers to an antibody that has variable regions within the same antibody molecule that recognize different epitopes. A bispecific or polyspecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.

In addition, polypeptides comprising antibody fragments include, for example, Fab fragments, F(ab')2 fragments, scFvs (Nat Biotechnol. 2005 Sep; 23(9): 1126-36), domain antibodies (dAb) (WO 2004/ 058821, WO 2003/002609), scFv-Fc (WO 2005/037989), dAb-Fc and Fc fusion proteins. The Fc domain can be used as a human FcRn binding domain if the molecule includes an Fc domain. Alternatively, the FcRn binding domain can be fused to these molecules.

Additionally, antigen binding molecules that are useful in the present invention may be antibody-like molecules. An antibody-like molecule (scaffold molecule, peptide molecule) is a molecule that can exert functions by binding to a target molecule (Current Opinion in Biotechnology (2006) 17: 653-658; Current Opinion in Biotechnology (2007) 18: 1-10; Current Opinion in Structural Biology (1997) 7: 463-469; Protein Science (2006) 15: 14-27) and includes, for example, DARPins (WO 2002/020565), Affitel (WO 1995/001937), Avimer (WO 2004/ 044011; WO 2005/040229) and Adnectin (WO 2002/032925). If these antibody-like molecules can bind the target molecule in a pH-dependent manner and/or have binding activity to human FcRn in the neutral pH range, it is possible to facilitate the uptake of antigen into cells through antigen-binding molecules, help reduce the concentration of antigen in plasma through the administration of antigen-binding molecules, improve the pharmacokinetics of antigen-binding molecules. molecules and increase the number of antigens to which one antigen-binding molecule can bind.

In addition, the antigen binding molecule may be a protein resulting from a fusion between a human FcRn binding domain and a receptor protein that binds to a target including a ligand and includes, for example, TNFR-Fc fusion proteins, ILIR-Fc fusion proteins, VEGFR-Fc and CTLA4-Fc fusion proteins (Nat Med. 2003, Jan; 9(1): 47-52; BioDrugs. (2006) 20(3): 151-60). If such receptor-human FcRn-binding domain fusion proteins bind the target ligand-containing molecule in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, it may be possible to facilitate antigen uptake into cells via antigen-binding molecules, promoting lower concentrations antigen in plasma by administering an antigen-binding molecule and improve the pharmacokinetics of antigen-binding molecules, and increase the number of antigens to which one antigen-binding molecule can bind. The receptor protein is suitably designed and modified to include the binding domain of the receptor protein to a target including a ligand. As set forth in the example above, including TNFR-Fc fusion proteins, ILIR-Fc fusion proteins, VEGFR-Fc fusion proteins and CTLA4-Fc fusion proteins, a soluble receptor molecule containing the extracellular domain of such receptor proteins that are required for binding to such targets, including ligands are preferably used in the present invention. Such a designed and modified receptor molecule is referred to in this application as an artificial receptor. The method used to design and modify a receptor molecule to construct an artificial receptor molecule is known in the art.

In addition, the antigen binding molecule may be a fusion protein in which an artificial ligand protein that binds to a target and has a neutralizing effect is fused to a human FcRn binding domain, and the artificial ligand proteins include, for example, mutant IL-6 ( EMBO J 1994 Dec 15;13(24):5863-70). If such artificial ligand fusion proteins can bind to target molecules in a pH-dependent manner and/or have binding activity to human FcRn in the neutral pH range, then it is possible to facilitate the uptake of antigen into cells through antigen-binding molecules, help reduce the concentration of antigen in plasma through the introduction of antigen-binding molecules molecules, improve the pharmacokinetics of antigen-binding molecules and increase the number of antigens to which one antigen-binding molecule can bind.

In addition, the antibodies of the present invention may include modified sugar chains. Antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (WO 99/54342), antibodies that lack fucose, which is attached to the sugar chain (WO 00/61739; WO 02/31140; WO 2006/067847; W02 006/ 067913), and GlcNAc separated antibodies (WO 02/79255).

Conditions used in the assay of antigen binding activity or human FcRn binding activity other than pH can be appropriately selected by those skilled in the art, and the conditions are not particularly limited. For example, the conditions of using MES buffer at 37 degrees C as described in WO 2009/125825 can be used to determine activity. Meanwhile, the antigen binding activity and human FcRn binding activity of an antigen binding molecule can be determined by methods known to those skilled in the art, for example, using Biacore (GE Healthcare) or the like. If the antigen is a soluble antigen, the activity of the antigen binding molecule to bind the soluble antigen can be determined by applying the antigen as an analyte to a chip immobilized with the antigen binding molecule. Alternatively, if the antigen is a membrane-type antigen, the activity of the antigen-binding molecule to bind to the membrane-type antigen can be determined by applying the antigen-binding molecule as an analyte to the antigen-immobilized chip. The human FcRn binding activity of an antigen binding molecule can be determined by applying the human FcRn or antigen binding molecule as an analyte to a chip immobilized with the antigen binding molecule or human FcRn, respectively.

The generation of chimeric antibodies is known. In the case of a human-mouse chimeric antibody, for example, the DNA encoding the V region of the antibody may be linked to the DNA encoding the C region of the human antibody; this construct can be inserted into an expression vector and introduced into a host to produce a chimeric antibody.

“Humanized antibodies” are also referred to as converted human antibodies, and are antibodies in which a complementarity determining region (CDR) from a non-human mammal, such as a mouse, is transplanted into the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General genetic recombination technologies suitable for this purpose are also known (see European patent application EP 125023; and WO 96/02576). Humanized antibodies can be produced by known methods, for example, the CDR of a mouse antibody can be determined and DNA encoding an antibody in which the CDR is linked to the framework region (FR) of a human antibody can be obtained. Humanized antibodies can then be produced using a system that uses traditional expression vectors. Such DNA can be synthesized by PCR, using as primers certain oligonucleotides prepared in such a way that they have regions that overlap the terminal regions of both the CDR and FR (see the method described in WO 98/13388). The human antibody FR linked to the CDR is selected such that the CDR forms a suitable antigen binding site. If desired, the amino acids in the FR variable region of an antibody can be changed such that the CDRs of the converted human antibody can form a suitable antigen binding region (Sato et al., Cancer Res. (1993) 53: 10.01-6). Amino acid residues in FR that can be modified include regions that directly bind antigen through non-covalent bonds (Amit et al., Science (1986) 233: 747-53), regions that influence or have an effect on the structure of the CDR (Chothia et al., J Mol Biol (1987) 196: 901-17), and regions involved in VH-VL interaction (EP 239400).

When the antigen binding molecules of the present invention are chimeric antibodies or humanized antibodies, the C regions of these antibodies are preferably derived from a human antibody. For example, C-gamma1, C-gamma2, C-gamma3 and C-gamma4 can be used for the H chain, while C-kappa and C-lambda can be used for the L chain. Additionally, if necessary, amino acid mutations can be introduced into the region With human antibodies to increase or decrease binding to the Fc-gamma receptor or to increase antibody stability or productivity. The chimeric antibody of the present invention preferably includes a variable region of an antibody derived from a non-human mammal and a constant region derived from a human antibody. Meanwhile, the humanized antibody preferably includes the CDR of an antibody derived from a non-human mammal, and the FR and C regions derived from a human antibody. Constant regions derived from human antibodies preferably include a human FcRn binding region. Such antibodies include, for example, IgG (IgG1, IgG2, IgG3 and IgG4). The constant regions used for the humanized antibodies of the present invention may be constant regions of antibodies of the same isotype. A constant region derived from human IgG1 is preferably used, although the constant regions are not limited to it. The human antibody-derived FRs that are used for humanized antibodies are not particularly limited and can be derived from any isotype antibody.

The variable and constant regions of the chimeric and humanized antibodies of the present invention can be modified by deletion, substitution, insertion and/or addition, and the like, provided that the original antibodies exhibit binding specificity.

Since immunogenicity in humans is reduced, chimeric and humanized antibodies using human-derived sequences are believed to be useful when administered to humans for therapeutic and similar purposes.

Such antigen binding molecules of the present invention can be produced by any method. For example, an antigen binding molecule that does not initially have binding activity to human FcRn in the acidic and neutral pH ranges, an antigen binding molecule that has stronger antigen binding activity in the acidic pH range than in the neutral pH range, or an antigen binding molecule that has comparable antigen-binding activity in acidic or neutral pH ranges, can be artificially converted into an antigen-binding molecule having the desired activity through the above amino acid changes or the like. Alternatively, an antibody having the desired activity can be selected by screening from a range of antibodies obtained from an antibody or hybridoma library described below.

If the amino acids in the antigen binding molecule are changed, it is possible to use a known sequence for the amino acid sequence of the antigen binding molecule before the change or the amino acid sequence of the antigen binding molecule newly identified by methods known to those skilled in the art. For example, if the antigen-binding molecule is an antibody, it can be obtained from an antibody library or by cloning the antibody-encoding gene from antibody-producing hybridomas.

Regarding antibody libraries, many antibody libraries are already known, and methods for preparing antibody libraries are also known; thus, antibody libraries can be prepared accordingly by those skilled in the art. For example, regarding phage libraries, reference may be made to literature such as Clackson et al., Nature (1991) 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 Biotechnology (1996) 14: 309-14; and Kohyo Japanese Patent Publication No. (JP-A) H20-504970 (unexamined Japanese national phase publication corresponding to non-Japanese international publication). In addition, it is possible to use known methods, such as methods using eukaryotic cells as a library (WO 95/15393), and methods based on ribosomal display. In addition, technologies for producing human antibodies by panning using human antibody libraries are also known. For example, human antibody variable regions can be expressed on the surface of phages as single chain antibodies (scFvs) using phage display methods, and phages that bind to antigens can be selected. By genetic analysis of selected phages, DNA sequences encoding the variable regions of human antibodies that bind antigens can be determined. By identifying the DNA sequences of scFvs that bind to antigens, suitable expression vectors for the production of human antibodies can be derived from these sequences. These methods are already well known and reference may be made to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388.

Regarding methods for obtaining genes encoding antibodies from hybridomas, generally known technologies can be used, which include using the desired antigens or cells expressing the desired antigens as sensitizing antigens, using them for immunizations according to conventional immunization methods, fusing the resulting immune cells with known parent cells through conventional cell fusion methods, screening cells producing monoclonal antibodies (hybridomas), conventional screening methods, synthesizing cDNA of antibody variable regions (region V) from hybridoma-derived mRNA using reverse transcriptase, and linking them to DNA encoding the required constants antibody regions (C regions).

More specifically, sensitizing antigens for producing the above-described H chain and L chain antigen binding molecule genes may include, for example, both complete antigens with immunogenicity and partial antigens including haptens and the like. without immunogenicity; however, antigens are not limited to these examples. For example, it is possible to use whole proteins and partial peptides of proteins of interest. In addition, it is known that agents containing polysaccharides, nucleic acids, lipids and the like may be antigens. Thus, the antigens of the antigen-binding molecules of the present invention are not particularly limited. Antigens can be produced by methods known to those skilled in the art, for example, baculovirus-based methods (eg WO 98/46777) and the like. Hybridomas can be obtained, for example, by the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. (1981) 73: 3-46) and the like. If the immunogenicity of the antigen is low, immunization can be carried out after the antigen has bound to a macromolecule having immunogenicity, such as albumin. Alternatively, if necessary, the antigens can be converted into soluble antigens by coupling to other molecules. If transmembrane molecules such as membrane antigens (eg, receptors) are used as antigens, portions of the extracellular regions of the membrane antigens can be used as a fragment, or cells expressing transmembrane molecules on their cell surface can be used as immunogens.

Cells producing antigen-binding molecules can be obtained by immunizing animals using the appropriate sensitizing antigens described above. Alternatively, cells that produce antigen-binding molecules can be obtained by in vitro immunization of lymphocytes that produce antigen-binding molecules. Various mammals can be used for immunization; so commonly used animals include rodents, lagomorphs and primates. Such animals include, for example, rodents such as mice, rats and hamsters; lagomorphs such as rabbits; and primates, including monkeys such as cynomolgus monkeys, rhesus monkeys, baboons and chimpanzees. In addition, the immune repertoire of transgenic animals carrying the human antibody gene is also known, and human antibodies can be obtained using such animals (see WO 96/34096; Mendez et al., Nat. Genet. (1997) 15: 146-56 ). Instead of using such transgenic animals, for example, desired human antibodies with antigen binding activity can be produced by sensitizing human lymphocytes in vitro with the desired antigens or cells expressing the desired antigens, and then fusing the sensitized lymphocytes with human myeloma cells, such as U266 (see patent publication Japan Kokoku Application No. (JP-B) H01-59878 (examined, approved Japanese patent application published for opposition)). In addition, the desired human antibodies can be obtained by immunizing transgenic animals carrying the full repertoire of human antibody genes with the desired antigens (see WO 93/12227, WO 92/03918, WO 94/02602, WO 96/34096 and WO 96/33735).

Immunization of animals can be accomplished by appropriately diluting and suspending the sensitizing antigen in phosphate-buffered saline (PBS), saline, or the like, and mixing them with an emulsification adjuvant, if necessary. It is then injected intraperitoneally or subcutaneously into animals. Then, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times over 21 days. Antibody production can be confirmed by measuring the titer of antibodies of interest in animal serum using conventional methods.

Cells producing antigen-binding molecules derived from lymphocytes or animals immunized with the desired antigen can be fused with myeloma cells to produce hybridomas using conventional fusion agents (eg, polyethylene glycol) (Goding, Monoclonal antibody: Principles and Practice, Academic Press, ( 1986) 59-103). If necessary, hybridoma cells can be cultured and grown, and the binding specificity of the antigen binding molecule obtained from such hybridomas can be measured using known assay methods such as immunoprecipitation, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Subsequently, if desired, hybridomas producing antigen binding molecules of interest whose specificity, affinity or activity have been determined can be subcloned by methods such as limiting dilution.

Genes encoding selected antigen-binding molecules can then be cloned from hybridomas or antigen-binding molecule-producing cells (sensitized lymphocytes and the like) using probes that specifically bind to antigen-binding molecules (eg, oligonucleotides complementary to sequences encoding antibody constant regions). It is also possible to clone genes from mRNA using RT-PCR. Immunoglobulins are classified into five different classes, IgA, IgD, IgE, IgG and IgM. These classes are further subdivided into several subclasses (isotypes) (eg, IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2; and the like). The H and L chains used in the present invention to produce antigen binding molecules are not particularly limited and may be derived from antibodies belonging to any of these classes or subclasses; however, IgG is particularly preferred.

Herein, it is possible to change the genes encoding the H chain and the genes encoding the L chain using genetic engineering techniques. Genetically modified antibodies, such as chimeric antibodies and humanized antibodies, which are artificially modified to reduce heterologous immunogenicity and the like in humans, can be suitably produced for antibodies such as mouse, rat, rabbit, hamster, sheep and camel antibodies. Chimeric antibodies are antibodies comprising the H and L chain variable regions of a non-human mammalian antibody, such as a mouse antibody, and the H and L chain constant regions of a human antibody. Chimeric antibodies can be produced by ligating DNA encoding the variable region of a mouse antibody with DNA encoding the constant region of a human antibody, inserting it into an expression vector, and introducing the vector into a host to produce antibodies. A humanized antibody, also called a "converted human antibody", can be synthesized by PCR using certain oligonucleotides designed to have overlapping regions at the ends of DNA sequences designed to link to the complementarity determining regions (CDRs) of a non-human mammalian antibody , such as a mouse. The resulting DNA can be ligated to DNA encoding the constant region of a human antibody. The ligated DNA can be inserted into an expression vector, and the vector can be introduced into an antibody-producing host (see EP 239400 and WO 96/02576). Human FR antibodies that ligate to CDRs are selected if the CDRs form a suitable antigen binding site. If desired, amino acids in the framework region of the antibody variable region can be replaced such that the CDR of the rearranged human antibody forms a suitable antigen binding region (K. Sato et al., Cancer Res. (1993) 53: 10.01-10.06).

In addition to the humanization described above, antibodies can be modified to enhance their biological properties, such as binding to antigens. In the present invention, such changes can be achieved through methods such as site-directed mutagenesis (see, for example, Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis and cassette mutagenesis. In general, mutant antibodies whose biological properties are improved exhibit amino acid sequence homology and/or similarity of 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 97% , 98% or 99%), compared to the amino acid sequence of the variable region of the parent antibody. As used herein, sequence homology and/or similarity is defined as the ratio of amino acid residues that are homologous (same residue) or similar (amino acid residues classified into the same group based on basic properties of amino acid side chains) to residues of the parent antibody, then the sequence homology value was maximized by sequence alignment and introducing skipping if necessary. In general, naturally occurring amino acid residues are classified into groups based on the characteristics of their side chains as follows:

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

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

(3) acidic: aspartic acid and glutamic acid;

(4) basic: arginine, histidine and lysine;

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

(6) aromatic: tyrosine, tryptophan and phenylalanine.

Basically, a total of six complementarity determining regions (CDRs; hypervariable regions) present in the variable regions of the H and L chains interact with each other to form the antigen binding region of an antibody. As is known, the variable region itself is also capable of recognizing and binding antigen, although its affinity is lower than that of the entire binding site. Thus, the genes encoding the H and L chains of the present invention may encode each fragment, including the antigen binding region of the H or L chain, provided that the polypeptide encoded by the gene retains binding activity to the desired antigen.

As described above, the heavy chain variable region mainly consists of three CDRs and four FRs. In a preferred embodiment of the present invention, the amino acid residues to be "changed" may be suitably selected from amino acid residues, for example, in the CDR or FR. In general, changes in amino acid residues in the CDR can reduce antigen-binding capacity. Thus, suitable amino acid residues to be “changed” in the present invention are preferably selected from, but are not particularly limited to, amino acid residues in FR. It is possible to select amino acids in the CDR provided that it is confirmed that the changes do not reduce binding activity. Alternatively, by using publicly available databases or the like, those skilled in the art can obtain suitable sequences that can be used as the FR of an antibody variable region of an organism such as a human or mouse.

In addition, the present invention relates to genes encoding the antigen binding molecules of the present invention. The genes encoding the antigen binding molecules of the present invention can be any genes and can be DNA, RNA, nucleic acid analogues or the like.

In addition, the present invention also relates to host cells carrying the genes described above. Host cells are not particularly limited and include, for example, E. coli and cells of various animals. Host cells can be used, for example, as a production system for the production and expression of antibodies of the present invention. In vitro and in vivo production systems are available for polypeptide production systems. Such in vitro production systems include, for example, production systems using eukaryotic or prokaryotic cells.

Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells and fungal cells. Animal cells include: mammalian cells, eg CHO (J. Exp. Med. (1995) 108:94.0), COS, HEK293, 3T3, myelomas, BHK (baby hamster kidney), HeLa and Vero; amphibian cells such as Xenopus laevis oocytes (Vaile et al., Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21 and Tn5. CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells and BHK cells are preferably used to express the antibodies of the present invention. Among animal cells, specifically CHO cells are preferred for expression on a large scale. Vectors can be introduced into host cells, for example, by calcium phosphate methods, DEAE-dextran methods, DOTAP cationic liposome methods (Boehringer-Mannheim), electroporation methods, and lipofection methods.

Regarding plant cells, for example, cells derived from Nicotiana tabacum and duckweed (Lemna minor) are known as protein production systems. To obtain the antigen-binding molecules of the present invention, calli can be cultured from these cells. Regarding fungal cells, known protein expression systems are those that use yeast cells, for example, cells of the Saccharomyces species (such as Saccharomyces cerevisiae and Saccharomyces pombe); and cells of filamentous fungi, for example, Aspergillus species (such as Aspergillus niger). These cells can be used as a host to produce the antigen binding molecules of the present invention.

Bacterial cells can be used in prokaryotic production systems. Regarding bacterial cells, production systems using Bacillus subtilis are known in addition to production systems using E. Coli described above. Such systems can be used in the production of antigen binding molecules of the present invention.

<Screening methods>

The present invention relates to methods for screening antigen-binding molecules having binding activity to human FcRn in acidic and neutral pH ranges. The present invention also provides methods for screening antigen binding molecules that have human FcRn binding activity in the acidic and neutral pH ranges and lower antigen binding activity in the acidic pH range than in the neutral pH range. The present invention also relates to methods for screening antigen-binding molecules capable of facilitating the uptake of antigen into cells. The present invention also relates to methods for screening antigen-binding molecules that are modified to be capable of binding to a large number of antigens per molecule. The present invention also provides methods for screening antigen-binding molecules capable of facilitating antigen clearance. The present invention further relates to methods for screening antigen-binding molecules with improved pharmacokinetics. The present invention also provides methods for screening antigen binding molecules with increased intracellular dissociation from their associated antigen outside of cells. The present invention also provides methods for screening antigen-binding molecules with increased extracellular release in an antigen-free form after uptake into cells in an antigen-bound form. The present invention further relates to methods for screening antigen-binding molecules that are particularly useful as pharmaceutical compositions. The methods described above are suitable for screening antigen-binding molecules that are particularly excellent in retention time and have excellent ability to eliminate antigens from plasma.

Specifically, the present invention relates to methods for screening antigen-binding molecules, where the methods include the following steps:

(a) selecting an antigen binding molecule that has higher human FcRn binding activity in the neutral pH range than that before changing at least one amino acid in the human FcRn binding domain of the antigen binding molecule having human FcRn binding activity in the acidic range pH; And

(b) changing at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule that has higher antigen binding activity in the neutral pH range than in the acidic pH range.

Steps (a) and (b) can be carried out in any order. In addition, each step can be repeated twice or more times. The number of repetitions for steps (a) and (b) is not particularly limited; however, the number is usually ten times or less.

In the screening methods of the present invention, the antigen binding activity of the antigen binding molecule in the neutral pH range is not particularly limited as long as it is antigen binding activity in the pH range of 6.7 to 10.0. For example, embodiments described in WO 2009/125825 are included. Preferred antigen binding activities include antigen binding activity in the pH range of 7.0 to 8.0. A more preferred antigen binding activity includes antigen binding activity at pH 7.4. Meanwhile, the antigen binding activity of the antigen binding molecules in the acidic pH range is not particularly limited as long as it is the antigen binding activity in the pH range of 4.0 to 6.5. Preferred antigen binding activity includes antigen binding activity in the pH range of 5.5 and 6.5. More preferred antigen binding activity includes antigen binding activity at pH 5.8 or pH 5.5.

The human FcRn binding activity of an antigen binding molecule in the neutral pH range is not particularly limited as long as it is human FcRn binding activity in the pH range of 6.7 to 10.0. Preferred human FcRn binding activity includes human FcRn binding activity in the pH range of 7.0 to 8.0. More preferred human FcRn binding activity includes human FcRn binding activity at pH 7.4.

The human FcRn binding activity of antigen binding molecules in the acidic pH range is not particularly limited as long as it is human FcRn binding activity in the pH range of 4.0 to 6.5. Preferred human FcRn binding activity includes human FcRn binding activity in the pH range of 5.5 to 6.5. More preferred human FcRn binding activity includes human FcRn binding activity in the pH range of 5.8 to 6.0.

As used herein, the acidic pH range generally refers to pH 4.0 to pH 6.5. Preferably, the acidic pH range is a range represented by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, particularly preferably a pH value of 5.8 to pH 6.0, which is a value close to the pH in the early endosome in vivo. Meanwhile, herein, the neutral pH range generally refers to pH 6.7 to pH 10.0. The neutral pH range is preferably a range represented by any pH value within pH 7.0 to pH 8.0, preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, more preferably pH 7.4, which is a value close to the pH of plasma (blood) in vivo. pH 7.0 can be used as an alternative to pH 7.4 if it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to the latter's low affinity at pH 7.4. Since temperature was used in the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature between 10 degrees C and 50 degrees C. Preferably, a temperature between 15 degrees C and 40 degrees C is used to determine the binding affinity between human FcRn-binding domain and human FcRn. More preferably, any temperature from 20 degrees C to 35 degrees C, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 degrees C is also used to determine the binding affinity between the human FcRn binding domain and human FcRn. The temperature of 25 degrees C described in Example 5 is one example of an embodiment of the present invention.

Antigen binding activity and human FcRn binding activity of antigen binding molecules can be determined by methods known to those skilled in the art. Suitable conditions, along with pH, can be selected by those skilled in the art. The antigen binding activity and human FcRn binding activity of antigen binding molecules can be assessed using KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate k _d (dissociation rate), apparent k _d (apparent dissociation: apparent dissociation rate), or t .P. They can be determined by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scatchard plot, flow cytometer or the like.

According to the Journal of Immunology (2009) 182: 7663-7671, the human FcRn binding activity of intact human IgG1 has a KD of 1.7 micromolar in the acidic pH range (pH 6.0), while the activity is almost undetectable in the pH range neutral pH values. Thus, in a preferred embodiment, antigen binding molecules of the present invention that have human FcRn binding activity in the acidic and neutral pH ranges can be screened, including antigen binding molecules that have human FcRn binding activity with a KD of 20 micromolar or greater in the acidic pH range, which is equal to or greater than that of intact human IgG in the neutral pH range. In a more preferred embodiment, antigen binding molecules of the present invention can be screened, including antigen binding molecules whose binding activity to human FcRn is a KD of 2.0 micromolar or greater in the acidic pH range and a KD of 40 micromolar or greater in range of neutral pH values. In an even more preferred embodiment, antigen binding molecules of the present invention can be screened, including antigen binding molecules whose binding activity to human FcRn is a KD value of 0.5 micromoles or greater in the acidic pH range, and a KD value of 15 micromoles or more more in the neutral pH range. The above KD values are determined by the method described in Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen binding molecule on a chip and applying human FcRn as an analyte).

The present invention relates to a method for screening an antigen binding molecule, where the method includes the steps of:

(a) selecting an antigen binding molecule that has a higher binding activity to human FcRn in the neutral pH range than a KD of 3.2 micromoles obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule,

(b) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in step (a) are linked; And

(c) producing an antigen binding molecule using the gene obtained in (b).

In one embodiment, an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain may be screened that has binding activity to human FcRn in the acidic and neutral pH ranges, where binding activity to human FcRn and lower antigen binding activity in the acidic range pH values than in the neutral pH range is greater than the KD of 3.2 micromoles, according to methods used by those skilled in the art as previously described herein. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 degrees C is higher than the KD of 3.2 micromolar.

The present invention relates to a method for screening an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is higher than KD, equal to 2.3 micromoles. The present invention also provides a method for screening an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein human FcRn binding activity in the neutral pH range is 38 times higher than for intact human IgG.

The antigen-binding molecules of the present invention having human FcRn binding activity in the neutral pH range are not particularly limited as long as they have human FcRn binding activity at a pH of 6.7 to 10.0. However, the binding activity to human FcRn at pH 6.7 to 10.0 of the preferred antigen-binding molecules is higher than that of intact human IgG.

The antigen-binding molecules of the present invention having human FcRn binding activity in the acidic pH range are not particularly limited as long as they have human FcRn binding activity in the pH range of 4.0 to 6.5. However, the binding activity to human FcRn at pH 5.5 to 6.5 of the preferred antigen-binding molecules is comparable to or greater than that of intact human IgG.

Herein, the step of selecting an antigen binding molecule that has higher antigen binding activity in the neutral pH range than in the acidic pH range is similar to the step of selecting an antigen binding molecule that has lower antigen binding activity in the acidic pH range than in the neutral pH range pH values.

The ratio of antigen binding activity between neutral and acidic pH ranges is not particularly limited as long as the antigen binding activity in the neutral pH range is higher than in the acidic pH range. However, the antigen binding activity at pH 6.7 to 10.0 is preferably two times or more, more preferably ten times or more, and even more preferably 40 times or more the antigen binding activity at pH 4.0 to 6. 5.

It is possible to use libraries, such as phage libraries, in the screening methods of the present invention.

In the methods of the present invention, the antigen and the antigen-binding molecule can be bound together in any state, and thus the state is not particularly limited. For example, an antigen can interact with an immobilized antigen-binding molecule to achieve binding. Alternatively, the antigen-binding molecule can interact with the immobilized antigen to achieve their binding. Alternatively, the antigen binding molecule and the antigen can interact with each other in solution to achieve their binding.

Antigen-binding molecules to be screened by the screening methods of the present invention can be produced by any method. For example, it is possible to use libraries of pre-existing antibodies, pre-existing antigen-binding domains (phage libraries, etc.), libraries of antibodies or antigen-binding domains obtained from B cells of immunized animals, or hybridomas obtained by immunizing animals, libraries of antibodies or antigen-binding domains, obtained by introducing random amino acid changes into the above libraries of antibodies or antigen binding domains, libraries of antibodies or antigen binding domains, with introduced mutations of histidine or unnatural amino acid (libraries with a high content of histidine or unnatural amino acid, libraries of antigen binding domains, with introduced mutation of histidine or unnatural amino acid mutation to specific areas, etc.), or the like.

Antigen-binding molecules that bind to the antigen many times, which are thus improved in plasma retention time, can be obtained by the screening method of the present invention. Thus, the screening methods of the present invention can be used as screening methods to obtain antigen binding molecules that have improved plasma retention time.

In addition, antigen-binding molecules that can bind to an antigen two or more times when administered to animals such as humans, mice or monkeys can be obtained by the screening methods of the present invention. Thus, the screening methods of the present invention can be used as screening methods to obtain antigen-binding molecules that can bind to an antigen two or more times.

In addition, antigen binding molecules that are capable of binding to a large number of antigens compared to the number of antigen binding sites when administered to animals such as humans, mice or monkeys can be obtained by the screening methods of the present invention. Thus, the screening methods of the present invention can be used as screening methods to obtain antigen binding molecules that are capable of binding a greater number of antigens relative to the number of their antigen binding sites. For example, if the antibody is a neutralizing antibody, the screening methods of the present invention can be used as screening methods to obtain antigen binding molecules that can neutralize a greater number of antigens compared to the number of antigen binding sites of the antigen binding molecules.

In addition, antigen binding molecules that are capable of dissociating intracellularly from extracellularly bound antigen when administered to animals such as humans, mice or monkeys can be obtained by the screening methods of the present invention.

Thus, the screening methods of the present invention can be used as screening methods to obtain antigen-binding molecules that are capable of dissociating within a cell from extracellularly bound antigen.

In addition, antigen-binding molecules that bind to antigen and are taken up by cells and released outside the cells in an antigen-free form when administered to animals such as humans, mice or monkeys can be obtained through the screening methods of the present invention. Thus, the screening methods of the present invention can be used as screening methods to obtain antigen-binding molecules that bind to an antigen and are taken up by cells and released outside the cells in an antigen-free form.

In addition, antigen binding molecules that can rapidly eliminate antigens in plasma when administered to animals such as humans, mice or monkeys can be produced by the screening methods of the present invention. Thus, the screening methods of the present invention can be used as screening methods to obtain antigen-binding molecules with increased ability to eliminate antigens in plasma.

In addition, such antigen-binding molecules are expected to be particularly excellent as drugs, since the dosage and frequency of administration to patients can be reduced, and as a result, the total dose can be reduced. Thus, the screening methods of the present invention can be used as methods for screening antigen binding molecules for use as pharmaceutical compositions.

<Methods of obtaining antigen-binding molecules>

The present invention relates to methods for producing antigen binding molecules that have human FcRn binding activity at endosomal and plasma pH, and lower antigen binding activity at endosomal pH than at plasma pH. The present invention also provides methods for producing antigen-binding molecules that are more advanced in pharmacokinetics and in accelerating the decline in plasma antigen concentration upon administration. The present invention also relates to methods for producing antigen-binding molecules that are particularly useful for use as pharmaceutical compositions.

Specifically, the present invention relates to methods for producing antigen-binding molecules, where the method includes the steps of:

(a) selecting an antigen binding molecule that has higher human FcRn binding activity in the neutral pH range than that before changing at least one amino acid in the human FcRn binding domain of the antigen binding molecule having human FcRn binding activity in the acidic range pH;

(b) changing at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule that has higher antigen binding activity in the neutral pH range than in the acidic pH range;

(c) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) are linked; And

(d) obtaining an antigen binding molecule using the gene obtained in step (c).

Steps (a) and (b) can be performed in any order. In addition, each step can be repeated twice or more. The number of repetitions for steps (a) and (b) is not particularly limited; however, the number is usually ten times or less.

The linker operably linking the human FcRn binding domain and the antigen binding domain obtained in steps (a) and (b) is not limited to any form. The human FcRn binding domain and the antigen binding domain can be linked through either covalent or non-covalent bonds. In particular, the linker may be a peptide linker or a chemical linker or linking pairs like a combination of biotin and streptavidin. Polypeptide modification including a human FcRn binding domain and an antigen binding domain is known in the art. In another embodiment, the human FcRn binding domain and the antigen binding domain of the present invention can be linked by forming a fusion protein between the human FcRn binding domain and the antigen binding domain. To construct a fusion protein between a human FcRn binding domain and an antigen binding domain, the genes encoding the human FcRn binding domain and the antigen binding domain can be operably linked so as to form an in-frame fusion polypeptide. Accordingly, a linker containing a multi-amino acid peptide can be inserted between the human FcRn binding domain and the antigen binding domain. Various flexible linkers, such as a linker whose sequence consists of (GGGGS) _n , are known in the art.

Antigen-binding molecules that are used in the production methods of the present invention can be produced by any method. For example, it is possible to use pre-existing antibodies, pre-existing libraries (phage libraries, etc.), antibodies and libraries that are obtained from hybridomas obtained by immunizing animals or from B cells of immunized animals, antibodies and libraries obtained by introducing random amino acid changes into the antibodies and libraries described above, antibodies and libraries obtained by introducing histidine or unnatural amino acid mutations into the antibodies described above, and libraries (libraries with a high content of histidine or unnatural amino acid, libraries with introduced histidine or unnatural amino acid at specific sites, etc. .) and similar ones.

In the above-described production methods, the human FcRn binding activity of antigen-binding molecules in the neutral pH range is not particularly limited as long as it is the human FcRn binding activity in the pH range of 6.7 to 10.0. Preferred human FcRn binding activity includes human FcRn binding activity in the pH range of 7.0 to 8.0. More preferred human FcRn binding activity includes human FcRn binding activity at pH 7.4.

The human FcRn binding activity of an antigen binding molecule in the acidic pH range is not particularly limited as long as it is the human FcRn binding activity in the pH range of 4.0 to 6.5. Preferred human FcRn binding activity includes human FcRn binding activity in the pH range of 5.5 to 6.5. More preferred human FcRn binding activity includes human FcRn binding activity at pH 6.0.

As used herein, the acidic pH range generally refers to pH 4.0 to pH 6.5. The acidic pH range is preferably a range represented by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6 .0, 6.1, 6.2, 6.3, 6.4 and 6.5, more preferably pH 5.8 to pH 6.0, which is a value close to the pH value of the early endosome in vivo . Meanwhile, herein, the neutral pH range generally refers to pH 6.7 to pH 10.0. The neutral pH range is preferably a range represented by any pH value within the range of pH 7.0 to pH 8.0, preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, more preferably pH 7.4, which is a value close to the pH value of plasma (blood) in vivo. pH 7.0 can be used as an alternative to pH 7.4 if it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to the latter's low affinity at pH 7.4. Since temperature is used as an assay condition, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature between 10 degrees C and 50 degrees C. Preferably, a temperature between 15 degrees C and 40 degrees C is used to determine the binding affinity between human FcRn-binding domain and human FcRn. More preferably, any temperature from 20 degrees C to 35 degrees C, such as one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 degrees C is also used to determine the binding affinity between the human FcRn binding domain and human FcRn. The temperature of 25 degrees C described in Example 5 is one example for an embodiment of the present invention.

The present invention relates to a method for producing an antigen binding molecule, where the method comprises the steps of:

(a) selecting an antigen-binding molecule that has higher binding activity to human FcRn in the neutral pH range than a KD of 3.2 micromolar, wherein the molecule is obtained by altering at least one amino acid in the human FcRn-binding domain of the antigen-binding molecule;

(b) obtaining a gene encoding an antigen binding molecule in which the human FcRn binding domain and the antigen binding domain obtained in step (a) are linked; And

(c) obtaining an antigen binding molecule using the gene obtained in step (b).

In a preferred embodiment, antigen binding molecules of the present invention having binding activity to human FcRn in the acidic and neutral pH ranges can be prepared, including antigen binding molecules that have binding activity to human FcRn with a KD value of 20 micromolar or higher in the acidic range pH values that are equal to or higher than that of intact human IgG in the neutral pH range. In a more preferred embodiment, antigen binding molecules of the present invention can also be prepared, including antigen binding molecules whose binding activity to human FcRn is a KD of 2.0 micromoles or higher in the acidic pH range and a KD of 40 micromoles or more high in the neutral pH range. In an even more preferred embodiment, the antigen binding molecules of the present invention may preferably be prepared including antigen binding molecules whose binding activity to human FcRn is a KD of 0.5 micromolar or higher in the acidic pH range and a KD of 15 micromolar or higher. higher in the range of neutral pH values. The KD values mentioned above are determined in the manner described in Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen binding molecule on a chip and applying human FcRn as the analyte). In one embodiment, an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen binding molecule has binding activity to human FcRn in the acidic and neutral pH ranges, wherein binding activity to human FcRn and lower antigen binding activity in the acidic range The pH, than in the neutral pH range, is higher than the KD of 3.2 micromolar, can be obtained according to methods used by those skilled in the art, as described herein above. In a more preferred embodiment, the human FcRn binding activity of the antigen binding molecules thus obtained at pH 7.0 and 25 degrees C is higher than the KD of 3.2 micromolar.

The present invention relates to a method for producing an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is higher than KD, equal to 2.3 micromoles. The present invention also provides a method for producing an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain that has human FcRn binding activity in the neutral pH range, wherein human FcRn binding activity in the neutral pH range is 38 times higher than that of intact human IgG.

In the production methods described above, the antigen binding activity of the antigen binding molecule in the neutral pH range is not particularly limited, provided that the antigen binding activity is an antigen binding activity at a pH between pH 6.7 and pH 10.0 and includes, for example, the embodiment described in WO 2009/125825. A preferred antigen binding activity is an antigen binding activity at a pH between pH 7.0 and a pH of 8.0, and a more preferred antigen binding activity is an antigen binding activity at a pH of 7.4. Alternatively, the antigen binding activity of the antigen binding molecule in the acidic pH range is not particularly limited, as long as the antigen binding activity is antigen binding activity at a pH between pH 4.0 and pH 6.5. Preferred antigen binding activity is antigen binding activity at a pH between pH 5.5 and pH 6.5, and a more preferred antigen binding activity is antigen binding activity at pH 5.8 or pH 5.5.

The antigen binding activity and human FcRn binding activity of an antigen binding molecule can be determined by methods known to those skilled in the art. Conditions other than pH can be determined accordingly by those skilled in the art.

In the production methods of the present invention, antigen-binding molecules having binding activity to human FcRn in the neutral pH range are not particularly limited as long as they have binding activity to human FcRn at a pH of 6.7 to 10.0. However, the binding activity of human FcRn antigen binding molecules at pH 6.7 to 10.0 is preferably higher than that of intact human IgG. More preferably, antigen binding molecules have a higher binding activity to human FcRn than a KD of 40 micromolar, even more preferably a KD of greater than 15 micromolar.

In the production methods of the present invention, antigen-binding molecules having human FcRn binding activity in the acidic pH range are not particularly limited as long as they have human FcRn binding activity in the pH range of 4.0 to 6.5. However, at a pH of 5.5 to 6.5, antigen binding molecules preferentially have human FcRn binding activity greater than the KD of 20 micromolar. The human FcRn binding activity is more preferably comparable to or greater than that of intact human IgG1 (higher than the KD of 1.7 micromolar), more preferably greater than the KD of 0.5 micromolar.

The KD values described above are determined in the manner described in The Journal of Immunology, (2009) 182: 7663-7671 (by immobilizing an antigen binding molecule on a chip and applying human FcRn as the analyte).

In the production methods of the present invention, the step of selecting antigen binding molecules whose antigen binding activity at pH 6.7 to pH 10.0 is higher than that at pH 4.0 to pH 6.5 is similar to the step of selecting antigen binding molecules whose antigen binding activity at pH 4.0 to pH 6.5 is lower than that at pH 6.7 to pH 10.0.

The relationship between antigen binding activity in the neutral pH range and in the acidic pH range is not particularly limited as long as the antigen binding activity in the neutral pH range is higher than that in the acidic pH range. The antigen binding activity at pH 6.7 to pH 10.0 is preferably two times or greater, more preferably ten times or greater, and even more preferably 40 times or greater than the antigen binding activity at pH 4.0 to pH 6.5.

In the above-described production methods, the antigen and the antigen binding molecule can bind to each other in any state, and the human FcRn and the antigen binding molecule can bind to each other in any state. The condition is not specifically limited; for example, a human antigen or FcRn can interact with an immobilized antigen binding molecule to bind to the antigen binding molecule. Alternatively, the antigen binding molecule may interact with an immobilized human antigen or FcRn to bind to the antigen binding molecule. Alternatively, the antigen binding molecule may react with a human antigen or FcRn in a solution to bind to the antigen binding molecule.

The antigen binding molecules produced by the methods described above may be an antigen binding molecule; and preferred antigen binding molecules include, for example, those having an antigen binding domain and a human FcRn binding domain that comprises a change in at least one amino acid in the human FcRn binding domain, and a histidine substitution with amino acid(s) or insertion of at least one histidine.

Amino acid changes in the human FcRn binding domain are not particularly limited so long as they increase binding activity to human FcRn in the neutral pH range. Variations include, for example, those amino acids located at positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, from 291 to 312, from 315 to 320, 324, 325, from 327 to 339, 341, 343, 345, 360, 362, 370, from 375 to 378, 380, 382, from 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440 and 442 (EU numbering) in the IgG Fc domain described above. More specifically, amino acid changes include those amino acids located at the amino acid positions presented in Tables 1, 2, 6-1 and 6-2 (EU numbering). Preferably, human FcRn binding activity can be increased in the neutral pH range by changing at least one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265 , 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 3 86 , 387, 389, 424, 428, 433, 434 and 436 (EU numbering). The number of amino acids to be changed is not particularly limited; and it is possible to change amino acids in only one region or two or more regions. Combinations of two or more amino acid changes include, for example, those presented in Tables 3, 4-1 to 4-5, 6-1 and 6-2.8

Meanwhile, the site at which the histidine mutation is introduced is not particularly limited, and thus the histidine mutation can be introduced into any site as long as the histidine mutation reduces the antigen binding activity in the acidic pH range to a value less than that in the neutral pH range . Such histidine mutations can be introduced into one or more sites.

Thus, the production methods of the present invention may further comprise the steps of changing the amino acids described above and a histidine substitution or histidine insertion. In the production methods of the present invention, unnatural amino acids can be used in place of histidine. Thus, the present invention can also be understood by replacing the above histidine with unnatural amino acids.

Moreover, in another embodiment, antigen binding molecules that are produced by the production methods described above include, for example, antigen binding molecules containing altered antibody constant regions. Thus, the production methods of the present invention may further comprise the step of changing the amino acids of the antibody constant regions.

Antigen-binding molecules produced by the production methods of the present invention are administered to accelerate the reduction of plasma antigen concentration. Thus, the production methods of the present invention can be used as a method for producing antigen-binding molecules to accelerate the reduction of plasma antigen concentration upon administration.

Alternatively, antigen binding molecules produced by the production methods of the present invention have improved pharmacokinetics. Thus, the production methods of the present invention can be used as a method for producing antigen-binding molecules with improved pharmacokinetics.

Alternatively, antigen binding molecules produced by the production methods of the present invention can increase the number of antigens to which one antigen binding molecule can bind when administered to an animal such as humans, mice and monkeys. Thus, the production methods of the present invention can be used as a method for producing antigen binding molecules that have an increased number of antigens to which one antigen binding molecule can bind.

In addition, antigen binding molecules produced by the production methods of the present invention are expected to be capable within a cell of dissociation from extracellularly bound antigen when administered to animals such as humans, mice or monkeys. Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules that are capable of dissociating within a cell from extracellular-bound antigen.

In addition, antigen-binding molecules produced by the production methods of the present invention are expected to be capable of binding to antigens and are taken up by cells and are also released outside the cells in an antigen-free form when administered to animals such as humans, mice or monkeys. . Thus, the production methods of the present invention can be used as methods for producing antigen-binding molecules that are capable of binding antigen and being taken up by cells and released outside the cells in an antigen-free form.

In addition, since when administered, such antigen-binding molecules have a higher activity for reducing the concentration of antigen in plasma, compared with typical antigen-binding molecules, they are expected to be particularly suitable as drugs. Thus, the production methods of the present invention can be used as methods for producing antigen binding molecules for use as pharmaceutical compositions.

Genes produced by the production methods of the present invention are typically carried (inserted) into appropriate vectors and then introduced into host cells. The vectors are not particularly limited as long as they stably retain the incorporated nucleic acids. For example, if E. coli is used as a host, preferred cloning vectors include the pBluescript vector (Stratagene); however, various commercially available vectors can be used. When using vectors to produce the antigen binding molecules of the present invention, expression vectors are particularly useful. Expression vectors are not particularly limited as long as the vectors express antigen binding molecules in vitro, in E. coli, in cell culture, or in vivo. For example, the pBEST vector (Promega) is preferred for in vitro expression; the pET vector (Invitrogen) is preferred for E. coli; the pME18S-FL3 vector (GenBank accession number AB009864) is preferred for cell culture; and the vector pME18S (Mol Cell Biol. (1988) 8:466-472) is preferred for organisms. The DNA of the present invention can be inserted into vectors by conventional methods, for example, by ligation using restriction enzyme recognition sites (Current Protocols in Molecular Biology, edit. Ausubel et al., (1987) Publish. John Wiley & Sons, Section 11.4-11.11) .

The host cells mentioned above are not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing antigen binding molecules include bacterial cells (such as Streptococcus, Staphylococcus, E. coli, Streptomyces and Bacillus subtilis), eukaryotic cells (such as yeast and Aspergillus), insect cells (such as Drosophila S2 and Spodoptera SF9), animal cells (such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293 and Bowes melanoma cells), and plant cells. Vectors can be introduced into the host cell by known methods, for example, calcium phosphate precipitation methods, electroporation methods (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons, Section 9.1-9.9), lipofection methods and microinjections.

Host cells can be cultured by known methods. For example, when using animal cells as the host, DMEM, MEM, RPMI1640 or IMDM can be used as the culture medium. Media can be used with serum supplements such as FBS or fetal calf serum (FCS). Cells can be cultured in serum-free cultures. The preferred pH is approximately 6 to 8 during the culture course. Incubation is typically carried out at 30 to 40 degrees C for approximately 15 to 200 hours. The medium is replaced, aerated or stirred as necessary.

Suitable secretion signals can be included in the polypeptides of interest, provided that the antigen binding molecules expressed by the host cell are secreted into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the antigen-binding molecules of interest or may be heterologous signals.

On the other hand, for example, production systems using animals or plants can be used as in vivo polypeptide production systems. The polynucleotide of interest is administered to an animal or plant, and the polypeptide is produced in the animal or plant and then collected. "Hosts" of the present invention include such animals and plants.

Animal-based production systems include mammalian or insect-based production systems. It is possible to use mammals such as goats, pigs, sheep, mice and bulls (Vicki Glaser SPECTRUM Biotechnology Applications (1993)). Mammals may be transgenic animals.

For example, a polynucleotide encoding an antigen binding molecule of the present invention is produced as a gene fused to a gene encoding a polypeptide produced, in particular, in milk, such as goat beta-casein. Next, polynucleotide fragments containing the fusion gene are injected into goat embryos and then transplanted into female goats. The desired antigen-binding molecules can be obtained from milk obtained from transgenic goats that are born to embryonated goats or their offspring. If necessary, hormones can be administered to increase the volume of milk containing the antigen binding molecule produced by the transgenic goat (Ebert et al., Bio/Technology (1994) 12: 699-702).

Insects such as silkworms can be used to produce the antigen binding molecules of the present invention. When using the silkworm, baculoviruses carrying a polynucleotide encoding an antigen binding molecule of interest can be used to infect the silkworm, and the antigen binding molecule of interest can be obtained from its body fluids.

In addition, when plants are used to produce the antigen binding molecules of the present invention, for example, tobacco can be used. If tobacco is used, the polynucleotide encoding the antigen binding molecule of interest is inserted into a plant expression vector, for example pMON 530, and then the vector is inserted into bacteria such as Agrobacterium tumefaciens. The bacteria are then allowed to infect tobacco, such as Nicotiana tabacum, and the required antigen binding molecules can be collected from its leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-138). Alternatively, it is possible to infect duckweed (Lemna minor) with similar bacteria. After cloning, the desired antigen-binding molecules can be obtained from duckweed cells (Cox KM et al., Nat. Biotechnol. 2006 Dec; 24(12): 1591-1597).

The antigen binding molecules thus obtained can be isolated from the interior or exterior of host cells (such as media and milk) and purified as substantially pure and homogeneous antigen binding molecules. Methods for isolating and purifying antigen-binding molecules are not particularly limited, and isolation and purification methods generally used for purifying polypeptides can be used. Antigen-binding molecules can be isolated and purified through suitable selection and combination, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis and recrystallization.

Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, reverse phase chromatography and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., (1996) Cold Spring Harbor Laboratory Press). Such chromatographic methods can be carried out using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include Protein A and Protein G columns. Columns using Protein A include, for example, Hyper D, POROS and Sepharose F. F. (Pharmacia).

If desired, the antigen binding molecule can be modified at will and the peptides can be partially deleted, providing a suitable protein modification of the enzyme to act before or after purification of the antigen binding molecule. Such protein modification enzymes include, for example, trypsin, chymotrypsin, lysyl endopeptidases, protein kinases and glucosidases.

<Pharmaceutical compositions>

The present invention also provides pharmaceutical compositions that include the antigen binding molecules of the present invention, antigen binding molecules isolated by the screening methods of the present invention, or antigen binding molecules obtained by the production methods of the present invention. The antigen-binding molecules of the present invention and the antigen-binding molecules produced by the production methods of the present invention have superior plasma antigen concentration-lowering activity upon administration compared to typical antigen-binding molecules, and are thus useful as pharmaceutical compositions. The pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers.

In the present invention, pharmaceutical compositions generally relate to agents for treating or preventing or testing and diagnosing a disease.

The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, the pharmaceutical compositions can be used parenterally, in the form of injections of sterile solutions or suspensions comprising water or other pharmaceutically acceptable liquid. For example, such compositions can be formulated by mixing in unit dose form generally required in an approved medical manufacturing technology by combining, as appropriate, with pharmaceutically acceptable carriers or media, particularly sterile water, saline, vegetable oil, emulsifier, suspension, surface -an active substance, stabilizer, flavoring agent, excipient, carrier, preservative, binding agent or the like. In such formulations, the amount of active ingredient is adjusted to obtain a suitable amount within a predetermined range.

Sterile injectable compositions can be formulated using vehicles such as distilled water for injection, according to standard formulation practices. Aqueous solutions for injection include, for example, saline and isotonic solutions containing dextrose or other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, and sodium chloride). It is also possible to use suitable solubilizers in combination, for example, alcohols (ethanol and the like), polyalcohols (propylene glycol, polyethylene glycol and the like), nonionic surfactants (polysorbate 80(TM), HCO-50 and the like).

Oils include sesame and soybean oils. Benzyl benzoate and/or benzyl alcohol can be used in combination as solubilizers. It is also possible to combine buffers (eg phosphate buffer and sodium acetate buffer), demulcents (eg procaine hydrochloride), stabilizers (eg benzyl alcohol and phenol) and/or antioxidants. Suitable ampoules are filled with the resulting injection solutions.

The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, the compositions may be in dosage form for injection, transnasal administration, transpulmonary administration or transdermal administration. For example, pharmaceutical compositions can be administered systemically or locally via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

The routes of administration can be appropriately selected based on the age and symptoms of the patient. The dose of the pharmaceutical composition containing the antigen binding molecule may be a dose of, for example, 0.0001 to 1000 mg/kg for each administration. Alternatively, the dose may be, for example, a dose of from 0.001 to 100,000 mg per patient. However, the present invention is not limited to the numerical values described above. Dosages and routes of administration vary depending on the weight, age, symptoms of the patient, etc. Those skilled in the art can determine appropriate doses and routes of administration, taking into account the factors described above.

The amino acids contained in the amino acid sequences of the present invention may be modified after translation. For example, modification of N-terminal glutamine to pyroglutamic acid through pyroglutamylation is well known to those skilled in the art. As would be expected, such post-translation amino acid modifications are included in the amino acid sequences of the present invention.

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

EXAMPLES

Hereinafter, the present invention will be specifically described with reference to examples, but these should not be construed as limiting.

[Example 1] Study on enhancing the accelerating effect of antibody on antigen elimination

Anti-IL-6 receptor antibody

Preparation of an antibody against the human IL-6 receptor with FcRn binding activity under neutral conditions

H54/L28-IgG1, containing H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), described in WO 2009/125825, is a humanized anti-IL-6 receptor antibody. A mutation was introduced into H54 (SEQ ID NO: 1) to increase binding to FcRn under neutral pH conditions (pH 7.4). Specifically, H54-IgG1-F14 (SEQ ID NO: 3) was obtained from the constant region of the heavy chain of IgG1 by replacing Trp with Met at position 252 and Trp with Asn at position 434 in EU numbering. Amino acid substitutions were introduced by methods known to those skilled in the art, as described in Reference Example 1.

H54/L28-IgG1 containing H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), and H54/L28-IgG1-F14 containing H54-IgG1-F14 (SEQ ID NO: 3) and L28 ( SEQ ID NO: 2), expressed and purified in a manner known to those skilled in the art, described in reference example 2.

In vivo antibody testing via a steady-state infusion model using the human FcRn transgenic mouse line 276

Using H54/L28-IgG1 and H54/L28-IgG1-F14 prepared as described above, in vivo testing was carried out through a steady-state infusion model using the human FcRn transgenic mouse line 276. An infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) containing a soluble human IL-6 receptor was implanted under the dorsal skin of human FcRn transgenic line 276 mice (B6.mFcRn-/-.hFcRn Tg line 27 6+/+ mouse ( B6.mFcRn-/-hFCRnTg276B6.Cg-Fcgrt<tm1Dcr>Tg(FCGRT)276Dcr(Jackson#4919)), Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104) to obtain an animal model in which the concentration in plasma, the soluble human IL-6 receptor was kept constant. Antibodies against the human IL-6 receptor were administered to animal models to evaluate in vivo dynamics following administration of soluble human IL-6 receptor. Monoclonal antibody against mouse CD4 (R&D) was administered at a dose of 20 mg/kg before implantation of the infusion pump and 14 days after injection of the antibody into the tail vein to suppress the production of neutralizing antibody against the soluble human IL-6 receptor. Next, an infusion pump containing 92.8 micrograms/ml of soluble human IL-6 receptor was implanted under the skin of the back of the mice. Three days after implantation of the infusion pump, antibodies against the human IL-6 receptor (H54/L28-IgG1 and H54/L28-IgG1-F14) were injected at a rate of 1 mg/kg once into the tail vein. Blood was collected 15 minutes, seven hours, one day, two days, three days, four days, seven days, 14 days, 21 days and 28 days after administration of the anti-human IL-6 receptor antibody. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -20 degrees C or below until analysis.

Determination of plasma hsIL-6R concentration by electrochemiluminescence assay

The concentration of hsIL-6R in mouse plasma was measured by electrochemiluminescence. hsIL-6R calibration curve samples were adjusted to concentrations of 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg/mL, and mouse plasma samples diluted 50-fold or more were obtained. Samples were mixed with a solution of ruthenium-labeled anti-human IL-6R (R&D) monoclonal antibody with sulfo-Tag-NHS ester (Meso Scale Discovery), biotinylated anti-human IL-6R (R&D) antibody and WT-IgG1, and then left for an overnight reaction at 37 degrees C. Final concentration of WT-IgG1 as an anti-human IL-6 receptor antibody containing H (WT) (SEQ ID NO: 4) and L (WT) (SEQ ID NO: 5), was 333 micrograms/ml, which exceeds the concentration of anti-human IL-6 receptor antibody contained in the samples to bind virtually all hsIL-6R molecules in the WT-IgG1 samples. Samples were then transferred to a MA400 PR streptavidin plate (Meso Scale Discovery) and allowed to react for one hour at room temperature and washed. Read Buffer T (×4) (Meso Scale Discovery) was then immediately added and measurements were taken using a Sector PR 400 Reader (Meso Scale Discovery). The concentration of hsIL-6R was calculated based on the calibration curve data using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma hsIL-6R concentrations following intravenous administration of H54/L28-IgG1 and H54/L28-IgG1-F14 as measured by this method is shown in FIG. 1.

As shown in Fig. 1, compared to a baseline hsIL-6R concentration without antibody, administration of H54/L28-IgG1 resulted in a significant increase in plasma hsIL-6R concentration. On the other hand, administration of H54/L28-IgG1-F14 resulted in a decrease in the increase in plasma hsIL-6R concentration compared to H54/L28-IgG1. This decrease in increase results from increased binding to human FcRn at neutral pH in H54/L28-IgG1-F14 compared to H54/L28-IgG1. This demonstrates that increasing antibody binding affinity to FcRn at neutral pH can increase antigen clearance, although the extent of increased antigen clearance was small for H54/L28-IgG1-F14 compared to H54/L28-IgG1.

[Example 2] Study on enhancing the accelerating effect of antigen elimination of antigen-binding antibodies dependent on pH (antibody production)

Consideration of pH-dependent antibodies binding the human IL-6 receptor

H54/L28-IgG1, containing H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), described in WO 2009/125825, is a humanized anti-IL-6 receptor antibody. Fv4-IgG1, containing VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7), is a humanized anti-IL-6 receptor antibody that results from imparting soluble soluble binding properties to H54/L28-IgG1. human IL-6 receptor in a pH-dependent manner (which binds at pH 7.4 but dissociates at pH 5.8). The in vivo test described in WO 2009/125825 using mice demonstrated that the elimination of soluble human IL-6 receptor was significantly increased in the group administered a mixture of Fv4-IgG1 and soluble human IL-6 receptor as antigen compared to the group , which was injected with a mixture of H54/L28-IgG1 and soluble human IL-6 receptor as antigen.

The soluble human IL-6 receptor binds to a generic antibody, which binds to the soluble human IL-6 receptor and is recycled into the plasma along with the antibody via FcRn. Meanwhile, the antibody that binds to the soluble human IL-6 receptor in a pH-dependent manner dissociates from the soluble human IL-6 receptor that binds to the antibody under acidic conditions in the endosome. The dissociated soluble human IL-6 receptor is degraded in the lysosome. This may greatly enhance the elimination of soluble human IL-6 receptor. The antibody, which binds to the soluble human IL-6 receptor in a pH-dependent manner, is then recycled into the plasma via FcRn. The recycled antibody can bind to another soluble human IL-6 receptor again. By repeating this cycle, one antibody molecule can repeatedly bind to the soluble human IL-6 receptor many times (Figure 2).

Antibodies that bind to antigens in a pH-dependent manner increase the elimination of soluble antigen. Antibodies work by repeatedly binding to a soluble antigen many times. Thus, such antibodies are very useful. A method of enhancing FcRn binding under neutral conditions (pH 7.4) was tested to further enhance the accelerating effect of antigen elimination.

Preparation of pH-dependent human IL-6 receptor binding antibodies with FcRn binding activity under neutral conditions

Mutations were introduced into Fv4-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) to increase binding to FcRn under neutral conditions (pH 7.4). Specifically, VH3-IgG1-v1 (SEQ ID NO: 8) was prepared from the IgG1 heavy chain constant region by replacing Tyr with Met at position 252, Thr with Ser at position 254, and Glu with Thr at position 256 in EU numbering, whereas VH3-IgG1-v2 (SEQ ID NO: 9) was constructed from the heavy chain constant region of IgG1 by replacing Trp with Asn at position 434 in EU numbering. Amino acid substitutions are introduced by a method known to those skilled in the art, as described in Reference Example 1.

H54/L28-IgG1 containing H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), Fv4-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7 ), Fv4-IgG1-v1 containing VH3-IgG1-v1 (SEQ ID NO: 8) and VL3-CK (SEQ ID NO: 7), and Fv4-IgG1-v2 containing VH3-IgG1-v2 (SEQ ID NO : 9) and VL3-CK (SEQ ID NO: 7), were expressed and purified in a manner known to those skilled in the art, as described in Reference Example 2.

[Example 3] Study on increasing the antigen elimination-accelerating effect of pH-dependent antigen-binding antibody (in vivo test)

In vivo test using human FcRn transgenic mice and normal mice

In vivo kinetics of hsIL-6R (soluble human IL-6 receptor: prepared as described in Reference Example 3) and anti-human IL-6 receptor antibodies were assessed following administration to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276 +/+ mouse, Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104) and normal mice (C57BL/6J mice; Charles River Japan) hsIL-6R alone or hsIL-6R and anti-IL-receptor antibodies 6 people in combination. A solution of hsIL-6R (5 micrograms/ml) or a solution of a mixture containing hsIL-6R and antibodies against the human IL-6 receptor (5 micrograms/ml and 0.1 mg/ml, respectively) was administered once at a dose of 10 ml/kg into the tail vein. In this case, antibodies against the human IL-6 receptor are present in excess over hsIL-6R, and thus almost every hsIL-6R is predicted to bind to the antibody. Blood was collected after 15 minutes, seven hours, one day, two days, three days, four days, seven days, 14 days, 21 days and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -20 degrees or below before analysis. Anti-human IL-6 receptor antibodies used are: H54/L28-IgG1, Fv4-IgG1 and Fv4-IgG1-v2 for human FcRn transgenic mice described above and H54/L28-IgG1, Fv4-IgG1, Fv4-IgG1 described above -v1 and Fv4-IgG1-v2 for normal mice.

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

The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA. The (Fab')2 fragment of anti-human IgG (gamma chain specific) antibody (Sigma) was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare immobilized anti-human IgG plates. Calibration curve samples having plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 microgram/mL and mouse plasma samples diluted 100-fold or more were prepared. 200 microliters (μl) of 20 ng/ml hsIL-6R was added to 100 microliters of calibration curve samples and plasma samples, and then the samples were left for one hour at room temperature. The samples were then transferred to anti-human IgG immobilized plates and left for one hour at room temperature. Biotinylated anti-human IL-6R (R&D) antibody was then added to react for one hour at room temperature. Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was then added to react for one hour at room temperature, and the chromogenic reaction was carried out using TMP One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After stopping the reaction 1 N. sulfuric acid (Showa Chemical), absorbance at 450 nm was measured using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma concentrations after intravenous administration, as measured by this method, is presented in Fig. 3 for human FcRn transgenic mice and in Fig. 5 for normal mice.

Measurement of plasma hsIL-6R concentration by electrochemiluminescence assay

The concentration of hsIL-6R in mouse plasma was measured by electrochemiluminescence. hsIL-6R calibration curve samples adjusted to concentrations of 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg/mL and mouse plasma samples diluted 50-fold or more were prepared. Samples were mixed with a solution of ruthenium-labeled anti-IL-6R monoclonal antibody (R&D), sulfo-Tag NHS ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and WT-IgG1, and then allowed to react overnight at 37 degrees C. The final concentration of WT-IgG1 as an anti-human IL-6 receptor antibody containing H (WT) (SEQ ID NO: 4) and L (WT) (SEQ ID NO: 5) was 333 microgram/ml, which is greater than the concentration of anti-human IL-6 receptor antibody contained in the samples to bind virtually all hsIL-6R molecules in WT-IgG1 samples. Samples were then transferred to a MA400 PR streptavidin plate (Meso Scale Discovery) and allowed to react for one hour at room temperature and washed. Read Buffer T (×4) (Meso Scale Discovery) was then immediately added and measurements were taken using a Sector PR 400 Reader (Meso Scale Discovery). The concentration of hsIL-6R was calculated based on the calibration curve data using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma hsIL-6R concentrations following intravenous administration of H54/L28-IgG1 and H54/L28-IgG1-F14 as measured by this method is shown in FIG. 4 for human FcRn transgenic mice and in FIG. 6 for normal mice.

Determination of plasma free hsIL-6R concentration by electrochemiluminescence assay

To assess the extent of neutralization of soluble human IL-6 receptor in plasma, the concentration of soluble human IL-6 receptor free from (non-neutralized by) anti-human IL-6 receptor antibody (free hsIL-6R concentration) in mouse plasma was determined by electrochemiluminescence assay. All IgG antibodies (mouse IgG, anti-human IL-6 receptor antibodies, and anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex) in plasma were adsorbed to protein A by adding 12 microliters of each hsIL-6R standard obtained at 10,000, 5,000, 2,500, 1,250, 625, 312.5, or 156.25 pg/mL and mouse plasma samples per appropriate amount of rProtein A Sepharose Fast Flow resin (GE Healthcare) dried on a 0.22 micrometer plate filter (Millipore). Next, the solution in the dish was pelleted using a high-speed centrifuge to collect the solution, which was passed through the resin. The resin solution did not contain the protein A-bound anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex. Thus, the concentration of free hsIL-6R in plasma can be determined by measuring the concentration of hsIL-6R in the resin solution.

The resin solution was then mixed with ruthenium-labeled anti-human IL-6R (R&D) monoclonal antibody with sulfo-Tag NHS ester (Meso Scale Discovery) and biotinylated anti-human IL-6R (R&D) antibody. The resulting mixture was incubated at room temperature for one hour and then added to a MA400 PR streptavidin plate and aliquoted (Meso Scale Discovery). After one hour of incubation at room temperature, the plate was washed and also aliquoted with Read Buffer T (×4) (Meso Scale Discovery). The plate reaction results were immediately measured in a SECTOR PR 400 Reader (Meso Scale Discovery). The concentration of hsIL-6R was calculated based on the data in the standard curve using analysis on SOFTmax PRO software (Molecular Devices). The dynamics of changes in the concentration of free hsIL-6R in the plasma of normal mice after intravenous administration was determined in the manner described above, as shown in Fig. 7.

Effect of pH-dependent binding to the human IL-6 receptor

H54/L28-IgG1 and Fv4-IgG1, which bind to the human IL-6 receptor in a pH-dependent manner, were tested in vivo and the results were compared with each other. As shown in Figures 3 and 5, antibody retention in plasma was comparable. Meanwhile, as shown in Figures 4 and 6, hsIL-6R coadministered with Fv4-IgG1, which binds to the human IL-6 receptor in a pH-dependent manner, was found to increase the elimination of hsIL-6R compared to hsIL-6R , administered simultaneously with H54/L28-IgG1. The trend described above was observed in both human FcRn transgenic mice and normal mice; Thus, it is shown that by imparting a pH-dependent binding ability to the human IL-6 receptor, the plasma concentration of hsIL-6R four days after administration can be reduced by approximately 17- and 34-fold, respectively.

Effect of binding to FcRn under neutral conditions (pH 7.4)

Intact human IgG1 has been reported to bind poorly to human FcRn (has very low binding affinity) under neutral conditions (pH 7.4). Binding to human FcRn under neutral conditions (pH 7.4) is reported to be increased by replacing Trp with Asn at position 434 (EU numbering) in intact human IgG1 (J Immunol. (2009) 182(12):7663-71) . Fv4-IgG1-v2, which is obtained by introducing the above amino acid substitution into Fv4-IgG1, was tested through an in vivo test using human FcRn transgenic mice. The test results were compared with those obtained for Fv4-IgG1. As shown in Figure 3, antibody retention time was comparable between the two antibodies. Meanwhile, as shown in Figure 4, hsIL-6R was coadministered with Fv4-IgG1-v2, which exhibits increased binding to human FcRn under neutral conditions (pH 7.4), was found to be eliminated faster compared to hsIL-6R administered simultaneously with Fv4-IgG1. Thus, it has been shown that by imparting the ability to bind to human FcRn under neutral conditions (pH 7.4), the plasma concentration of hsIL-6R four days after administration can be reduced approximately fourfold.

Based on the homology between human FcRn and mouse FcRn, the substitution of Trp with Asn at position 434 in EU numbering is predicted to increase binding to mouse FcRn under neutral conditions (pH 7.4). Meanwhile, binding to mouse FcRn under neutral conditions (pH 7.4) has been reported to be increased by substitution of Tyr to Met at position 252, Thr to Ser at position 254, and Glu to Thr at position 256 in EU numbering (J Immunol. (2002) 169(9): 5171-80). Fv4-IgG1-v1 and Fv4-IgG1-v2, which are obtained by introducing the amino acid substitutions described above into Fv4-IgG1, were tested in vivo using normal mice. The test results were compared with the results obtained for Fv4-IgG1. As shown in Figure 5, the plasma retention time of Fv4-IgG1-v1 and Fv4-IgG1-v2, which were enhanced to increase binding to mouse FcRn under neutral conditions (pH 7.4), was slightly shortened (plasma concentrations of neutralizing antibody one day after administration decreased by approximately 1.5 and 1.9 times, respectively) compared to Fv4-IgG1.

As shown in Fig. 6, hsIL-6R coadministered with Fv4-IgG1-v1 or Fv4-IgG1-v2, which were modified to increase binding to mouse FcRn under neutral conditions (pH 7.4), was shown to be cleared clearly faster compared with hsIL-6R administered simultaneously with Fv4-IgG1. Fv4-IgG1-v1 and Fv4-IgG1-v2 reduced the concentration of hsIL-6R in plasma one day after administration by approximately 32 and 80 times, respectively. Thus, it was found that plasma concentrations can be reduced by imparting the ability to bind to mouse FcRn under neutral conditions (pH 7.4). As described above, by imparting binding ability to mouse FcRn under neutral conditions (pH 7.4), plasma antibody concentrations were slightly reduced; however, they obtained an effect of reducing plasma hsIL-6R concentrations that was significantly superior to the reduction in antibody concentrations. In addition, hsIL-6R administered simultaneously with Fv4-IgG1-v1 or Fv4-IgG1-v2 was found to be eliminated even faster compared to the group in which hsIL-6R was administered alone. As shown in Fig. 6, it is shown that hsIL-6R, administered simultaneously with Fv4-IgG1-v1 or Fv4-IgG1-v2, can reduce the plasma concentration of hsIL-6R one day after administration by approximately 4-fold or 11-fold, respectively. , compared to hsIL-6R alone. Specifically, this means that elimination of soluble IL-6 receptor can be accelerated by administration of an antibody that binds to soluble IL-6 receptor in a pH-dependent manner and is rendered capable of binding to mouse FcRn under neutral conditions (pH 7.4). Specifically, the plasma concentration of an antigen can be reduced in vivo by introducing such an antibody into the body.

As shown in FIG. 7, free hsIL-6R was present in a detectable concentration range seven days after H54/L28-IgG1 administration, while hsIL-6R was undetectable one day after Fv4-IgG1 administration. On the other hand, free hsIL-6R was undetectable seven hours after administration of Fv4-IgG1-v1 or Fv4-IgG1-v2. Specifically, the concentration of free hsIL-6R was lower in the presence of Fv4-IgG1, which binds to hsIL-6R in a pH-dependent manner, compared to H54/L28-IgG1, suggesting a stronger neutralizing effect of hsIL-6R that is obtained by providing pH-dependent binding ability to hsIL-6R. In addition, the free concentration of hsIL-6R was lower in the presence of Fv4-IgG1-v1 or Fv4-IgG1-v2, both of which are modified from Fv4-IgG1 to increase binding ability to FcRn at pH 7.4. This demonstrates that a stronger neutralizing effect of hsIL-6R can be obtained by increasing FcRn binding activity at pH 7.4.

When administered, a common neutralizing antibody, such as H54/L28-IgG1, reduces the clearance of the bound antigen, resulting in a prolonged plasma retention time of the antigen. It is not preferred that the administered antibodies prolong the retention time of the antigen, whose action should be neutralized by the antibodies. The antigen retention time can be shortened by making the antigen binding process pH dependent (the antibody binds under neutral conditions but dissociates under acidic conditions). In the present invention, the retention time of the antigen in plasma could be further shortened by further providing binding ability to human FcRn under neutral conditions (pH 7.4). In addition, it has been shown that, compared to antigen clearance alone, antigen clearance could be further increased by administration of an antibody that binds to antigen in a pH-dependent manner and that is provided with FcRn binding ability under neutral conditions (pH 7.4). Currently, there is no method suitable for increasing the clearance of an antigen by administering an antibody relative to the clearance of the antigen alone. Thus, the methods carried out as described in this EXAMPLE are very useful as a method for eliminating antigens from plasma by administering antibodies. In addition, the present inventors were the first to discover the benefit of increasing FcRn binding capacity under neutral conditions (pH 7.4). In addition, both v4-IgG1-v1 and Fv4-IgG1-v2, which have different amino acid substitutions that increase FcRn binding ability under neutral conditions (pH 7.4), produced comparable effects. This suggests that, regardless of the type of amino acid substitution, each amino acid substitution that increases binding capacity to human FcRn under neutral conditions (pH 7.4) potentially has an effect that increases antigen clearance. Specifically, antibody molecules that, when administered, eliminate antigens from plasma, can be prepared using the following amino acid substitutions, either individually or in combination:

an Ile to Pro amino acid change at position 257 and an Ile to Gln amino acid change at position 311 in EU numbering, each described in J Biol Chem. 2007, 282(3): 1709-17; replacement of the amino acid Ala, Tyr or Trp with Asn at position 434, replacement of the amino acid Tyr with Met at position 252, replacement of the amino acid Gln with Thr at position 307, replacement of the amino acid Pro with Val at position 308, replacement of the amino acid Gln with Thr at position 250, replacement Leu to Met amino acid at position 428, Ala to Glu amino acid change at position 380, Val to Ala amino acid change at position 378, Ile to Tyr amino acid change at position 436 in EU numbering, each of which is described in J Immunol. (2009) 182(12): 7663-71; Tyr to Met amino acid change at position 252, Thr to Ser amino acid change at position 254, Glu to Thr amino acid change at position 256 in EU numbering, each described in J Biol Chem. 2006 Aug. 18, 281(33): 23514-24; a Lys to His amino acid change at position 433, a Phe to Asn amino acid change at position 434, and a His to Tyr amino acid change at position 436 in EU numbering, each of which is described in Nat Biotechnol. 2005 Oct 23(10): 1283-8; and so on.

[Example 4] Evaluation of human FcRn binding activity

For an assay system based on Biacore for testing the interaction between antibody and FcRn, a system that immobilizes the antibody onto a sensor chip and uses human FcRn as the analyte is described in J Immunol. (2009) 182(12): 7663-71. For this purpose, human FcRn was prepared as described in Reference Example 4. Fv4-IgG1, Fv4-IgG1-v1 and Fv4-IgG1-v2 were assessed for human FcRn binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 using the system described above. Antibodies were tested as a test substance after direct immobilization onto the Series S CM5 sensor chip. Using an amine coupled kit according to the manufacturer's instructions, antibodies were immobilized on the sensor chip so as to ensure an immobilization amount of 500 RU (resonance units). The running buffer used was 50 mmol/1 Na-phosphate/150 mmol/1 NaCl containing 0.05% (v/v%) surfactant P20 (pH 6.0).

After obtaining the sensor chips, the analysis was carried out using 50 mmol/1 Na-phosphate/150 mmol/1 NaCl containing 0.05% surfactant P20 (pH 6.0) or 50 mmol/1 Na as a running buffer -phosphate/150 mmol/1 NaCl containing 0.05% surfactant P20 (pH 7.4). Assays were performed exclusively at 25 degrees C. Diluted human FcRn solutions and a running buffer control were injected at a flow rate of 5 microliters/min for ten minutes to allow the human FcRn to interact with the antibody on the chip. The running buffer was then injected at a flow rate of 5 microliters/min for one minute to monitor FcRn dissociation. The sensor chip was then regenerated through two rounds of injection with 20 mmol/1 Tris-HCl/150 mmol/1 NaCl (pH 8.1) at a flow rate of 30 microliters/min for 15 seconds.

The results were analyzed using Biacore T100 Evaluation software (Ver. 2.0.1). Using the steady-state affinity method, the dissociation constant (KD) was calculated from analysis of the results at six different concentrations of FcRn. The human FcRn binding activity results (dissociation constants (KD)) of Fv4-IgG1, Fv4-IgG1-v1 and Fv4-IgG1-v2 at pH 6.0 and pH 7.4 are presented in Table 5 below.

Table 5 KD (µM) pH 6.0 pH 7.4 Fv4-IgG1 1.99 N.A. Fv4-IgG1-v1 0.32 36.55 Fv4-IgG1-v2 0.11 11.03

At pH 7.4, binding of human FcRn to Fv4-IgG1 was too weak to determine the KD (NA) value. Meanwhile, Fv4-IgG1-v1 and Fv4-IgG1-v2 were observed to bind to human FcRn at pH 7.4, and the KD value was determined to be 36.55 and 11.03 micromolar, respectively. KD values for human FcRn at pH 6.0 were determined to be 1.99, 0.32, and 0.11 micromolar. As shown in Fig. 3, when compared with Fv4-IgG1, Fv4-IgG1-v2 enhanced the elimination of hsIL-6R in human FcRn transgenic mice. Thus, it can be predicted that antigen elimination is accelerated by increasing binding to human FcRn at pH 7.4, at least higher than 11.03 micromolar by changing human IgG1. Meanwhile, as described in J Immunol. (2002) 169(9): 5171-80, human IgG1 binds approximately ten times more strongly to mouse FcRn than to human FcRn. For this reason, Fv4-IgG1-v1 and Fv4-IgG1-v2 are also predicted to bind approximately ten times more strongly to mouse FcRn than to human FcR at pH 7.4. The enhanced clearance of hsIL-6R by Fv4-IgG1-v1 or Fv4-IgG1-v2 in the normal mouse shown in Fig. 6 is more significant than the enhanced clearance by Fv4-IgG1-v2 in the human FcRn transgenic mice shown in Fig. .4. This suggests that the increase in hsIL-6R clearance is enhanced by the strength of binding to FcRn at pH 7.4.

[Example 5] Preparation of pH-dependent human IL-6 receptor binding antibodies with increased human FcRn binding under neutral conditions

Various changes to increase binding to human FcRn under neutral conditions were introduced into Fv4-IgG1 to further enhance the antigen clearance effect of pH-dependent human IL-6 receptor binding antibodies in human FcRn transgenic mice. Specifically, the amino acid changes presented in Tables 6-1 and 6-2 were introduced into the heavy chain constant region of Fv4-IgG1 to produce various mutants (the number of amino acid mutation sites is presented according to EU numbering). Amino acid substitutions were introduced by methods known to those skilled in the art, as described in Reference Example 1.

Table 6-2 is a continuation of Table 6-1.

Each variant containing the resulting heavy chain and L(WT) (SEQ ID NO: 5) was expressed and purified by methods known to those skilled in the art, as described in Reference Example 2.

Assessment of binding to human FcRn

Binding between antibody and human FcRn was analyzed kinetically using Biacore T100 (GE Healthcare). For this purpose, human FcRn was prepared as described in Reference Example 4. An appropriate amount of protein L (ACTIGEN) was immobilized onto a CM4 sensor chip (GE Healthcare) via a coupled amine method and the antibodies of interest were retained on the chip. Next, diluted FcRn solutions and running buffer (as a control solution) were injected to allow human FcRn to interact with the antibody held on the sensor chip. The running buffer used contained 50 mmol/1 sodium phosphate, 150 mmol/1 NaCl, and 0.05% (w/v) Tween20 (pH 7.0). FcRn was diluted using each buffer. The chip was regenerated using 10 mmol/1 glycine-HCl (pH 1.5). Assays were performed exclusively at 25 degrees C. The association rate constant ka (1/Ms) and the dissociation rate constant _kd (1/s), each of which are kinetic parameters, were calculated based on the sensorgrams obtained in the assays and KD ( M) of each antibody for human FcRn was determined from these values. Each parameter was calculated using Biacore T100 Evaluation software (GE Healthcare).

The result of assessing human FcRn binding under neutral conditions (pH 7.0) by Biacore is presented in Tables 6-1 and 6-2. The KD of intact IgG1 could not be calculated as it showed only very weak binding. Thus, KD is represented as ND in Table 6-1.

[Example 6] In vivo testing of pH-dependent human IL-6 receptor binding antibodies with increased human FcRn binding under neutral conditions

pH-dependent human IL-6 receptor binding antibodies with human FcRn binding ability under neutral conditions were prepared using the heavy chains prepared as described in Example 4 to impart human FcRn binding ability under neutral conditions. Antibodies were evaluated for their antigen clearance effect in vivo. Specifically, the antibodies listed below were expressed and purified by methods known to those skilled in the art, as described in Reference Example 2:

Fv4-IgG1, containing VH3-IgG1 and VL3-CK;

Fv4-IgG1-v2, containing VH3-IgG1-v2 and VL3-CK;

Fv4-IgG1-F14, containing VH3-IgG1-F14 and VL3-CK;

Fv4-IgG1-F20, containing VH3-IgG1-F20 and VL3-CK;

Fv4-IgG1-F21, containing VH3-IgG1-F21 and VL3-CK;

Fv4-IgG1-F25, containing VH3-IgG1-F25 and VL3-CK;

Fv4-IgG1-F29, containing VH3-IgG1-F29 and VL3-CK;

Fv4-IgG1-F35, containing VH3-IgG1-F35 and VL3-CK;

Fv4-IgG1-F48, containing VH3-IgG1-F48 and VL3-CK;

Fv4-IgG1-F93, containing VH3-IgG1-F93 and VL3-CK; And

Fv4-IgG1-F94, containing VH3-IgG1-F94 and VL3-CK.

By such methods as described in Example 3, the resulting pH-dependent human IL-6 receptor binding antibodies were tested in vivo using human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276 +/+ mouse, Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104).

The dynamics of the plasma concentration of soluble human IL-6 receptor after intravenous administration to transgenic mice with human FcRn is presented in Fig. 8. Test results showed that plasma concentrations of soluble human IL-6 receptor were maintained at low levels over time in the presence of any of the pH-dependent human IL-6 receptor binding antibodies, with increased human FcRn binding under neutral conditions compared to binding in the presence of Fv4-IgG1, which has almost no binding ability to human FcRn under neutral conditions. Among others, antibodies for which significant effects have been obtained include, for example, Fv4-IgG1-F14. The plasma concentration of soluble human IL-6 receptor coadministered with Fv4-IgG1-F14 showed an approximately 54-fold decrease one day after administration compared with that of soluble human IL-6 receptor coadministered with Fv4-IgG1. In addition, plasma concentrations of soluble human IL-6 receptor coadministered with Fv4-IgG1-F21 showed an approximately 24-fold decrease seven hours after administration compared with that of soluble human IL-6 receptor coadministered with Fv4-IgG1 . In addition, the plasma concentration of soluble human IL-6 receptor coadministered with Fv4-IgG1-F25 seven hours after administration was below the detection limit (1.56 ng/ml). Thus, Fv4-IgG1-F25, as expected, promotes a striking 200-fold or more reduction in the concentration of soluble human IL-6 receptor relative to the concentration of soluble human IL-6 receptor administered simultaneously with Fv4-IgG1. The results described above demonstrated that increasing the binding of pH-dependent antigen-binding antibodies to human FcRn under neutral conditions is highly effective in enhancing the antigen elimination effect. Meanwhile, the type of amino acid changes for enhancing human FcRn binding under neutral conditions that are introduced to enhance the antigen elimination effect is not particularly limited; and such changes include those presented in Tables 6-1 and 6-2. The effect of antigen elimination can be predicted to be increased by any introduced change in vivo.

In addition, plasma concentrations of soluble human IL-6 receptor were coadministered with one of four types of pH-dependent human IL-6 receptor binding antibodies, Fv4-IgG1-F14, Fv4-IgG1-F21, Fv4-IgG1-F25, and Fv4 -IgG1-F48, maintained at a lower level over time than that of soluble human IL-6 receptor administered alone. Such pH-dependent human IL-6 receptor binding antibodies can be administered to a body where the plasma concentration of the soluble human IL-6 receptor is kept constant to maintain the plasma concentration of the soluble human IL-6 receptor at a lower level than steady-state plasma concentration. Specifically, the concentration of an antigen in plasma in vivo can be reduced by introducing such an antibody into the body.

[Example 7] Evaluation of the effectiveness of low doses (0.01 mg/kg) Fv4-IgG1-F14

Fv4-IgG1-F14 prepared as described in Example 6 was tested at low doses (0.01 mg/kg) using the same in vivo testing method as described in Example 6. The results (shown in FIG. 9) were compared with the results described in example 6, which were obtained by administering Fv4-IgG1 and Fv4-IgG1-F14 at 1 mg/kg.

The results demonstrated that although the plasma antibody concentration in the group administered Fv4-IgG1-F14 at 0.01 mg/kg was approximately 100 times lower than that in the group administered 1 mg/kg (FIG. 10) , the dynamics of plasma concentrations of the soluble human IL-6 receptor were comparable to each other. In addition, it was shown that the plasma concentration of soluble human IL-6 receptor seven hours after administration in the group that was administered Fv4-IgG1-F14 at a rate of 0.01 mg/kg was reduced by approximately three times compared with that in the group , which was administered Fv4-IgG1 at a rate of 1 mg/kg. In addition, in the presence of Fv4-IgG1-F14, plasma concentrations of soluble human IL-6 receptor administered at different doses were reduced over time in both groups compared with the group administered soluble human IL-6 receptor at separately.

The results demonstrate that even when administered at one hundredth of a dose, Fv4-IgG1, Fv4-IgG1-F14, which is derived from modifying Fv4-IgG1 to enhance binding to human FcRn under neutral conditions, effectively reduces plasma concentrations of soluble IL-1 receptor. 6 people. Specifically, it is believed that antigens can be effectively eliminated even at a lower dose if pH-dependent antigen-binding antibodies are modified to enhance their ability to bind FcRn under neutral conditions.

[Example 8] In vivo test based on a stationary model using normal mice

Evaluation of binding to mouse FcRn under neutral conditions

VH3/L (WT)-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and L (WT) (SEQ ID NO: 5), VH3/L (WT)-IgG1-v2 containing VH3-IgG1-v2 (SEQ ID NO: 9) and L (WT) (SEQ ID NO: 5), and VH3/L (WT)-IgG1-F20 containing VH3-IgG1-F20 (SEQ ID NO: 10) and L (WT) (SEQ ID NO: 5), each prepared as described in Example 5, were assessed for binding to mouse FcRn under neutral conditions (pH 7.4) by the method described below.

Binding between antibody and mouse FcRn was analyzed kinetically using Biacore T100 (GE Healthcare). An appropriate amount of protein L (ACTIGEN) was immobilized onto a CM4 sensor chip (GE Healthcare) via a coupled amine method and ensured that the antigen of interest was captured on the chip. Diluted FcRn solutions and running buffer (as a control solution) were then injected to allow the mouse FcRn to interact with the antibody fixed to the sensor chip. The running buffer used contained 50 mmol/1 sodium phosphate, 150 mmol/1 NaCl, and 0.05% (w/v) Tween 20 (pH 7.4). FcRn was diluted using each buffer. The chip was regenerated using 10 mmol/1 glycine-HCl (pH 1.5). Assays were performed exclusively at 25 degrees C. The association rate constant ka (1/Ms) and the dissociation rate constant _kd (1/s), each of which are kinetic parameters, were calculated based on the sensorgrams obtained in the assays and KD ( M) of each mouse FcRn antibody was determined from these values. Each parameter was calculated using Biacore T100 Evaluation software (GE Healthcare).

The results are presented in Table 7 (affinity for mouse FcRn at pH 7.4). VH3/L (WT)-IgG1 (IgG1 in Table 7), whose constant region is intact IgG1, which shows only very weak binding to mouse FcRn. Thus, the KD cannot be calculated and is referred to as ND in Table 7. Analysis of the results showed that the modified antibodies with increased binding to human FcRn under neutral conditions also showed increased binding to mouse FcRn under neutral conditions.

Table 7 KD (M) IgG1 ND IgG1-v2 1.04E-06 IgG1-F20 1.17E-07

In vivo testing using normal mice with constant plasma concentrations of soluble human IL-6 receptor

Using H54/L28-IgG1, Fv4-IgG1, Fv4-IgG1-v2 and Fv4-IgG1-F20 prepared as described in Examples 1 and 5, in vivo testing was carried out in the manner described below.

In vivo infusion test using normal mice

An infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) containing soluble human IL-6 receptor was implanted under the dorsal skin of normal mice (C57BL/6J mice; Charles River Japan) to obtain an animal model where the plasma concentration of soluble IL-6 receptor 6 people are kept constant. Antibodies against the human IL-6 receptor were administered to animal models to evaluate in vivo dynamics following administration of soluble human IL-6 receptor. Anti-mouse CD4 monoclonal antibody (R&D) was administered at a dose of 20 mg/kg once into the tail vein to suppress the production of neutralizing antibody against soluble human IL-6 receptor. Next, an infusion pump containing 92.8 micrograms/ml of soluble human IL-6 receptor was implanted under the skin of the mouse's back. Three days after implantation of the infusion pump, antibodies against the human IL-6 receptor were injected at a rate of 1 mg/kg once into the tail vein. Blood was collected 15 minutes, seven hours, one day, two days, three days, four days, seven days, 14 days, 21 days and 28 days after administration of the anti-human IL-6 receptor antibody. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. Before analysis, separated plasma was stored in a refrigerator at -20 degrees C or colder.

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

The method used was the same as described in example 3.

Determination of hsIL-6R plasma concentration by electrochemiluminescence assay

The method used was the same as described in example 1.

As shown in FIG. 11, the plasma concentration of soluble human IL-6 receptor was increased to 650 ng/ml (15-fold predose) when H54/L28-IgG1, a neutralizing antibody against soluble human IL-6 receptor, was administered to normal mice. (hsIL-6R group), in which the plasma concentration of soluble human IL-6 receptor remained constant at approximately 40 ng/ml. On the other hand, the plasma concentration of soluble human IL-6 receptor was maintained at approximately 70 ng/ml in the Fv4-IgG1-administered group, which is derived from providing H54/L28-IgG1 with a pH-dependent antigen binding ability. This suggests that the increase in plasma concentration of soluble human IL-6 receptor caused by administration of H54/L28-IgG1, a typical neutralizing antibody, can be reduced to approximately one-tenth by providing pH-dependent binding capacity.

In addition, the plasma concentration of soluble human IL-6 receptor has been shown to be maintained constant at one-tenth or less than one-tenth of the steady-state concentration by administration of Fv-IgG1-v2 or Fv-IgG1-F20, each resulting from the administration of a change into pH-dependent IL-6 receptor binding antibodies to reduce FcRn binding under neutral conditions. When Fv-IgG1-v2 was administered, the plasma concentration of soluble human IL-6 receptor 14 days after administration was approximately 2 ng/ml. Thus, Fv-IgG1-v2 could reduce the concentration to 1/20 of the level before administration. Meanwhile, if Fv-IgG1-F20 was administered, the plasma concentration of soluble human IL-6 receptor seven hours, one day, two days and four days after administration was below the detection limit (1.56 ng/ml). This suggests that Fv-IgG1-F20 reduced the concentration to 1/25 or below 1/25 of the pre-administration level.

The results described above demonstrate that the concentration of antigen in plasma can be significantly reduced by increasing the rate of elimination of antigen in plasma, through the administration of an antibody having both pH-dependent antigen-binding ability and FcRn binding activity under neutral conditions in an animal model for which the concentration antigen levels in plasma remain constant.

Typical antibodies such as H54/L28-IgG1 can only neutralize the effect of the target antigen by binding to the target antigen, and even more, they increase the concentration of the antigen in the plasma. In contrast, antibodies having both pH-dependent antigen-binding capacity and FcRn-binding activity under neutral conditions have been found to not only neutralize the target antigen, but also reduce the plasma concentration of the target antigen. It can be expected that the effect of removal of antigen from plasma is more significant than neutralization. In addition, antigen removal may also promote target antigens for which removal is not sufficiently effective through neutralization alone.

[Example 9] Identification of the threshold level of human FcRn binding affinity at neutral pH required to increase antigen elimination and the relationship between antigen elimination and human FcRn binding affinity at neutral pH

Preparation of antibodies for in vivo studies

Fc variants Fv4-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) were generated with increased binding to FcRn under neutral pH conditions. Specifically, VH3-M73 (SEQ ID NO: 15) and VH3-IgG1-v1 (SEQ ID NO: 8) were prepared. Amino acid substitutions were introduced by methods known to those skilled in the art, as described in Reference Example 1.

H54/L28-IgG1 containing H54 (SEQ ID NO: 1) and L28 (SEQ ID NO: 2), Fv4-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7 ), Fv4-M73 containing VH3-M73 (SEQ ID NO: 15) and VL3-CK (SEQ ID NO: 7), Fv4-IgG1-v1 containing VH3-IgG1-v1 (SEQ ID NO: 8) and VL3 -CK (SEQ ID NO: 7), and Fv4-IgG1-v2 containing VH3-IgG1-v2 (SEQ ID NO: 9) and VL3-CK (SEQ ID NO: 7) were expressed and purified in a manner known to those skilled in the art. this area described in reference example 2.

Evaluation of antibody binding affinity to human FcRn under neutral pH conditions

VH3/L (WT)-IgG1 containing VH3-IgG1 (SEQ ID NO: 6) and L (WT) (SEQ ID NO: 5), VH3/L (WT)-M73 containing VH3-M73 (SEQ ID NO : 15) and L (WT) (SEQ ID NO: 5), VH3/L (WT)-IgG1-v1 containing VH3-IgG1-v1 (SEQ ID NO: 8) and L (WT) (SEQ ID NO: 5), and VH3/L (WT)-IgG1-v2 containing VH3-IgG1-v2 (SEQ ID NO: 9) and L (WT) (SEQ ID NO: 5), each of which was prepared as described in the example 2 were assessed for binding to human FcRn under neutral pH conditions (pH 7.0).

The binding activity of VH3/L (WT)-IgG1-v1 and VH3/L (WT)-IgG1-v2 to human FcRn was measured using the method described in Example 5. Due to the low binding activity of VH3/L (WT)-IgG1 and VH3 /L(WT)-M73 c human FcRn, human FcRn binding activity could not be measured using the method described in Example 5, so these antibodies were assessed using the method described below. Binding between antibody and human FcRn was analyzed kinetically using Biacore T100 (GE Healthcare). An appropriate amount of protein L (ACTIGEN) was immobilized onto a CM4 sensor chip (GE Healthcare) via a coupled amine method and allowed to capture the antibody of interest on the chip. Then, diluted FcRn solutions and running buffer as a control solution were injected to allow human FcRn to interact with the antibody fixed on the sensor chip. The running buffer used contained 50 mmol/1 sodium phosphate, 150 mmol/1 NaCl, and 0.05% (w/v) Tween 20 (pH 7.0). FcRn was diluted using each buffer. The chip was regenerated using 10 mmol/1 glycine-HCl (pH 1.5). Analyzes were performed at 25 degrees C.

The KD (M) of each antibody was obtained from the sensorgram data using Biacore T100 Evaluation software (GE Healthcare), which simultaneously corresponds to the association and dissociation phases of the sensorgrams and generally matches all curves in the working set. The sensorgrams conformed to a 1:1 binding model, the Langmuir binding model, specified by Biacore T100 Evaluation software. For some of the binding interactions, KD was obtained by nonlinear regression analysis of plots of R _eq , the equilibrium binding reaction, as a function of log analyte concentration using an equilibrium approach.

The results of binding to human FcRn under neutral conditions (pH 7.0) by Biacore are presented in Table 8.

Table 8 KD (M) IgG1 8.8E-05 M73 1.4E-05 IgG1-v1 3.2E-06 IgG1-v2 8.1E-07

In vivo study of the effect of antibody on antigen elimination in a co-injection model using the human FcRn transgenic mouse line 276

An in vivo antibody study using a co-injection model was performed as described in Example 3. Anti-human IL-6 receptor antibodies used in this study are the above-described H54/L28-IgG1, Fv4-IgG1, Fv4-M73, Fv4- IgG1-v1 and Fv4-IgG1-v2. The mice used in this study are human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276 +/+ mouse, Jackson Laboratories; Methods Mol Biol. (2010) 602:93-104).

As shown in Figure 12, the pharmacokinetics of H54/L28-IgG1, Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1 and Fv4-IgG1-v2 were comparable, and these antibodies were maintained at similar plasma concentrations throughout the study.

The dynamics of the concentration of hsIL-6R in plasma is presented in Fig. 13. Compared with hsIl-6R administered with Fv4-IgG1, hsIL-6R administered with Fv4-IgG1-v2 demonstrated increased clearance, while hsIL-6R administered with Fv4-M73 and Fv4-IgG1-v1 demonstrated reduced clearance. Although all Fc variants, M73, v1 and v2 have increased binding affinity to human FcRn under neutral pH conditions (pH 7.0), only Fv4-IgG1-v2, but not Fv4-M73 and Fv4-IgG1-v1, have been shown to demonstrated increased clearance of hsIL-6R. This shows that to increase antigen clearance, the binding affinity of the antibody to human FcRn at pH 7.0 must be at least greater than that of IgG1-v1, whose binding affinity to human FcRn at pH 7.0 is KD 3.2 micromoles or 28 times higher than that of intact human IgG1 (binding affinity for human FcRn is represented by a KD of 88 micromoles).

Figure 14 describes the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the plasma concentration of hsIL-6R at day 1 after co-injection of hsIL-6R and Fc variants. Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4 -IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93 and Fv4-IgG1-F94) are presented in the graph. By increasing the binding affinity of the antibody to human FcRn at pH 7.0, the plasma concentration of hsIL-6R, which reflects antigen clearance, initially increases but then rapidly decreases. This shows that to increase antigen clearance compared to intact human IgG1, the binding affinity of the antibody to human FcRn at pH 7.0 should preferably be higher than the KD of 2.3 micromoles (value obtained from curve fitting, Fig. .14). An antibody binding affinity to human FcRn between a KD of 88 micromolar and a KD of 2.3 micromolar would be more likely to reduce antigen clearance (higher concentration of hsIL-6R). In other words, the binding affinity of the antibody to human FcRn at pH 7.0 should preferably be 38 times higher than for intact human IgG1 to enhance antigen clearance or otherwise reduce antigen clearance.

Figure 15 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the plasma antibody concentration day 1 after co-injection of hsIL-6R and Fc variants. Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4 -IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93 and Fv4-IgG1-F94) are plotted. By increasing the binding affinity of the antibody to human FcRn at pH 7.0, the plasma concentration of the antibody, which reflects antibody pharmacokinetics (clearance), initially increases but then rapidly decreases. This demonstrates that to maintain antibody pharmacokinetics similar to intact human IgG1 (binding affinity for human FcRn is a KD of 88 micromolar), the antibody's affinity for human FcRn at pH 7.0 must be weaker than the KD of 0 .2 micromoles (value obtained from the fitting curve in Fig. 15). An antibody binding affinity for human FcRn greater than the KD of 0.2 micromolar increased antibody clearance (ie, faster elimination of antibody from plasma). In other words, the binding affinity of the antibody to human FcRn, which at pH 7.0 would have to be 440 times higher than that of intact human IgG1 to demonstrate similar antibody pharmacokinetics as intact human IgG1, or otherwise, would result in rapid elimination antibodies from plasma.

Taking into account both FIGS. 14 and 15, to increase antigen clearance (i.e., decrease plasma antigen concentration) relative to IgG1, while maintaining antibody pharmacokinetics similar to intact human IgG1, the binding affinity of the antibody to human FcRn at pH 7.0 should be between 2.3 micromolar and 0.2 micromolar, or in other words, the binding affinity of the antibody to human FcRn at pH 7.0 should be within the range of 38 times to 440 times higher than for intact IgG1 person. Such an antibody with similar pharmacokinetics as IgG1 with long-lasting antigen elimination activity may be useful for antibody therapeutics that require longer dosing intervals, as in the case of a chronic disease, due to its prolonged action.

On the other hand, by increasing the binding affinity of the antibody to human FcRn at pH 7.0 by more than a KD of 0.2 micromolar, or in other words, by increasing the binding affinity of the antibody to human FcRn at pH 7.0 by more than 440 times Compared with intact human IgG1, antigen clearance is increased to a greater extent within the short term, although the antibody is eliminated from plasma more quickly than for intact human IgG1. Such an antibody with the ability to induce a rapid and potent reduction in antigen concentration would be useful for an antibody therapeutic, such as for an acute disease where the disease-associated antigen needs to be removed from the plasma due to its rapid-acting property.

The amount of antigen eliminated from plasma by an antibody is an important factor in assessing the efficiency of antigen elimination by administration of antibody Fc variants having increased binding affinity to human FcRn at pH 7.0. To evaluate the antigen elimination efficiency of the antibody, the following calculation was performed at each time point of the in vivo study described in this example and Example 6.

Value A: molar concentration of antigen at each time point;

B value: molar concentration of antigen at each time point;

C-value: molar concentration of antigen relative to molar concentration of antibody (molar antigen/antibody ratio) at each time point

C=A/B

The time dependence of the C value (molar ratio of antigen/antibody) for each antibody is described in Fig. 16. Low C values indicate high antigen elimination by the antibody, whereas high C values indicate low antigen elimination efficiency by the antibody. Low C values compared to IgG1 indicate that higher antigen elimination efficiency is achieved by Fc variants, while high C values compared to IgG1 indicate that Fc variants have a negative effect on antigen elimination efficiency. All Fc variants except Fv4-M73 and Fv4-IgG1-v1 showed increased antigen clearance efficiency compared to Fv4-IgG1. Fv4-M73 and Fv4-IgG1-v1 showed a negative effect on antigen elimination efficiency, which is consistent with Fig. 14.

Figure 17 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the C value (molar ratio of antigen/antibody) at day 1 after co-injection of hsIL-6R and Fc variants. Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4 -IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93, and Fv4-IgG1-F94) are presented graphically. This shows that to achieve higher antigen elimination efficiency compared to intact human IgG1, the affinity of the antibody for human FcRn at pH 7.0 must be higher than the KD of 3.0 micromoles (value obtained from the fitting curve in FIG. .17). In other words, the binding affinity of the antibody to human FcRn at pH 7.0 must be at least 29 times higher than that of intact human IgG1 to achieve higher antigen elimination efficiency compared to intact human IgG1.

In conclusion, a group of antibody variants having a binding affinity for FcRn at pH 7.0 between a KD of 3.0 micromolar and 0.2 micromolar, or in other words, a group of antibody variants having a binding affinity for FcRn at pH 7.0 in range from 29 times to 440 times higher than for intact human IgG1, has pharmacokinetics similar to those of IgG1 antibodies, but has an increased ability to eliminate antibodies from plasma. Thus, such an antibody demonstrates increased antigen elimination efficiency compared to IgG1. Pharmacokinetics similar to IgG1 will allow for prolonged clearance of antigen from plasma (long-acting antigen clearance), and thus allows for longer dosing intervals, which may be an advantage for antibody therapeutics intended to treat chronic diseases. A group of antibody variants having a binding affinity to FcRn at pH 7.0 higher than the KD of 0.2 micromolar, or in other words, a group of antibody variants having a binding affinity to FcRn at pH 7.0 440 times higher than for an intact human IgG1, has accelerated antigen clearance (short-term elimination of the antibody). However, since such an antibody allows for even more accelerated antibody clearance (rapid antigen elimination), such an antibody also exhibits increased antigen elimination efficiency compared to IgG1. As shown in Example 8, Fv4-IgG1-F20 in normal mice will induce extensive plasma antigen clearance in a very short period, but the antigen clearance effect is not long-lasting. This profile would be advantageous for targeting acute disease, where the disease-associated antigen needs to be rapidly and extensively cleared from the plasma in a very short period.

[Example 10] In vivo study of Fv4-IgG1-F14 through a steady-state infusion model using the human FcRn transgenic mouse line 276

An in vivo study of Fv4-IgG1-F14 through a steady-state infusion model using human FcRn 276 transgenic mice was performed as described in Example 1. The study group consists of a control group (without antibody), Fv4-IgG1 at a dose of 1 mg/kg and Fv4-IgG1-F14 at a dose of 1 mg/kg, 0.2 mg/kg and 0.01 mg/kg.

FIG. 18 describes the time course of plasma concentration of hsIL-6R after antibody administration. Compared to baseline hsIL-6R levels without antibody, administration of 1 mg/kg Fv4-IgG1 resulted in a several-fold increase in plasma hsIL-6R concentrations. On the other hand, administration of 1 mg/kg Fv4-IgG1-F14 resulted in a significant decrease in plasma concentration compared to the Fv4-IgG1 group and the control group. After day 2, the plasma concentration of hsIL-6R was undetectable (the lower limit of determination of the plasma concentration of hsIL-6R is 1.56 ng/ml in this measurement system), and this continued for up to 14 days.

As shown in Example 1, H54/L28-IgG1-F14 showed a decrease in plasma hsIL-6R concentration compared to H54/L28-IgG1, but the degree of decrease was small. The degree of reduction was greater for the Fv4 variable region, which had a pH-dependent binding property to hsIL-6R. This demonstrates that although increasing binding affinity to human FcRn at pH 7.0 is effective in reducing plasma antigen concentrations, the combination of pH-dependent antigen binding and increased binding affinity to human FcRn at neutral pH significantly increases antigen clearance.

A study using lower doses of Fv4-IgG1-F14 demonstrated that even at 0.01 mg/kg, 1/100 of 1 mg/kg reduced plasma antigen concentrations below baseline, demonstrating significant effectiveness of the molecule in removing antigen from plasma.

[Example 11] Comparison of transgenic line 276 and line 32 of mice with human FcRn in a co-injection model

A previous in vivo study was performed using the human FcRn transgenic mouse line 276 (Jackson Laboratories). To compare the differences between the human FcRn transgenic mouse line 276 and another transgenic line, line 32, a co-injection study of H54/L28-IgG1, Fv4-IgG1 and Fv4-IgG1-v2 was undertaken using the human FcRn transgenic mouse line 32 (B6.mFcRn -/-.hFcRn Tg line 32 +/+ mouse (B6.mFcRn-/- hFCRN Tg32; B6.Cg-Fcgrt<tmlDcr> Tg(FCGRT)32Dcr) (Jackson #4915)), Jackson Laboratories; Mehtods Mol Biol. (2010) 602:93-104). The test method was similar to that of Example 3, but the human FcRn transgenic mouse line 32 was used instead of the human FcRn transgenic mouse line 276.

19 describes the time dependence of plasma concentrations of hsIL-6R and the human FcRn transgenic mouse line 276 and the human FcRn transgenic mouse line 32. H54/L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 showed similar time courses of hsIL-6R plasma concentrations. In both mouse strains, increasing binding affinity to human FcRn at pH 7.0 increased antigen clearance from plasma (comparing Fv4-IgG1 and Fv4-IgG1-v2) to a similar extent.

Figure 20 describes the time course of antibody concentration in plasma and in human FcRn transgenic mouse line 276 and in human FcRn transgenic mouse line 32. H54/L28-IgG1, Fv4-IgG1 and Fv4-IgG1-v2 showed similar time courses of plasma antibody concentrations.

In conclusion, a nonsignificant difference was observed between lines 276 and 32, demonstrating that the Fc variant to increase binding affinity to human FcRn at pH 7.0 was more effective in two different transgenic mouse lines expressing human FcRn to increase the elimination of plasma antigen concentrations.

[Example 12] Generation of Various Fc Antibody Variants Having Increased Binding Affinity to Human FcRn at Neutral pH

Creating Fc variants

Various mutations to increase binding affinity to human FcRn in the neutral pH range were introduced into Fv4-IgG1 to further increase the antigen clearance profile. Specifically, the amino acid mutations presented in Tables 9-1 to 9-14 were introduced into the heavy chain constant region of Fv4-IgG1 to create Fc variants (the number of amino acid mutation sites are described according to EU numbering). Amino acid substitutions were introduced in a manner known to those skilled in the art, as described in Reference Example 1.

Additional variants (IgG1-F100 to IgG1-F599), each containing the resulting heavy chain and L (WT) (SEQ ID NO: 5), were expressed and purified by methods known to those skilled in the art, as described in Reference Example 2.

Human FcRn binding assessment

Binding between antibody and human FcRn was kinetically analyzed as described in Example 5 for IgG1-v1, IgG1-v2 and IgG1-F2 to IgG1-F599 or Example 9 for IgG1 and M73. Results for binding to human FcRn under neutral conditions (pH 7.0) are presented by Biacore in Tables 9-1 to 9-14.

Table 9-2 is a continuation of Table 9-1.

Table 9-3 is a continuation of Table 9-2.

Table 9-4 is a continuation of Table 9-3.

Table 9-5 is a continuation of Table 9-4.

Table 9-6 is a continuation of Table 9-5.

Table 9-7 is a continuation of Table 9-6.

Table 9-8 is a continuation of Table 9-7.

Table 9-9 is a continuation of Table 9-8.

Table 9-10 is a continuation of Table 9-9.

Table 9-11 is a continuation of Table 9-10.

Table 9-12 is a continuation of Table 9-11.

Table 9-13 is a continuation of Table 9-12.

Table 9-14 is a continuation of Table 9-13.

[Example 13] In vivo study of various Fc antibody variants via a steady-state infusion model using human FcRn transgenic mouse line 32

The Fc variants produced in Example 12 were tested for their ability to clear antigen from plasma in a steady-state infusion model using the human FcRn transgenic mouse line 32. The in vivo steady-state infusion model study was performed as described in Example 1, but the human FcRn transgenic mouse line 32 was used instead of the human FcRn line 276, and mouse anti-CD4 monoclonal antibodies were injected twice (before implantation of the infusion pump and 14 days after antibody injection) or three times (before implantation of the infusion pump and 10 and 20 days after injection of the antibody).

From the Fc variants described in Tables 9-1 to 9-14, selected Fc antibody variants listed below were expressed and purified by methods known to those skilled in the art, as described in Reference Example 2:

Fv4-IgG1, containing VH3-IgG1 and VL3-CK;

Fv4-IgG1-F11, containing VH3-IgG1-F11 and VL3-CK;

Fv4-IgG1-F14, containing VH3-IgG1-F14 and VL3-CK;

Fv4-IgG1-F39, containing VH3-IgG1-F39 and VL3-CK;

Fv4-IgG1-F48, containing VH3-IgG1-F48 and VL3-CK;

Fv4-IgG1-F140, containing VH3-IgG1-F140 and VL3-CK;

Fv4-IgG1-F157, containing VH3-IgG1-F157 and VL3-CK;

Fv4-IgG1-F194, containing VH3-IgG1-F194 and VL3-CK;

Fv4-IgG1-F196, containing VH3-IgG1-F196 and VL3-CK;

Fv4-IgG1-F198, containing VH3-IgG1-F198 and VL3-CK;

Fv4-IgG1-F262, containing VH3-IgG1-F262 and VL3-CK;

Fv4-IgG1-F264, containing VH3-IgG1-F264 and VL3-CK;

Fv4-IgG1-F393, containing VH3-IgG1-F393 and VL3-CK;

Fv4-IgG1-F424, containing VH3-IgG1-F434 and VL3-CK; And

Fv4-IgG1-F447, containing VH3-IgG1-F447 and VL3-CK.

These antibodies were administered to transgenic mouse line 32 with human FcRn at a dose of 1 mg/kg.

Figure 21 describes the time course of plasma concentration of hsIL-6R in mice. Compared to Fv4-IgG1, all Fc variants with increased binding affinity to human FcRn at pH 7.0 showed a decrease in plasma hsIL-6R concentrations, thereby increasing antigen clearance from plasma. Although the extent and duration of reduction in antigen concentrations varied among Fc variants, all variants consistently reduced plasma concentrations of hsIL-6R relative to IgG1, demonstrating that increased binding affinity to human FcRn at pH 7.0 will universally increase antigen clearance from plasma. FIG. 22 describes the time course of plasma antibody concentration in mice. Antibody pharmacokinetics were different among Fc variants.

As described in Example 9, the amount of antigen eliminated from plasma by an antibody is an important factor in assessing the efficiency of antigen elimination by administration of Fc antibody variants having increased binding affinity to human FcRn at pH 7.0. Thus, the time dependence of the C value (molar ratio of antigen/antibody) for each antibody is described in Fig. 23. Figure 24 shows the relationship between the binding affinity of Fc variants to human FcRn at pH 7.0 and the C value (molar ratio of antigen/antibody) at day 1 after antibody administration. This shows that all Fc antibody variants tested in this study have a lower C value compared to Fv4-IgG1. Because all Fc variants tested in this study have a binding affinity for human FcRn at pH 7.0 higher than the KD of 3.0 micromolar, they achieve higher antigen clearance efficiencies compared to intact human IgG1. This is consistent with the results obtained in example 9 (Fig. 17).

Figure 25 describes that among the Fc variants tested in this study, antibodies having the Fc variants F11, F39, F48 and F264 showed similar pharmacokinetics to IgG1. Because this study is performed using human FcRn transgenic mice, these Fc variants are expected to have a long half-life similar to human IgG1. Figure 26 describes the time course of plasma concentration of hsIL-6R in a mouse injected with antibodies having pharmacokinetics similar to intact human IgG1 (F11, F39, F48 and F264). These variants reduced plasma hsIL-6R concentrations relative to IgG1 by approximately 10-fold. In addition, these antibodies reduced the concentration of hsIL-6R below the baseline concentration of hsIL-6R (concentration without antibody). Thus, these antibodies will allow for prolonged clearance of antigen from plasma, and thus provide long dosing intervals that will be advantageous for antibody therapeutics needed to treat chronic diseases.

FIGS. 27 and 28 describe the time course of plasma antibody concentration and hsIL-6R plasma concentration for IgG1 and Fc variant F157, F196 and F262, respectively. Surprisingly, although the pharmacokinetics of antibodies F157 and F262 showed significantly accelerated clearance from plasma compared with intact human IgG1, F157 and F262 demonstrated very intense and prolonged elimination of hsIL-6R from plasma. Specifically, the plasma concentration of hsIL-6R for F157 was below the detection limit (1.56 ng/mL) from days 1 to 28 (except for day 14), and the plasma concentration of hsIL-6R for F262 was below the detection limit (1 .56 ng/ml) from 14 to 28 days. On the other hand, for F196, with slower antibody clearance compared to F157, the antigen concentration began to increase on day 14 and returned back to baseline on day 28. Among the Fc variants tested in this study, F157 and F262 were the only Fc variants that were capable of reducing plasma hsIL-6R concentrations below 1.56 ng/ml at day 28.

This long-lasting effect of F157 and F262 is unexpected from the pharmacokinetics of the antibody, since the antibodies are eliminated from plasma very quickly compared to intact human IgG1. In particular, the concentration of antibodies in plasma F157 was not determined on day 21. However, plasma hsIL-6R concentrations continued to decline to levels below the detection limit of 1.56 ng/mL on days 21 and 28. This unexpected effect is believed to exist as a result of the presence of the antibody on the surface of vascular endothelial cells as FcRn-related form. Although these antibodies showed low plasma concentrations, these antibodies are still present in the vascular compartment in the FcRn-bound form (which cannot be measured as plasma antibody concentration). This FcRn-bound antibody may still be bound to the antigen in the plasma, and the FcRn then mediates the uptake of the antigen/antibody complex, the antigen is released within the endosome and degraded by the lysosome, while the antibody is recycled back to the cell surface in the FcRn-bound form. Thus, these FcRn-associated antibodies contribute to antigen elimination. This explains the fact that these antibodies retain the ability to eliminate antigen even after the antibody concentration in plasma becomes low.

[Example 14] In silico comparative study of a conventional antibody and an antigen eliminating antibody

Example 13 shows that an antibody that binds antigen in a pH-dependent manner and increases binding affinity to human FcRn at neutral pH is capable of eliminating antigen from plasma. Thus, such antigen-eliminating antibodies are useful for antigen-antibody targeting in which simple binding and neutralization is insufficient to treat the disease and removal of the antigen from the plasma is required.

Antigen-eliminating antibodies are also useful for antigen-antibody targeting, for which simple binding and neutralization is sufficient. Antibodies that bind and neutralize an antigen require at least a molar amount of antibody similar to the amount of antigen in the plasma (if the antibody has infinitely high affinity for the antigen, the antigen can be neutralized by a molar amount of antibody similar to the antigen). Unlike a conventional antibody (an antibody without pH-dependent antigen binding and Fc construction), antigen-eliminating antibodies can reduce plasma antigen concentrations. This means that the concentration of antibody required to neutralize the antigen can be reduced. If an antigen-eliminating antibody reduced the plasma concentration of antigen by 10-fold compared to a conventional antibody, the concentration of antibody required to neutralize the antigen could also be reduced by 10-fold. Thus, therapeutically, an antigen eliminating antibody may reduce the dose of the antigen or increase the dosing interval compared to a conventional antibody.

Fc variants, such as F11, F39, F48 and F264, are capable of reducing plasma antigen concentrations compared to IgG1 by approximately 10-fold. To evaluate the effect of such antigen-eliminating antibodies compared to a conventional antibody, an in silico assessment of the dose of antibody required to maintain antigen neutralization therapeutically for both the conventional antibody and the antigen-eliminating antibody was performed. The dose required to maintain neutralization was determined using a 3-month dosing interval. (i.e. dose required for Q3M).

Construction of a pharmacokinetic model

A pharmacokinetic (PK) model was constructed using SAAM II PK analysis software (The SAAM Institute, Inc.). The PK model was constructed as described in Pharmacokinet Pharmacodyn. 2001 Dec; 28(6): 507-32 and Br J Clin Pharmacol. May 2007; 63(5): 548-61. The concept of the PK model is shown in Fig. 29. The amount of each compartment was described by the following differential equations.

[Math.1]

Xsc: amount of antibody in subcutaneous tissue

Xmab: amount of free antibody in serum

Xcom: amount of immune complex antibody and antigen (=complex)

Xag: amount of free antigen in serum

ka: suction rate constant

In this model, bioavailability (F) is estimated as 1 for all antibodies, and the rate of antigen biosynthesis (R) is estimated using the following equation.

[Math.2]

Cpre: steady-state serum antigen concentration.

The pharmacokinetic parameters and antigen binding kinetic parameters used in this in silico study are described in Table 10.

Table 10 CLmab L/day/kg 0.0025 CLag L/day/kg 0.0243 CLcom L/day/kg 0.0045 Vmab=Vag L/kg 0.0843 Vcom L/kg 0.0519 ka 1/day 0.4800 koff 1/day 53.0496 kon 1/nM/day 53.0496 L/mcg/day 0.353664

Simulation to calculate antigen-eliminating antibody effect and affinity maturation

The steady-state concentration (Cpre) before antibody administration was set at 2400 ng/ml. With the construction of the PK model, the minimum dose of antibody to maintain the free antigen concentration below 35 ng/ml was assessed 84 days after a single subcutaneous injection. The molecular weight of the antigen was set to 190 kDa and the molecular weight of the therapeutic antibody was set to 150 kDa for all.

As the antibody, a conventional antibody and an antigen elimination antibody with different binding affinities (different degrees of affinity maturation from the parent antibody with a KD of 1 nM) were used in this in silico study. The effect of an antigen-eliminating antibody is reflected by faster clearance of the antigen-antibody complex than for a conventional antibody. The clearance parameters of the antigen-antibody complex (CLcom) are described in Table 11.

Table 11 Traditional Ab Antigen elimination Ab CLcom L/day/kg 0.0045 0.0729

The effect of affinity maturation of the parent antibody with a KD of 1 nM was also considered (affinities varied over a 100-fold range). KDs of 1 nM, 300 pM, 100 pM, 30 pM and 10 pM are used in this in silico study. The effect of affinity maturation is reflected as a decrease in koff. The koff values vary in a 100-fold range (koff = 53.05, 17.68, 5.30, 1.77, 0.53 [1/day]).

For conventional and antigen eliminating antibodies with binding affinities (KDs) of 1 nM, 300 pM, 100 pM, 30 pM, and 10 pM, the antibody was dosed to maintain the free antigen concentration below 35 ng/mL after 84 days. after a single subcutaneous injection, the results are described in table 12.

Table 12 Dose (mg/body) 1 nM 333 pM 100 pM 33 pM 10 pM Traditional Ab 2868 1256 692 532 475 Antigen elimination Ab 180 81 46 36 33

For a parent conventional antibody with a binding affinity of 1 nM, 2.868 mg is required to achieve the Q3M dose. Although the dose of the antibody can be reduced by increasing the binding affinity to the antigen, the dose reduction reaches a maximum value. This maximum value is derived from the fact that an antibody that binds and neutralizes an antigen requires at least the same molar amount of antibody relative to the antigen in the plasma. Even with a binding affinity of 10 pM, the conventional antibody requires 475 mg to achieve the Q3M dose, which is a dose that cannot be administered subcutaneously by injection due to the limitation of the concentration of the formulated antibody and the volume administered subcutaneously.

On the other hand, by engineering a conventional antibody into an antigen-eliminating antibody, by engineering a pH-dependent antigen binding (or by directly engineering an antibody with pH-dependent binding), and by engineering the Fc region to obtain increased binding affinity to FcRn at neutral pH, the dose antibodies can be significantly reduced. For an antibody that eliminates antigen with a binding affinity of 1 nM, only 180 mg is required to achieve the Q3M dose. This dose level cannot be achieved with a conventional antibody even with unlimited affinity. By increasing the binding affinity of the antigen eliminating antibody to 10 pM, the dose can be reduced to 33 mg, which is a dose that can be easily administered subcutaneously.

Thus, this in silico study demonstrated that the antigen elimination antibody has a significant advantage over the conventional antibody. The dose of the antibody can be reduced to a level that is unattainable for a conventional antibody even with unlimited affinity. With respect to the dosing interval, if the antigen eliminating antibody is administered at the same dose as the dose of the conventional antibody, the antigen eliminating antibody will have a longer lasting effect, thereby allowing for a significantly longer dosing interval. Both lowering the dose and increasing the duration of the dosing interval by means of an antigen-eliminating antibody will provide a significant advantage over the conventional antibody.

It should be noted that, as described in Example 1, the antigen eliminating antibody does not necessarily require pH-dependent binding to the antigen. pH-dependent binding to antigen can significantly enhance the antigen elimination activity of the antibody. In addition, the pH-dependent binding property can be replaced by the use of other factors, the concentration of which is different within the plasma and endosome. Such a factor can also be used to produce an antibody that binds to an antigen within the plasma but dissociates from the antigen within the endosome.

[Example 15] Study on the enhancement effect of human IL-6 elimination enhancement of pH-dependent anti-human IL-6 antibodies

Generation of pH-dependent antibodies that bind human IL-6

CLB8-IgG1, containing CLB8H-IgG1 (SEQ ID NO: 16) and CLB8L-CK (SEQ ID NO: 17), described in WO 2009/125825, is a chimeric antibody against IL-6. H16/L13-IgG1, containing H16-IgG1 (SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19), is a chimeric anti-IL-6 antibody that is obtained by providing CLB8-IgG1 with the ability to bind IL -6 human in a pH-dependent manner (which binds at pH 7.4 but dissociates at pH 5.8).

Evaluation of the pH-dependent binding activity of a chimeric anti-IL-6 antibody to human IL-6

CLB8-IgG1 and H16/L13-IgG1 were assessed for binding activity to human IL-6 (dissociation constant (KD)) at pH 5.5 and pH 7.4 using Biacore T100 (GE Healthcare). The assay was performed using 10 mmol/1 ACES/150 mmol/1 NaCl containing 0.05% P20 surfactant (pH 7.4 and pH 6.0) as running buffer. Antibodies were then coupled to recombinant protein A/G (Thermo Scientific) immobilized on sensor chips using a coupled amine method and appropriate concentrations of human IL-6 (TORAY) were injected as the analyte. Assays were performed at 37 degrees C. Results were analyzed using Biacore T100 Evaluation software (GE Healthcare), and the association rate constant, ka (1/Ms), and dissociation rate constant, k _d (1/s), were calculated from the assay results . KD(M) was then calculated from ka and k _d (Table 13). In addition, pH-dependent binding was assessed to calculate the KD ratio between pH 7.4 and pH 6.0 for each antibody.

Table 13 sample pH ka (1/Ms) kd (1/s) KD(M) KD (pH 5.5)/KD (pH 7.4) CLB8-IgGl pH 7.4 3.6E+06 8.0E-04 2.2E-10 0.8 pH 5.5 3.7E+06 6.6E-04 1.8E-10 H16/L13-IgGl pH 7.4 2.1E+06 4.6E-03 2.2E-09 7.4 pH 5.5 3.7E+05 5.9E-03 1.6E-08

Generation of pH-dependent anti-human IL-6 antibodies with FcRn binding activity under neutral conditions

Mutations were introduced into H16/L13-IgG1, containing H16-IgG1 (SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19), to increase FcRn binding under neutral conditions (pH 7.4). Specifically, H16-IgG1-v2 (SEQ ID NO: 20) was constructed from the heavy chain constant region of IgG1 by replacing Trp with Asn at position 434 in EU numbering, while H16-F14 (SEQ ID NO: 21) was constructed from the heavy chain constant region IgG1 chain by replacing Tyr with Met at position 252 and Trp with Asn at position 434 in EU numbering. Amino acid substitutions were introduced in a manner known to those skilled in the art, as described in Reference Example 1.

CLB8-IgG1 containing CLB8H-IgG1 (SEQ ID NO: 16) and CLB8L-CK (SEQ ID NO: 17), H16/L13-IgG1 containing H16-IgG1 (SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19), H16/L13-IgG1-v2 containing H16-IgG1-v2 (SEQ ID NO: 20) and L13-CK (SEQ ID NO: 19), and H16/L13-F14 containing H16-F14 (SEQ ID NO: 21) and L13-CK (SEQ ID NO: 19), were expressed and purified in a manner known to those skilled in the art, as described in Reference Example 2.

Evaluation of the binding activity of Fc variants to mouse FcRn at neutral pH

VH3/L(WT)-IgG1 containing VH3-IgG1 and L (WT), VH3/L(WT)-IgG1-v2 containing VH3-IgG1-v2 and L (WT), and VH3/L (WT)- IgG1-F14 containing VH3-IgG1-F14 and L (WT), each prepared as described in Example 5, was assessed for binding to mouse FcRn under neutral conditions (pH 7.4) using the method described in Example 8.

The results are presented in Table 14. IgG1 showed very weak binding activity, while IgG1-v2 and IgG1-F14 showed higher binding affinity to mouse FcRn at pH 7.4.

Table 14 KD IgG1 not determined IgG1-v2 1.0E-06 IgG1-F14 1.3E-07

In vivo test using normal mice

In vivo kinetics for human IL-6 (hIL-6; TORAY) and anti-human IL-6 antibody were assessed following administration of a normal mouse (C57BL/6J mouse; Charles River Japan) with hIL-6 alone or hIL-6 and anti-IL-6 antibody. 6 people. hIL-6 solution (5 microgram/ml) or mixture solution containing hIL-6 and anti-human IL-6 antibody (CLB8-IgG1 group; 5 microgram/ml hIL-6 and 0.025 mg/ml CLB8-IgG1, H16/L13 -IgG1, H16/L13-IgG1-v2 and H16/L13-IgG1-F14 group; 5 microgram/ml hIL-6 and 0.14 mg/ml H16/L13-IgG1, H16/L13-IgG1-v2 and H16/ L13-IgG1-F14, respectively) was administered once at a dose of 10 ml/kg into the tail vein. The dose of antibody was adjusted such that more than 99.8% of human IL-6 bound to the antibody in the administration solution. Blood was collected after 5 minutes, 30 minutes, two hours, four hours, seven hours, one day after administration of hIL-6 alone and after 5 minutes, seven hours, one day, two days, three days, four days, seven days, 14 days, 21 days and 30 days after administration of hIL-6 and a solution of a mixture with an antibody against human IL-6. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -20 degrees C or below until analysis.

Measuring human plasma IL-6 concentration by ELISA

The concentration of human IL-6 in mouse plasma was measured using the Quantikine HS ELISA kit (R&D). Calibration curve samples having plasma concentrations of 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 ng/mL and mouse plasma samples diluted 100-fold or more were prepared. To obtain human IL-6 bound entirely to CLB8-IgG1 in a sample, 150 microliters of 5 microgram/ml CLB8-IgG1 was added to 150 microliters of calibration curve samples and to plasma samples, and then the samples were left for one hour at room temperature. Samples were then transferred to the plates provided in the ELISA kit (R&D) and left for one hour at room temperature. Then, the IL-6 conjugate provided in the ELISA kit (R&D) was added to react for one hour at room temperature, and the substrate solution provided in the ELISA kit (R&D) was added to react for one hour at room temperature. Next, a chromogenic reaction was carried out to carry out the reaction for half an hour at room temperature using the Amplifier solution provided in the ELISA (R&D) kit as the substrate. After stopping the reaction with the stopping solution provided in the ELISA kit (R&D), the absorbance was measured at 490 nm using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of hIL-6 plasma concentration after intravenous administration as measured by this method is shown in FIG. 30 for normal mice.

Measurement of plasma concentration of anti-IL-6 antibody by ELISA

The concentration of anti-human IL-6 antibody in mouse plasma was measured by ELISA. The F(ab')2 fragment of anti-human IgG (gamma chain specific) (Sigma) was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare immobilized anti-IgG plates. Calibration curve samples were obtained with plasma concentrations of 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0.025 microgram/ml, and mouse plasma samples diluted 100-fold or more. To obtain anti-human IL-6 antibody fully bound to human IL-6 in the sample, 200 microliters of human IL-6 at 1 microgram/ml was added to 100 microliters of calibration curve samples and plasma samples, and then the samples were left for one hour at room temperature. Samples were then transferred to anti-human IgG antibody-coated plates and left for one hour at room temperature. Next, goat anti-human IgG (gamma chain specific) biotin-conjugated (BIOT) antibody (Southern Biotech Association) was added to react for one hour at room temperature. Next, streptavidin-polyHRP80 (Stereospecific Detection Technologies) was added to react for one hour at room temperature, and a chromogenic reaction was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After stopping the reaction 1 N. sulfuric acid (Showa Chemical), absorbance at 450 nm was measured using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma antibody concentration after intravenous administration as measured by this method is presented for normal mice in FIG. 31.

Effect of pH-dependent binding to human IL-6

CLB8-IgG1 and H16/L13-IgG1, which bind to human IL-6 in a pH-dependent manner, were tested in vivo and the results were compared with each other. As shown in Fig. 31, the pharmacokinetics of the antibody demonstrated linear clearance. Meanwhile, as shown in FIG. 30, hIL-6 coadministered with H16/L13-IgG1, which binds to human IL-6 in a pH-dependent manner, was found to accelerate the elimination of hIL-6 compared with hIL-6. administered simultaneously with CLB8-IgG1. Thus, it was shown that by imparting a pH-dependent binding ability to human IL-6, the plasma concentration of hIL-6 four days after administration could be reduced by approximately 76-fold.

Effect of binding to FcRn under neutral conditions (pH 7.4)

In addition to H16/L13-IgG1, H16/L13-IgG1-v2 and H16/L13-F14, which are derived from the amino acid substitutions described above in H16/L13-IgG1, were tested in vivo using normal mice. The test results were compared with those obtained for H16/L13-IgG1. As shown in Figure 31, the plasma concentration of antibody H16/L13-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), was 2.9-fold lower compared to H16/L13-IgG1 per day one after administration. Alternatively, the plasma concentration of antibody H16/L13-F14, which further increased binding to mouse FcRn under neutral conditions (pH 7.4), was 21-fold lower than that for H16/L13-IgG1 at 7 hours post-administration.

As shown in FIG. 30, hIL-6 coadministered with H16/L13-IgG1-v2 or H16/L13-F14, which increased binding to mouse FcRn under neutral conditions (pH 7.4), showed faster clearance by compared with hIL-6 administered simultaneously with H16/L13-IgG1. H16/L13-IgG1-v2 reduced the plasma concentration of hIL-6 by approximately 10-fold compared with H16/L13-IgG1 on day 1. H16/L13-F14 reduced the plasma concentration of hIL-6 by approximately 38-fold compared with H16 /L13-IgG1 in seven hours. Thus, it was found that the plasma concentration of human IL-6 can be reduced by imparting the ability to bind to mouse FcRn under neutral conditions (pH 7.4). As described above, by allowing binding to mouse FcRn under neutral conditions (pH 7.4), the plasma concentration of the antibody is reduced; however, there was an effect of reducing hIL-6 plasma concentrations that greatly exceeded the reduction in antibody concentrations. Specifically, this means that the elimination of human IL-6 can be increased by administering an antibody that binds to human IL-6 in a pH-dependent manner and is rendered capable of binding to mouse FcRn under neutral conditions (pH 7.4).

The results obtained above demonstrated that the plasma concentration of antigen, not only soluble human IL-6 receptor, but also antigen such as human IL-6, can be significantly reduced by administering an antibody having pH-dependent antigen binding ability and the ability to bind to FcRn under neutral conditions.

[Example 16] Study on enhancing the elimination-accelerating effect of human IgA Fc receptor fusion protein that binds to human IgA in a pH-dependent manner

Preparation of an Fc receptor fusion protein that binds to human IgA in a pH-dependent manner

A0-IgG1 containing dimer A0H-IgG1 (SEQ ID NO: 22) is a human CD89-Fc fusion protein. As described in J. Mol. Biol. (2003) 324: 645-657, human CD89, also known as human Fc alpha receptor I, binds to human IgA in a pH-dependent manner (i.e., binds strongly to human IgA at neutral pH, but weakly binds to human IgA at acidic pH values).

Evaluation of pH-dependent binding activity of CD89-Fc fusion protein to human IgA

A0-IgG1 was assessed against human IgA binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 using Biacore T100 (GE Healthcare). The assay was performed using 10 mmol/1 ACES/150 mmol/1 NaCl containing 0.05% P20 surfactant (pH 7.4 and pH 6.0) as running buffer. The CD89-Fc fusion protein was then coupled to recombinant protein A/G (Thermo Scientific) immobilized on sensor chips using a coupled amine method, and appropriate concentrations of hIgA were injected as the analyte (human IgA: prepared as described in Reference Example 5 ). Assays were performed at 37 degrees C. Results were analyzed using Biacore T100 Evaluation software (GE Healthcare) and the resulting sensorgrams are presented in FIG. 32. The CD89-Fc fusion protein is clearly shown to have pH-dependent human IgA binding activity, where the fusion protein binds strongly to human IgA at neutral pH, but weakly binds to human IgA at acidic pH.

Preparation of a pH-dependent Fc receptor fusion protein having FcRn binding activity under neutral conditions

Mutations were introduced into A0-IgG1 containing the A0H-IgG1 dimer (SEQ ID NO: 22) to increase binding to FcRn under neutral conditions (pH 7.4). Specifically, A0-IgG1-v2 was derived from the heavy chain constant region of IgG1 by replacing Trp with Asn at position 426 in A0-IgG1. Amino acid substitutions were introduced in a manner known to those skilled in the art, as described in Reference Example 1.

A0-IgG1 containing the A0H-IgG1 dimer (SEQ ID NO: 22) and A0-IgG1-v2 containing the A0H-IgG1-v2 dimer (SEQ ID NO: 23) were expressed and purified in a manner known to those skilled in the art, described in reference example 2.

In vivo test using normal mice

The in vivo kinetics of human IgA (hIgA) and CD89-Fc fusion protein were assessed after administration of hIgA or hIgA and CD89-Fc fusion protein (A0H-IgG1 or A0H-IgG1-v2) separately in a normal mouse (C57BL/6J mice; Charles River Japan ). An hIgA solution (80 microgram/ml) or a mixture solution containing hIgA and a CD89-Fc fusion protein (80 microgram/ml and 1.5 mg/ml, respectively, in which the majority of the hIgA is bound to the CD89-Fc fusion protein) was administered once at a dose of 10 ml/kg into the tail vein. Blood was collected 15 minutes, seven hours, one day, two days, four days and seven days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -20 degrees C or below until analysis.

Measuring human plasma IgA concentration by ELISA

The concentration of human IgA in mouse plasma was measured by ELISA using hsIL-6R, since recombinant human IgA has a variable region against hsIL-6R. Goat anti-human IgA antibody (Bethyl Laboratories) was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare immobilized anti-human IgA plates. Calibration curve samples having plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, or 0.00625 microgram/mL and mouse plasma samples diluted 100-fold or more were prepared. To obtain human IgA completely bound to hsIL-6R in the sample, 200 microliters of 10 microgram/ml hsIL-6R was added to 100 microliters of calibration curve samples and plasma samples, and then the samples were left for one hour at room temperature. Then, the samples were transferred to plates with immobilized anti-human IgA antibody and left for one hour at room temperature. Biotinylated anti-human IL-6R (R&D) antibody was then added to react for one hour at room temperature. Streptavidin-polyHRP80 (Stereospecific Detection Technologies) was then added to react for one hour at room temperature, and a chromogenic reaction was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After stopping the reaction 1 N. sulfuric acid (Showa Chemical), absorbance was measured at 450 nm using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of hIgA plasma concentration after intravenous administration measured by this method is shown in FIG. 33 for normal mice.

Measurement of plasma concentration of CD89-Fc fusion protein by ELISA

The concentration of CD89-Fc fusion protein in mouse plasma was measured by ELISA. The F(ab')2 fragment of anti-human IgG (specific gamma chain) (Sigma) was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare immobilized anti-human IgG plates. Calibration curve samples were prepared with plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, and 0.4 microgram/ml, and mouse plasma samples diluted 100-fold and more. To obtain a CD89-Fc fusion protein in a sample fully bound to human IgA, 200 microliters of 5 microgram/ml human IgA was added to 100 microliters of calibration curve samples and plasma samples, and then the samples were left for one hour at room temperature. The samples were then transferred to anti-human IgG immobilized plates and left for one hour at room temperature. Goat anti-human IgG (Fc specific)-alkaline phosphatase (SIGMA) conjugate was then added to react for one hour at room temperature. The chromogenic reaction was then carried out using the BluePhos Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as the substrate, and the absorbance at 650 nm was measured using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of the plasma concentration of the CD89-Fc fusion protein after intravenous administration as measured by this method is presented in FIG. 34 for normal mice.

Effect of binding to FcRn under neutral conditions (pH 7.4)

In addition to A0-IgG1, A0-IgG1-v2, which is derived from introducing the above amino acid substitutions into A0-IgG1, was tested in vivo using normal mice. The test results were compared with the results of A0-IgG1. As shown in Fig. 34, the plasma concentration of A0-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), was 1.8 times less than for A0-IgG1 two days after administration .

As shown in FIG. 33, hIgA coadministered with A0-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), showed faster clearance compared to hIgA coadministered with A0-IgG1. A0-IgG1-v2 reduced the plasma concentration of hIgA by approximately 5.7 times compared to A0-IgG1 after two days. As described above, by imparting binding ability to mouse FcRn under neutral conditions (pH 7.4), the plasma concentration of the antibody was reduced; however, the effect of reducing plasma hIgA concentrations was greater than the reduction in antibody concentrations. Specifically, this means that the elimination of human IgA can be accelerated by introducing an Fc receptor fusion protein that binds to human IgA in a pH-dependent manner and is rendered capable of binding to mouse FcRn under neutral conditions (pH 7.4).

The results described above indicate that the plasma concentration of an antigen, such as that of human IgA, can also be significantly reduced by administering an Fc receptor fusion protein having both pH-dependent antigen-binding ability and FcRn-binding ability under neutral conditions. Thus, an Fc receptor fusion protein can also be engineered to impart the ability to eliminate plasma concentrations of antigen (or ligand) from plasma.

[Example 17] Study on increasing the effect of accelerating the elimination of plexin A1 of pH-dependent antibodies against plexin A1 (antibody production)

Consideration of a pH-dependent antibody that binds human plexin A1

PX268-IgG1, containing PX268H-IgG1 (SEQ ID NO: 24) and PX268L-CK (SEQ ID NO: 25), is a chimeric antibody against plexin A1. PX141-IgG1, containing PX141H-IgG1 (SEQ ID NO: 26) and PX141L-CK (SEQ ID NO: 27), is a chimeric anti-plexin A1 antibody that binds to soluble human plexin A1 in a pH-dependent manner (i.e. binds strongly to soluble human plexin A1 at neutral pH, but binds weakly to soluble human plexin A1 at acidic pH).

Evaluation of pH-dependent binding activity of anti-human plexin A1 antibody

PX268-IgG1 and PX141-IgG1 were assessed against human plexin A1 binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 with Biacore T100 (GE Healthcare). The assay was performed using 10 mmol/1 ACES/150 mmol/1 NaCl containing 0.05% P20 surfactant (pH 7.4 and pH 6.0) as running buffer. Antibodies were then coupled to recombinant protein A/G (Thermo Scientific) immobilized on sensor chips using a coupled amine method, suitable concentrations of hsPlexin A1 (soluble human plexin A1: prepared as described in Reference Example 5) were injected as analyte . Assays were performed at 37 degrees C. Results were analyzed using Biacore T100 Evaluation software (GE Healthcare), and the association rate constant, ka (1/Ms), and dissociation rate constant, k _d (1/s), were calculated from the assay results . KD(M) was then calculated from ka and k _d (Table 15). In addition, pH-dependent binding was assessed to calculate the KD ratio between pH 7.4 and pH 6.0 for each antibody.

Table 15 Ligand Sample_pH ka (1/Ms) kd (1/s) KD(M) KD (pH 6.0)/KD (pH 7.4) PX268-IgGl pH 7.4 5.2E+04 2.8E-04 5.4E-09 0.8 pH 6.0 6.3E+04 2.7E-04 4.4E-09 PX141-IgGl pH 7.4 1.5E+05 6.4E-04 4.2E-09 14.9 pH 6.0 7.9E+04 4.9E-03 6.3E-08

Generation of pH-dependent antibodies against human plexin A1 with FcRn binding activity under neutral conditions

Mutations were introduced into PX141-IgG1, containing PX141H-IgG1 (SEQ ID NO: 26) and PX141L-CK (SEQ ID NO: 27), to increase binding to FcRn under neutral conditions (pH 7.4). Specifically, PX141H-IgG1-v2 (SEQ ID NO: 28) was obtained from the constant region of the heavy chain of IgG1 by replacing Trp with Asn at position 434 in the EU numbering. Amino acid substitutions were introduced in a manner known to those skilled in the art, as described in Reference Example 1.

PX268-IgG1 containing PX268H-IgG1 (SEQ ID NO: 24) and PX268L-CK (SEQ ID NO: 25), PX141-IgG1 containing PX141H-IgG1 (SEQ ID NO: 26) and PX141L-CK (SEQ ID NO : 27), and PX141-IgG1-v2, containing PX141H-IgG1-v2 (SEQ ID NO: 28) and PX141L-CK (SEQ ID NO: 27), were expressed and purified in a manner known to those skilled in the art, described in ref. example 2.

In vivo testing using normal mice

In vivo kinetics of soluble human plexin A1 (hsPlexin A1) and anti-plexin A1 antibody were assessed following administration of hsPlexin A1 alone or hsPlexin A1 and anti-plexin A1 antibody to normal mice (C57BL/6J mice; Charles River Japan). hsPlexin A1 solution (100 micrograms/ml) or a mixture solution containing hsPlexin A1 and anti-plexin A1 antibody (group PX268-IgG1; 100 micrograms/ml hsPlexin A1 and 1.2 mg/ml PX268-IgG1, group PX141-IgG1 and PX141 -IgG1-v2; 100 microgram/ml hsPlexin A1 and 1.0 mg/ml PX141-IgG1 and PX141-IgG1-v2, respectively) were administered as a single dose of 10 ml/kg into the tail vein.

The dose of antibody was adjusted such that more than 99.9% of the soluble human plexin A1 was bound to the antibody in the administration solution. Blood was collected 15 minutes, seven hours, one day, two days, four days, seven days after administration of hsPlexin A1 and a mixture of solution with anti-plexin A1 antibody. The collected blood was immediately centrifuged at 15,000 rpm and 4 degrees C for 15 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -20 degrees C or below until analysis.

Measurement of human plasma plexin A1 concentration by ELISA after administration of hsPlexin A1 alone

The concentration of human plexin A1 in mouse plasma was measured by ELISA using biotinylated anti-FLAG antibody M2 (Sigma), since recombinant human plexin A1 has a terminal FLAG tag sequence at the C terminus. Rabbit polyclonal anti-human plexin A1 antibody generated by immunization with rabbit plexin A1 was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare anti-plexin A1 antibody immobilized plates. Calibration curve samples having plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6, and 0.8 microgram/ml and mouse plasma samples diluted 100-fold or more were prepared. Samples were then transferred to anti-human plexin A1 antibody-immobilized plates and left for one hour at room temperature. Biotinylated anti-FLAG M2 antibody (Sigma) was then added to react for one hour at room temperature. Streptavidin-polyHRP80 (Stereospecific Detection Technologies) was then added to react for one hour at room temperature, and a chromogenic reaction was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as the substrate. After stopping the reaction 1 N. sulfuric acid (Showa Chemical) absorbance was measured at 450 nm using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma concentration of hsPlexin A1 after intravenous administration measured by this method is shown in FIG. 35.

Measurement of human plasma plexin A1 concentration in the PX268-IgG1 group by ELISA

The concentration of human plexin A1 in mouse plasma was measured by ELISA using biotinylated anti-FLAG antibody M2 (Sigma), since recombinant human plexin A1 has a terminal FLAG tag sequence at the C terminus. Rabbit polyclonal anti-human plexin A1 antibody generated by immunization with rabbit plexin A1 was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare anti-human plexin A1 immobilized antibody plates. Calibration curve samples having plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6 and 0.8 microgram/ml and mouse plasma samples diluted 50-fold or more were prepared. To ensure that human plexin A1 was fully bound to PX268-IgG1 in the sample, 150 microliters of PX268-IgG1 at a concentration of 40 micrograms/ml was added to 150 microliters of calibration curve samples and plasma samples, and then the samples were left overnight at 37 degrees C. The samples were then transferred to human plexin A1 antibody-immobilized plates and left for one hour at room temperature (or 4 degrees C). Biotinylated anti-FLAG M2 antibody (Sigma) was then added to react for one hour at room temperature (or 4 degrees C). Streptavidin-polyHRP80 (Stereospecific Detection Technologies) was then added to react for one hour at room temperature (or 4 degrees C) and a chromogenic reaction was carried out using TMB One Component HRP Microwell Subsrate (BioFX Laboratories) as the substrate. After stopping the reaction 1 N. sulfuric acid (Showa Chemical) absorbance was measured at 450 nm using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The time course of plasma concentrations of hsPlexin A1 after intravenous administration measured by this method is shown in FIG. 35.

Measurement of human plasma plexin A1 concentration in PX141-IgG1 and PX141-IgG1-v2 group by ELISA

The concentration of human plexin A1 in mouse plasma was measured by ELISA using a biotinylated anti-FLAG antibody M2 (Sigma), since recombinant human plexin A1 has a terminal FLAG tag sequence at the C terminus. PX268-IgG1 was transferred to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4 degrees C to prepare anti-plexin A1 antibody-immobilized plates. Calibration curve samples having plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6 and 0.8 microgram/ml and mouse plasma samples diluted 50-fold or more were prepared. To ensure that human plexin A1 is completely bound to PX141-IgG1 or PX141-IgG1-v2 in a sample, 150 microliters of PX141-IgG1 or PX141-IgG1-v2 at a concentration of 40 micrograms/ml was added to 150 microliters of calibration curve samples and plasma samples, and the samples were then left overnight at 37 degrees C. The samples were then transferred to human plexin A1 antibody-coated plates and left for one hour at room temperature (or 4 degrees C). Biotinylated anti-FLAG M2 antibody (Sigma) was then added to react for one hour at room temperature (or 4 degrees C). Streptavidin-polyHRP80 (Stereospecific Detection Technologies) was then added to react for one hour at room temperature (or 4 degrees C) and a chromogenic reaction was carried out using TMB One Component HRP Microwell Subsrate (BioFX Laboratories) as the substrate. After stopping the reaction with 1 N sulfuric acid (Showa Chemical), absorbance was measured at 450 nm using a microplate reader. Mouse plasma concentration was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). The plasma concentration of hsPlexin A1 7 hours after intravenous administration measured by this method is presented in Fig. 35.

Effect of pH-dependent binding to soluble human plexin A1

PX268-IgG1 and PX141-IgG1, which bind to human IL-6 in a pH-dependent manner, were tested in vivo and the results were compared with each other. Meanwhile, as shown in FIG. 35, hsPlexin A1 co-administered with PX141-IgG1, which binds to soluble human plexin A1 in a pH-dependent manner, showed a decrease in the total plasma concentration of hsPlexin A1 compared with hsPlexin A1 co-administered with PX268-IgG1.

Effect of binding to FcRn under neutral conditions (pH 7.4)

In addition to PX141-IgG1, PX141-IgG1-v2, which is derived from introducing the amino acid substitutions described above into PX141-IgG1, was tested in vivo using normal mice. The test results were compared with the results obtained for PX 141-IgG1.

As shown in FIG. 35, hsPlexin A1 coadministered with PX141-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), showed a decrease in the total plasma concentration of hsPlexin A1 to undetectable levels (detection limit represents is 0.8 micrograms/ml). Thus, it is shown that the concentration of soluble human plexin A1 can be reduced by imparting binding ability to mouse FcRn under neutral conditions (pH 7.4). Specifically, this means that the elimination of soluble human plexin A1 can be increased by administering an antibody that binds to human plexin A1 in a pH-dependent manner and is rendered capable of binding to mouse FcRn under neutral conditions (pH 7.4).

The results described above demonstrate that the concentration of antigen in plasma, not only soluble human IL-6 receptor, but also antigen such as human IL-6, human IgA and soluble human plexin A1 can also be significantly reduced by administering an antibody having and pH-dependent antigen-binding ability and FcRn binding ability under neutral conditions.

[Reference Example 1] Construction of Expression Vectors for IgG Antibodies with Amino Acid Substitutions Introduced

Mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) or the In-Fusion HD cloning kit (Clontech) according to the method described in the instructions provided, and the resulting plasmid fragments were inserted into a mammalian cell expression vector to obtain the desired vectors , expressing the H chain, and vectors expressing the L chain. The nucleotide sequences of the resulting expression vectors were determined using conventional methods known to those skilled in the art.

[Reference Example 2] Expression and Purification of an IgG Antibody

Antibodies were expressed in the manner described below. Antibodies were expressed in FreestyleHEK293 (Invitrogen) as described in the protocol provided by the manufacturer or in the HEK293H cell line (Invitrogen). The HEK293H cell line derived from human fetal renal carcinoma cells (Invitrogen) was suspended in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Cells were seeded at 10 ml per dish in cell attachment dishes (10 cm in diameter; CORNING) at a cell density of 5-6 x 10 ⁵ cells/ml and cultured in a CO ₂ incubator (37 degrees C, 5% CO ₂ ) during the day and night. The medium was then removed by aspiration and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The resulting plasmid was introduced into cells by lipofection. The resulting supernatant cultures were harvested, centrifuged (approximately 2000×g, 5 min, room temperature) to remove cells, and filter sterilized through a 0.22 micrometer MIILEX (registered trademark)-GV filter (Millipore) to obtain supernatants . Antibodies were purified from the resulting culture supernatants in a manner known to those skilled in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine the concentration of purified antibody, absorbance was measured at 280 nm using a spectrophotometer. Antibody concentrations were calculated from the determined values using the optical intensity coefficient calculated by the method described in Protein Science (1995) 4: 2411-2423.

[Reference Example 3] Preparation of Soluble Human IL-6 Receptor (hsIL-6R)

Recombinant human IL-6 receptor as an antigen was obtained by the following method. A cell line constitutively expressing soluble human IL-6 receptor (hereinafter referred to as hsIL-6R) having the amino acid sequence at positions 1 to 357 from the N-terminus, as described in J. Immunol. 152: 4958-4968 (1994), were created in a manner known to those skilled in the art. Cells were cultured to express hsIL-6R. hsIL-6R was purified from the culture supernatant by two steps: chromatography on a Blue Sepharose 6 FF column and gel filtration. The fractions eluted as the main peak in the final step were used as the final purification product.

[Reference Example 4] Preparation of Human FcRn

FcRn is a complex of FcRn and beta2-microglobulin. Oligo-DNA primers were prepared based on the published sequence of the human FcRn gene (J Exp Med. 1994 Dec 1; 180(6): 2377-81). A DNA fragment encoding the complete gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the resulting primers. Using the resulting DNA fragment as a template, the DNA fragment encoding the extracellular domain containing the signal sequence (Metl-Leu290) was amplified by PCR and inserted into a mammalian cell expression vector. In addition, oligo-DNA primers were prepared based on the published sequence of the human beta2-microglobulin gene (Proc. Natl. Acad. Sci. U.S.A. 99 (26): 16899-16903 (2002)). A DNA fragment encoding the complete gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the resulting primers. Using the resulting DNA fragment as a template, the DNA fragment encoding the complete protein containing the signal sequence (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

Soluble human FcRn was expressed by the following method. Plasmids engineered to express human FcRn (SEQ ID NO: 30) and beta2-microglobulin (SEQ ID NO: 31) were introduced into the HEK293H cell line derived from human embryonal renal carcinoma cells (Invitrogen) by a lipofection method using PEI ( Polyscience). The resulting culture supernatants were collected and FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences), followed by further purification using HiTrap Q HP (GE Healthcare) (J Immunol. 2002 Nov 1; 169(9): 5171- 80).

[Reference Example 5] Production of human IgA (hIgA)

hIgA containing H (WT)-IgA1 (SEQ ID NO: 29) and L (WT) (SEQ ID NO: 5) was expressed and purified in a manner known to those skilled in the art using rProtein L-agarose (ACTIgen) followed by gel filtration.

[Reference Example 6] Preparation of Soluble Human Plexin A1 (hsPlexin A1)

Recombinant soluble human plexin A1 antigen (hereinafter referred to as hsPlexin A1) was prepared as follows. hsPlexin A1 was constructed by consulting the NCBI Reference Sequence (NP_115618). Specifically, hsPlexin A1 contained an amino acid sequence at positions 27-1243 from the above NCBI Reference FLAG-tag (DYKDDDDK), which was connected to its C terminus. hsPlexin A1 was transiently expressed using FreeStyle293 (Invitrogen) and purified from the culture supernatant through two steps: anti-FLAG column chromatography and gel filtration. The fractions eluted as the main peak in the final step were used as the final purification product.

--->

LIST OF SEQUENCES

<110> CHUGAI SEIYAKU KABUSHIKI KAISHA

<120> MOLECULES THAT BIND ANTIGENS THAT INCREASE CLEARANCE

ANTIGENS

<130> C1-A1004Y1P

<150> JP 2010-079667

<151> 2010-03-30

<150> JP 2010-250830

<151> 2010-11-09

<160> 31

<170> Patent In version 3.4

<210> 1

<211> 447

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<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 2

<211> 214

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 3

<211> 447

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<213> Artificial

<220>

<223> Artificially synthesized sequence

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Trp Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 4

<211> 449

<212> PROTEIN

<213> Homo sapiens

<400> 4

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 5

<211> 214

<212> PROTEIN

<213> Homo sapiens

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 7

<211> 214

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 7

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 8

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr

245 250 255

Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 9

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 10

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Phe Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 11

<211> 324

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 12

<211> 324

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 13

<211> 326

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 14

<211> 324

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 15

<211> 443

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 15

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 16

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Ala Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Trp Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 17

<211> 213

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 17

Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 18

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Ala Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr His Pro His Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Trp Gly His Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 19

<211> 213

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 19

Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser His His Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Gly His Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 20

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Ala Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr His Pro His Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Trp Gly His Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 21

<211> 447

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Ala Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr His Pro His Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Trp Gly His Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Trp Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 22

<211> 436

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 22

Gln Glu Gly Asp Phe Pro Met Pro Phe Ile Ser Ala Lys Ser Ser Pro

1 5 10 15

Val Ile Pro Leu Asp Gly Ser Val Lys Ile Gln Cys Gln Ala Ile Arg

20 25 30

Glu Ala Tyr Leu Thr Gln Leu Met Ile Ile Lys Asn Ser Thr Tyr Arg

35 40 45

Glu Ile Gly Arg Arg Leu Lys Phe Trp Asn Glu Thr Asp Pro Glu Phe

50 55 60

Val Ile Asp His Met Asp Ala Asn Lys Ala Gly Arg Tyr Gln Cys Gln

65 70 75 80

Tyr Arg Ile Gly His Tyr Arg Phe Arg Tyr Ser Asp Thr Leu Glu Leu

85 90 95

Val Val Thr Gly Leu Tyr Gly Lys Pro Phe Leu Ser Ala Asp Arg Gly

100 105 110

Leu Val Leu Met Pro Gly Glu Asn Ile Ser Leu Thr Cys Ser Ser Ala

115 120 125

His Ile Pro Phe Asp Arg Phe Ser Leu Ala Lys Glu Gly Glu Leu Ser

130 135 140

Leu Pro Gln His Gln Ser Gly Glu His Pro Ala Asn Phe Ser Leu Gly

145 150 155 160

Pro Val Asp Leu Asn Val Ser Gly Ile Tyr Arg Cys Tyr Gly Trp Tyr

165 170 175

Asn Arg Ser Pro Tyr Leu Trp Ser Phe Pro Ser Asn Ala Leu Glu Leu

180 185 190

Val Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser His Thr Ser Pro Pro Ser Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser

435

<210> 23

<211> 436

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 23

Gln Glu Gly Asp Phe Pro Met Pro Phe Ile Ser Ala Lys Ser Ser Pro

1 5 10 15

Val Ile Pro Leu Asp Gly Ser Val Lys Ile Gln Cys Gln Ala Ile Arg

20 25 30

Glu Ala Tyr Leu Thr Gln Leu Met Ile Ile Lys Asn Ser Thr Tyr Arg

35 40 45

Glu Ile Gly Arg Arg Leu Lys Phe Trp Asn Glu Thr Asp Pro Glu Phe

50 55 60

Val Ile Asp His Met Asp Ala Asn Lys Ala Gly Arg Tyr Gln Cys Gln

65 70 75 80

Tyr Arg Ile Gly His Tyr Arg Phe Arg Tyr Ser Asp Thr Leu Glu Leu

85 90 95

Val Val Thr Gly Leu Tyr Gly Lys Pro Phe Leu Ser Ala Asp Arg Gly

100 105 110

Leu Val Leu Met Pro Gly Glu Asn Ile Ser Leu Thr Cys Ser Ser Ala

115 120 125

His Ile Pro Phe Asp Arg Phe Ser Leu Ala Lys Glu Gly Glu Leu Ser

130 135 140

Leu Pro Gln His Gln Ser Gly Glu His Pro Ala Asn Phe Ser Leu Gly

145 150 155 160

Pro Val Asp Leu Asn Val Ser Gly Ile Tyr Arg Cys Tyr Gly Trp Tyr

165 170 175

Asn Arg Ser Pro Tyr Leu Trp Ser Phe Pro Ser Asn Ala Leu Glu Leu

180 185 190

Val Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser His Thr Ser Pro Pro Ser Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Trp His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser

435

<210> 24

<211> 448

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 24

Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ser Asn Tyr

20 25 30

Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Cys Ile Tyr Ile Gly Ser Asp Ser Ala Tyr Tyr Ala Ser Trp Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu

65 70 75 80

Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Arg Trp Asp Asn Ser Gly Arg Ala Leu Lys Leu Trp Gly Pro Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 25

<211> 219

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 25

Gln Met Leu Thr Gln Thr Ala Ser Pro Val Ser Ala Ala Val Gly Gly

1 5 10 15

Thr Val Thr Ile Lys Cys Gln Ser Ser Gln Ser Val Ala Asp Asn Asn

20 25 30

His Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Phe Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys

50 55 60

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Gln

65 70 75 80

Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Asn Tyr Asp Cys Gly

85 90 95

Ser Ala Asp Cys His Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 26

<211> 454

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 26

Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Ile Val Thr Gly Ser Tyr Gly Arg Ser Trp Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Thr Arg Ser Thr Ser Leu Asn Thr

65 70 75 80

Val Thr Leu Gln Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr

85 90 95

Phe Cys Ala Arg Asp Pro Phe Val Ile Ala Ser Ser His Tyr Gln Asn

100 105 110

Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 27

<211> 218

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 27

Ala Leu Val Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Pro Asn Ile Tyr Asn Asn

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Gln

65 70 75 80

Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Ser Tyr Asp

85 90 95

Asn Asp Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 28

<211> 454

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 28

Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Ile Val Thr Gly Ser Tyr Gly Arg Ser Trp Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Thr Arg Ser Thr Ser Leu Asn Thr

65 70 75 80

Val Thr Leu Gln Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr

85 90 95

Phe Cys Ala Arg Asp Pro Phe Val Ile Ala Ser Ser His Tyr Gln Asn

100 105 110

Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 29

<211> 472

<212> PROTEIN

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 29

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Pro Thr Ser Pro Lys Val Phe

115 120 125

Pro Leu Ser Leu Cys Ser Thr Gln Pro Asp Gly Asn Val Val Ile Ala

130 135 140

Cys Leu Val Gln Gly Phe Phe Pro Gln Glu Pro Leu Ser Val Thr Trp

145 150 155 160

Ser Glu Ser Gly Gln Gly Val Thr Ala Arg Asn Phe Pro Pro Ser Gln

165 170 175

Asp Ala Ser Gly Asp Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu Pro

180 185 190

Ala Thr Gln Cys Leu Ala Gly Lys Ser Val Thr Cys His Val Lys His

195 200 205

Tyr Thr Asn Pro Ser Gln Asp Val Thr Val Pro Cys Pro Val Pro Ser

210 215 220

Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

225 230 235 240

Cys Cys His Pro Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu

245 250 255

Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg

260 265 270

Asp Ala Ser Gly Val Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser

275 280 285

Ala Val Gln Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val

290 295 300

Ser Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys Thr

305 310 315 320

Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro Leu Thr Ala

325 330 335

Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu

340 345 350

Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr

355 360 365

Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu

370 375 380

Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser

385 390 395 400

Arg Gln Glu Pro Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile

405 410 415

Leu Arg Val Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys

420 425 430

Met Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile

435 440 445

Asp Arg Leu Ala Gly Lys Pro Thr His Val Asn Val Ser Val Val Met

450 455 460

Ala Glu Val Asp Gly Thr Cys Tyr

465 470

<210> 30

<211> 365

<212> PROTEIN

<213> Homo sapiens

<400> 30

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr

20 25 30

His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp

35 40 45

Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu

50 55 60

Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val

65 70 75 80

Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys

85 90 95

Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr

100 105 110

Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val

115 120 125

Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp

130 135 140

Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile

145 150 155 160

Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr

165 170 175

Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg

180 185 190

Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys

195 200 205

Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe

210 215 220

Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu

225 230 235 240

Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser

245 250 255

Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His

260 265 270

Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val

275 280 285

Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val

290 295 300

Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu

305 310 315 320

Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg

325 330 335

Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp

340 345 350

Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala

355 360 365

<210> 31

<211> 119

<212> PROTEIN

<213> Homo sapiens

<400> 31

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

35 40 45

Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met

115

<---

Claims (102)

1. A method for producing an antibody, comprising: (a) selecting an antibody that contains an antigen binding domain and a human FcRn binding domain, wherein the antibody has human FcRn binding activity at pH 5.8 and 7.4 and lower antigen binding activity at pH 5.8 than at pH 7, 4, wherein the binding activity to human FcRn at pH 7.4 is higher than that of intact human IgG, and the ratio of antigen binding activity at pH 5.8 to 7.4 is at least 2 for the KD value at pH 5.8/ KD at pH 7.4, wherein the human FcRn-binding domain contains the human IgG Fc domain, (b) culturing a host cell containing a vector into which a polynucleotide encoding the antibody selected in step (a) is introduced, and (c) releasing the antibody from the host cell. 2. The method of claim 1, wherein the antibody comprises an amino acid mutation of the antigen binding domain that comprises a substitution of at least one amino acid in the antigen binding domain for histidine or an insertion of at least one histidine. 3. The method of claim 1, wherein the antigen binding domain is obtained from a library of antigen binding domains. 4. Method according to any one of paragraphs. 1-3, wherein the antibody contains, as a human FcRn binding domain, an Fc domain resulting from the substitution of various amino acids with at least one amino acid in the Fc domain of the parent human IgG. 5. Method according to any one of paragraphs. 1-4, wherein the human FcRn binding domain is a human FcRn binding domain comprising an amino acid sequence with various amino acids replaced by at least one amino acid selected from amino acids at positions 237, 238, 239, 248, 250, 252, 254 , 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 3 76 , 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 EU numbering in the Fc domain of parental human IgG. 6. Method according to any one of paragraphs. 1-5, wherein the antibody comprises a human FcRn binding domain comprising an amino acid substitution in the Fc domain of a parental human IgG that includes at least one amino acid substitution selected from: replacing the amino acid Met with Gly at position 237; replacing the amino acid Ala with Pro at position 238; replacing the amino acid Lys with Ser at position 239; replacing the amino acid Ile with Lys at position 248; substitution of amino acids Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr with Thr at position 250; replacing the amino acids Phe, Trp or Tyr with Met at position 252; replacing the amino acid Thr with Ser at position 254; replacing the amino acid Glu with Arg at position 255; substitution of amino acids Asp, Glu or Gln with Thr at position 256; substitution of amino acids Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val with Pro at position 257; amino acid replacements of His with Glu at position 258; replacing the amino acid Ala with Asp at position 265; replacing the amino acid Phe with Asp at position 270; replacing the amino acid Ala or Glu with Asn at position 286; replacing the amino acid His with Thr at position 289; replacing the amino acid Ala with Asn at position 297; replacing the amino acid Gly with Ser at position 298; replacing the amino acid Ala with Val at position 303; replacing the amino acid Ala with Val at position 305; replacing the amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr with Thr at position 307; substitution of amino acids Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr with Val at position 308; substitution of amino acids Ala, Asp, Glu, Pro or Arg with Leu or Val at position 309; replacing the amino acid Ala, His or Ile with Gln at position 311; replacing amino acids Ala or His with Asp at position 312; replacing the amino acid Lys or Arg with Leu at position 314; replacing the amino acid Ala or His with Asn at position 315; replacing the amino acid Ala with Lys at position 317; replacing the amino acid Gly with Asn at position 325; replacing the amino acid Val with Ile at position 332; replacing the amino acid Leu with Lys at position 334; replacing the amino acid His with Lys at position 360; replacing the amino acid Ala with Asp at position 376; replacing the amino acid Ala with Glu at position 380; replacing the amino acid Ala with Glu at position 382; replacing the amino acid Ala with Asn or Ser at position 384; replacing the amino acid Asp or His with Gly at position 385; replacing the amino acid Pro with Gln at position 386; replacing the amino acid Glu with Pro at position 387; replacing the amino acid Ala or Ser with Asn at position 389; replacing the amino acid Ala with Ser at position 424; replacing the amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr with Met at position 428; replacing the amino acid Lys with His at position 433; replacing the amino acid Ala, Phe, His, Ser, Trp or Tyr with Asn at position 434; and replacing the amino acid His or Phe with Tyr at position 436 according to EU numbering. 7. Method according to any one of paragraphs. 1-5, wherein the human FcRn binding domain contains at least one amino acid selected from: Met at amino acid position 237; Ala at amino acid position 238; Lys at amino acid position 239; Ile at amino acid position 248; Ala, Phe, He, Met, Gln, Ser, Val, Trp or Tyr at amino acid position 250; Phe, Trp or Tyr at amino acid position 252; Thr at amino acid position 254; Glu at amino acid position 255; Asp, Glu or Gln at amino acid position 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val at amino acid position 257; His at amino acid position 258; Ala at amino acid position 265; Phe at amino acid position 270; Ala or Glu at amino acid position 286; His at amino acid position 289; Ala at amino acid position 297; Gly at amino acid position 298; Ala at amino acid position 303; Ala at amino acid position 305; Ala, Asp, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr at amino acid position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr at amino acid position 308; Ala, Asp, Glu, Pro or Arg at amino acid position 309; Ala, His or Ile at amino acid position 311; Ala or His at amino acid position 312; Lys or Arg at amino acid position 314; Ala or His at amino acid position 315; Ala at amino acid position 317; Gly at amino acid position 325; Val at amino acid position 332; Leu at amino acid position 334; His at amino acid position 360; Ala at amino acid position 376; Ala at amino acid position 380; Ala at amino acid position 382; Ala at amino acid position 384; Asp or His at amino acid position 385; Pro at amino acid position 386; Glu at amino acid position 387; Ala or Ser at amino acid position 389; Ala at amino acid position 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr at amino acid position 428; Lys at amino acid position 433; Ala, Phe, His, Ser, Trp or Tyr at amino acid position 434; And His or Phe as amino acid at position 436 according to the EU numbering in the Fc domain of the parental human IgG. 8. Method according to any one of paragraphs. 1-7, where the antibody has antagonistic activity. 9. Method according to any one of paragraphs. 1-8, where the antibody binds to a membrane antigen or soluble antigen. 10. Method according to any one of paragraphs. 1-9, wherein the antibody is selected from a chimeric antibody, a humanized antibody, or a human antibody.