KR101468271B1 - Pharmaceutical formulation containing improved antibody molecules - Google Patents

Abstract

It is an object of the present invention to provide a pharmaceutical preparation containing an improved antibody molecule. The present invention modifies the amino acid sequence of the variable region and the normal region of TOCILIZUMAB, which is a humanized anti-IL-6 receptor IgG1 antibody, to improve the pharmacokinetics while enhancing antigen neutralization ability, And also to improve the immunogenicity, safety and physical properties (stability and uniformity), and to provide a pharmaceutical preparation containing a second generation molecule superior to TOCILIZUMAB. The preparation contains arginine, preferably as a stabilizer, and is provided in liquid or solid form.

Description

Pharmaceutical compositions containing improved antibody molecules,

The present invention relates to stable liquid preparations containing high concentrations of polypeptide and / or antibody-containing preparations, especially modified anti-IL-6 receptor antibodies.

Recently, various antibody preparations have been developed and actually used. Many of these formulations are used for intravenous use. It is essential to increase the antibody concentration in the liquid because the design of subcutaneous injectable antibody-containing preparations is generally limited in dose for subcutaneous injection (100 mg to 200 mg) and antibody content per dose .

In solutions containing high concentrations of antibody, undesirable degradation occurs, which involves the formation of insoluble and / or soluble aggregates. Such insoluble and soluble aggregates will be formed in a liquid state by association of antibody molecules. In cases where the liquid formulation is stored for a long period of time, the bioactivity of the antibody molecule may be lost or reduced by deamination of the asparagine residue. The circulation of freezing and thawing is also reduced and leads to the formation of aggregated antibody molecules.

Various ideas have been proposed to provide stabilized formulations in which the loss of active ingredient is reduced even after the formulation is stored for a longer period of time. These preparations are obtained by dissolving the active ingredient and various additives in a buffer solution. There is a need to provide highly concentrated antibody-containing preparations which are capable of inhibiting dimerization and deamination during long term storage and which are stable and suitable for use in subcutaneous administration.

Antibodies are attracting attention as drugs that are very stable in plasma and have little side effects. Among them, many IgG type antibody drugs are commercially available, and numerous antibody drugs have been developed (Non-Patent Documents 1 and 2). IL-6 is a cytokine (non-patent document 3) that is involved in various autoimmune diseases, inflammatory diseases, and malignant tumors. The humanized anti-IL-6 receptor IgG1 antibody, TOCILIZUMAB (tocilizumab), specifically binds to the IL-6 receptor. Since TOCILIZUMAB neutralizes the biological action of IL-6, it can be used as a therapeutic agent for IL-6 related diseases such as arthritic rheumatism (Patent Documents 1 to 3 and Non-Patent Document 4). TOCILIZUMAB has been approved in Japan as a treatment for castleman's disease and joint rheumatism (Non-Patent Document 5).

Humanized antibodies such as TOCILIZUMAB are first-generation antibody drugs. Second generation antibody drugs are currently being developed by improving the drug efficacy, convenience and cost of first generation antibody drugs. Various technologies applicable to second-generation antibody drugs are being developed. Techniques for improving effector function, antigen-binding ability, pharmacokinetics and stability as well as techniques for reducing immunogenicity risk have been reported. As a method for enhancing the drug efficacy or reducing the dose, there has been reported a technique for enhancing antibody-dependent cytotoxic activity (ADCC activity) or complement dependent cytotoxic activity (CDC activity) through amino acid substitution in the Fc region of an IgG antibody (Non-Patent Document 6). In addition, affinity maturation has been reported as a technique for enhancing antigen binding ability or antigen neutralizing ability (Non-Patent Document 7). This technique can enhance the antigen binding activity by introducing amino acid mutations into the complementarity determining region (CDR) of the variable region. Enhancement of antigen binding ability improves in vitro bioactivity, decreases dose, and thus improves drug efficacy in vivo (Non-Patent Document 8). Currently, clinical trials have been conducted to evaluate motavizumab (potabimu mab) (produced by affinity maturation), which is expected to have superior efficacy than the first-generation anti-RSV antibody drug palivizumab (palivizumab) (non-patent 9). An anti-IL-6 receptor antibody with an affinity of about 0.05 nM (i.e., more affinity than TOCILIZUMAB) has been reported (Patent Document 4). However, there is no report describing human, humanized or chimeric antibodies with an affinity stronger than 0.05 nM.

The current problem with antibody drugs is the high production costs associated with the administration of extremely large amounts of protein. For example, the dose of TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, is estimated to be about 8 mg / kg / month by intravenous injection (Non-Patent Document 4). Preferred dosage forms thereof are subcutaneous preparations in chronic autoimmune diseases. In general, the subcutaneous preparation should be a high-concentration preparation. From the standpoint of stability and the like, the limit for an IgG-type antibody preparation is generally about 100 mg / mL (Non-Patent Document 10). The low-cost, convenient second-generation antibody drugs that can be administered subcutaneously at even longer intervals can be used to prolong the therapeutic effect thereof and thereby increase the half-life in plasma to reduce the amount of protein administered, .

FcRn is closely involved in the pharmacokinetics of antibodies. Regarding the difference in plasma half-life of antibody isotype, it is known that IgG1 and IgG2 have an excellent half-life in plasma compared to IgG3 and IgG4 (Non-Patent Document 11). Amino acid substitutions in the constant region that enhance binding to FcRn have been reported as methods for further enhancing plasma half-life of IgG1 and IgG2 antibodies with good plasma half-life (Non-Patent Documents 12 and 13) . From the viewpoint of immunogenicity, half-life in plasma is preferably further improved by substituting the amino acid in the variable region rather than the normal region (Patent Document 5). However, until now there has been no report on the improvement of plasma half-life of IL-6 receptor antibodies through modification of the variable region.

Another important issue in developing biopharmaceuticals is immunogenicity. Generally, the immunogenicity of mouse antibodies is reduced by antibody humanization. It is estimated that immunogenicity risk can be further alleviated by using a framework germline sequence as a template in antibody humanization (Non-Patent Document 14). However, Adalimumab (Adalimumab), a fully human anti-TNF antibody, also showed high frequency (13% to 17%) immunogenicity and the therapeutic effect was found to be alleviated in immunogenicity patients 16). T-cell epitopes can exist even in the CDRs of human antibodies, and such T-cell epitopes in CDRs are possible causes of immunogenicity. Methods for predicting T-cell epitopes in silico and in vivo have been reported (Non-Patent Documents 17 and 18). It is presumed that the immunogenicity risk can be alleviated by removing the T-cell epitope using the above method (Non-Patent Document 19).

TOCILIZUMAB, a humanized anti IL-6 receptor IgG1 antibody, is an IgG1 antibody obtained by humanizing a mouse PM1 antibody. CDR grafting is performed using NEW and REI human sequences as the template framework for each of the H and L chains, but 5 mouse sequence amino acids remain in the framework as essential amino acids for maintaining activity (Non-Patent Document 20) . There is no report so far that has completely humanized the remaining mouse sequence in the framework of the humanized antibody TOCILIZUMAB without degrading its activity. In addition, the CDR sequence of TOCILIZUMAB is a mouse sequence, and thus, like Adalimumab, it can have a T-cell epitope on the CDR and can have potential immunogenicity risk. In a clinical trial of TOCILIZUMAB, the anti-TOCILIZUMAB antibody was not detected at the effective dose of 8 mg / kg, but it was observed at contents of 2 mg / kg and 4 mg / kg (Patent Document 6). This suggests that there is still room for improvement in the immunogenicity of TOCILIZUMAB. However, until now there has been no report on mitigating the immunogenicity risk of TOCILIZUMAB by amino acid substitution.

The isotype of TOCILIZUMAB is IgG1. The isotype difference is the difference in the normal region sequence. The normal region sequence is presumed to have a strong influence on the effect function, pharmacokinetics, physical properties and the like, and selection of the normal region sequence is very important in the development of an antibody drug (Non-Patent Document 11). In recent years, the safety of antibody drugs has become very important. The interaction between the antibody Fc portion and the Fc gamma receptor (effector function) could cause significant side effects in Phase I clinical trials of TGN1412 (non-patent document 21). In antibody medicines designed to neutralize the biological action of an antigen, binding to an Fc gamma receptor important for effector function, such as ADCC, is unnecessary. Binding to the Fc gamma receptor may be rather undesirable in terms of side effects. The method of lowering the binding to Fc gamma receptor is to change the isotype of IgG antibody from IgG1 to IgG2 or IgG4 (Non-Patent Document 22). IgG2 is preferable to IgG4 in terms of pharmacokinetics and binding to Fc gamma receptor I (Non-Patent Document 11). TOCILIZUMAB is an IL-6 receptor neutralizing antibody, and its isotype is IgG1. Thus, in view of potential side effects, IgG2 may be the preferred isotype, since an effector function such as ADCC is not required.

On the other hand, when developing an antibody drug, the physical properties of the protein, particularly the uniformity and stability, are very important. In the case of IgG2 isotype, it has been reported that there is a remarkable heterogeneity derived from disulfide bonds in the hinge region (Non-Patent Document 23). It is not easy and expensive to produce them as pharmaceuticals while maintaining the heterogeneity associated with the target substance / related substances derived from interdispersal disulfide bonds. Therefore, it is desirable to be a single substance as much as possible. Further, amidation of the C-terminal carboxyl group by deletion of the lysine residue of the C-terminal amino acid and deletion of glycine and lysine of the 2-amino acid at the C-terminal has been reported with respect to the heterogeneity of the H chain C-terminal sequence of the antibody 24). In developing an IgG2 isotype antibody as a medicine, it is preferable to reduce the heterogeneity and maintain high stability. In order to produce a convenient and stable high-concentration subcutaneous preparation, it is preferable that not only the stability is high but also the plasma half-life is better than that of TOCILIZUMAB isotype. However, there has been no report of normal region sequences for antibodies having an IgG2-isotype normal region with reduced heterogeneity, high stability, and half-life in plasma, which is superior to antibodies having an IgG1 isotype normal region.

WO 92/19759 WO 96/11020 WO 96/12503 WO 2007/143168 WO 2007/114319 WO 2004/096273

Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnology 23, 1073-1078 (2005) Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59 (3): 389-96. Nishimoto N, Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2 (11): 619-26. Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson D, Kishimoto T; CHARISMA Study Group, Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, Tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate, Arthritis Rheum. 2006 Sep; 54 (9): 2817-29 Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, Nishioka K, Hara M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S, , Nakahara H, Hagihara K, Yoshizaki K, Kishimoto T. Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease. Blood. 2005 Oct 15; 106 (8): 2627-32. Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20 (1): 17-29. Review. Rothea, Hosse RJ, Power BE. Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther. 2006 Feb; 6 (2): 177-87. Rajpala, White N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R. A general method for greatly improving the affinity of antibodies using using combinatorial libraries, Proc Natl Acad Sci USA 2005 Jun 14; 102 (24): 8466-71. Epub 2005 Jun 6. 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 Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93 (6): 1390-402. Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25 (12): 1369-72. Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life. 2006 Jan 1; 176 (1): 346-56 Gattie 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. 1997 Jul; 15 (7): 637-40 Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods. 2005 May; 36 (1): 35-42 Bartelds GM, Wijbrandtsca, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wolbink GJ. Clinical response to adalimumab: The relationship with anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; [Epub ahead of print] Bender NK, Heilig CE, Doll B, Wohlgemuth J, Armbruster FP, Heilig B. Immunogenicity, efficacy and adverse events of adalimumab in RA patients. Rheumatol Int. 2007 Jan; 27 (3): 269-74. Van Walle I, Gansemans Y, Parren PW, Stas P, Lasters I. Immunogenicity screening in protein drug development. Expert Opin Biol Ther. 2007 Mar; 7 (3): 405-18. Jones TD, Phillips WJ, Smith BJ, Bamford CA, Nayee PD, Baglin TP, Gaston JS, Baker MP. Identification and removal of a promiscuous CD4 + T cell epitope from the C1 domain of factor VIII. J Thromb Haemost. 2005 May; 3 (5): 991-1000. Chirino AJ, Ary ML, Marshall SA. Minimizing the immunogenicity of protein therapeutics. Drug Discov Today. 2004 Jan 15; 9 (2): 82-90. Sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig MM. Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth. Cancer Res. 1993 Feb 15; 53 (4): 851-6. Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned future directions. Nat Rev Drug Discov. 2007 Jan; 6 (1): 75-92. Gessner JE, Heiken H, Tamme, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1998 Jun; 76 (6): 231-48. Dillon TM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, Plant M, Henkle B, Li Y, Deechongkit S, Varnum B, Wypych J, Ballande, Bondarenko PV. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008 Jun 6; 283 (23): 16206-15. Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007 Jan 1; 360 (1): 75-83.

[Summary of the Invention]

It is an object of the present invention to improve the antigenic neutralization ability and the pharmacokinetics by modifying the amino acid sequence of the variable region and the normal region of TOCILIZUMAB, thereby decreasing the frequency of administration and continuously exhibiting the therapeutic effect and exhibiting immunogenicity, safety, (Hereinafter also referred to as a " medicament "or" medicinal composition ") comprising a second generation molecule that is superior to TOCILIZUMAB, which is a humanized anti IL-6 receptor IgG1 antibody, .

In addition, the formulations of the present invention are highly concentrated polypeptides and / or antibody-containing preparations which are suitable for use in subcutaneous administration and which are stable and preferably inhibit dimerization and deamination during long-term storage or several cycles of freezing / thawing Lt; / RTI >

Concentrated studies have been conducted to solve the above-mentioned problems, and it has been found that a stable highly concentrated polypeptide and / or an antibody-containing preparation can be obtained by adding amino acid arginine or a salt thereof as a stabilizer to a solution.

The present invention will now be described in detail. This study focused on acquiring a stable medicinal product from the viewpoint of second-generation molecules superior to TOCILIZUMAB, the first-generation humanized anti IL-6 receptor IgG1 antibody. Consequently, with respect to the above discussed antibodies, the present inventors have found a plurality of CDR mutations in the variable region of TOCILIZUMAB that enhance binding ability (affinity) to the antigen. Thus, the present inventors have successfully improved the affinity by using a combination of the above mutations. The present inventors have also succeeded in improving pharmacokinetics by introducing an alteration which lowers the isoelectric point of the variable region sequence. The present inventors have also succeeded in enhancing pharmacokinetics by allowing a single antibody molecule to neutralize the antigen multiple times by imparting a pH dependency to the binding to the IL-6 receptor antigens. In addition, they successfully reduced immunogenicity risk by fully humanizing the mouse-derived sequence remaining in the framework of TOCILIZUMAB and reducing the number of T-cell epitope peptides predicted by inositol in the variable region. The present inventors have also found that, in order to enhance safety, the binding to the Fc gamma receptor is lowered than that of IgG1, the pharmacokinetics of IgG1 are improved and the stability is not deteriorated, and the heterogeneity due to the disulfide bond of the hinge region of IgG2 and the Lt; RTI ID = 0.0 > of TOCILIZUMAB < / RTI > SUMMARY OF THE INVENTION Generation of second generation molecules superior to TOCILIZUMAB has been successfully made by properly combining the modifications of the amino acid sequence in the cleavage region and the normal region.

The present invention is based on the finding that the ability to bind to an excellent antigen (IL-6 receptor), excellent pharmacokinetics, excellent safety and physical properties (stability and / or stability), and the like are improved by the modification of the amino acid sequence of the variable region and the normal region of TOCILIZUMAB which is a humanized anti- Uniformity), and a humanized anti-IL-6 receptor IgG antibody having a further reduced immunogenicity risk, and a method for producing the pharmaceutical composition. More specifically, the present invention provides the following (1) to (21).

(1) A pharmaceutical preparation comprising at least one polypeptide selected from the following (a) to (f):

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: A polypeptide comprising CDR3 having a sequence;

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: A polypeptide comprising CDR3 having a sequence;

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: A polypeptide comprising CDR3 having a sequence;

(d) CDR1 having the sequence of SEQ ID NO: 10 (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and CDR3 having the sequence of SEQ ID NO: 12 ≪ / RTI >

(e) CDR1 having the sequence of SEQ ID NO: 13 (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR3 having the sequence of SEQ ID NO: 15 ≪ / RTI > And

(f) CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and CDR3 having the sequence of SEQ ID NO: 18 Lt; / RTI >

(2) A pharmaceutical preparation comprising one or more antibodies selected from the following (a) to (i):

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and a heavy chain variable region comprising CDR3 having the sequence of SEQ ID NO: 10 An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 having the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR2 having the sequence of SEQ ID NO: 13 (VL3 of VL3), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(d) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73), and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region having a sequence of SEQ ID NO: 20 (a variable region of VH3-M73), and a light chain variable region having a sequence of SEQ ID NO: 23 (a variable region of VL3);

(f) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83) and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);

(g) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3); And

(i) an antibody comprising a light chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5).

(3) A stable pharmaceutical preparation according to (1) or (2), which comprises histidine and / or citrate buffer.

(4) A stable pharmaceutical preparation according to any one of (1) to (3), which comprises at least one cationic amino acid.

(5) any one of (1) to (4), which comprises 1-500 mM of histidine and / or citrate, 1-1500 mM of at least one cationic amino acid, 1-200 mg / mL of antibody and 1-400 mM of carbohydrate ≪ / RTI >

(6) The agent according to (4) or (5), wherein the cationic amino acid is arginine.

(7) The preparation according to (5) or (6), wherein the carbohydrate is sucrose or trihalose.

(8) The preparation according to any one of (1) to (7), further comprising a surfactant.

(9) The agent according to any one of (1) to (8), which contains a polypeptide and / or an antibody at a content of 10 mg / ml or more.

(10) The agent according to any one of (1) to (9), which contains a polypeptide and / or an antibody in a content of 50 mg / ml or more.

(11) The agent according to any one of (1) to (10), which contains a polypeptide and / or an antibody in an amount of 80 mg / ml or more.

(12) An agent according to any one of (1) to (11), which contains a polypeptide and / or an antibody at a content of 240 mg / ml or less.

(13) A preparation according to any one of (1) to (12), having a pH in the range of 4.5 to 7.0.

(14) The preparation according to (13), which has a pH in the range of 5.5 to 6.6.

(15) The preparation according to any one of (1) to (14), which is in a liquid phase.

(16) An agent according to (15), which was not applied to freeze drying during manufacture of the agent.

(17) A preparation according to any one of (1) to (16), wherein the dimerization of the polypeptide and / or the antibody molecule is reduced.

(18) The agent according to any one of (1) to (17), wherein the dimerization of the polypeptide and / or the antibody molecule is inhibited.

(19) The preparation according to any one of (1) to (18) for subcutaneous administration.

(20) A method for stabilizing a solution containing an antibody selected from the following (a) to (i), which comprises adding at least one cationic amino acid.

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and a heavy chain variable region comprising CDR3 having the sequence of SEQ ID NO: 10 An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 having the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR2 having the sequence of SEQ ID NO: 13 (VL3 of VL3), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(d) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83) and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);

(g) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3); And

(i) an antibody comprising a light chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5).

(21) A method for stabilizing an antibody during a cooling / thaw cycle of a solution containing an antibody selected from the following (a) to (i), comprising adding at least one cationic amino acid.

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and a heavy chain variable region comprising CDR3 having the sequence of SEQ ID NO: 10 An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 having the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR2 having the sequence of SEQ ID NO: 13 (VL3 of VL3), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(d) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83) and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);

(g) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3); And

(i) an antibody comprising a light chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5).

The above-described humanized anti-IL-6 receptor IgG antibodies have increased pharmacological activity and improved pharmacokinetics, and thus can exhibit a therapeutic effect continuously with a reduced administration frequency.

[Figure 1]
Figure 1 shows a list of mutation sites that improve the affinity of TOCILIZUMAB for the IL-6 receptor. SEQ ID NO: 81 of HCDR2 of TOCILIZUMAB, SEQ ID NO: 82 of HCDR2, SEQ ID NO: 83 of HCDR2, SEQ ID NO: 83 of HCDR2, HCDR3 sequence of TOCILIZUMAB to SEQ ID NO: 84 of HCDR3 SEQ ID NO: 85 after the side of HCDR3, SEQ ID NO: 86 after the side of HCDR3, SEQ ID NO: 87 of the LCDR1 sequence of TOCILIZUMAB, SEQ ID NO: 88 (SEQ ID NO: 89), the LCDR3 sequence of TOCILIZUMAB is represented by SEQ ID NO: 90, the sequence after the side of LCDR3 (top) is represented by SEQ ID NO: 91, the sequence after the side of LCDR3 : 92.
[Figure 2]
Figure 2 shows the neutralization activity of TOCILIZUMAB and RDC-23 in BaF / gp130.
[Figure 3]
Figure 3 lists mutation sites that may degrade the isoelectric point of the variable region without significantly degrading the binding of TOCILIZUMAB to the IL-6 receptor. The asterisk in the figure is a point that did not affect the isoelectric point but was changed for conversion to a human sequence. The HFR1 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 93, the sequence after HFR1 is referred to as SEQ ID NO: 94, the HCDR1 sequence of TOCILIZUMAB as SEQ ID NO: 95, the sequence after HCDR1 as SEQ ID NO: 96, and the sequence of HFR2 of TOCILIZUMAB as SEQ ID NO: : SEQ ID NO: 98 after the side of HFR2, HCDR2 sequence of TOCILIZUMAB, SEQ ID NO: 81 of HCDR2, SEQ ID NO: 99 of HCDR2, HFR4 sequence of TOCILIZUMAB, SEQ ID NO: 100 of HCDR2, SEQ ID NO: 101 of TOCILIZUMAB, SEQ ID NO: 102 of TOCILIZUMAB, SEQ ID NO: 103 of LFR1 side, SEQ ID NO: 87 of TOCILIZUMAB, SEQ ID NO: 87 of TOCILIZUMAB, SEQ ID NO: 104 of TOCILIZUMAB The LFR2 sequence is referred to as SEQ ID NO: 105, the sequence after LFR2 is referred to as SEQ ID NO: 106, the LCDR2 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 107, the sequence after LCDR2 as SEQ ID NO: 108, 109 and the LFR3 sequence of TOCILIZUMAB as SEQ ID NO: 110, the sequence after the side of LFR3 is referred to as SEQ ID NO: 111, the LFR4 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 112, LFR4 Since the change sequence SEQ ID NO: 113 represents the.
[Figure 4]
Figure 4 shows the neutralization activity of TOCILIZUMAB and H53 / L28 in BaF / gp130.
[Figure 5]
FIG. 5 shows changes in plasma concentrations of TOCILIZUMAB and H53 / L28 after intravenous administration in mice.
[Figure 6]
FIG. 6 shows changes in plasma concentrations of TOCILIZUMAB and H53 / L28 after subcutaneous administration in mice.
[Figure 7]
FIG. 7 is a schematic diagram showing that an IgG molecule can be re-bound to another antigen by dissociation from a solid antigen in the endosome. FIG.
[Figure 8]
Figure 8 shows a list of mutation sites that are capable of conferring a pH-dependent (binding at pH 7.4 and dissociated at pH 5.8) binding of TOCILIZUMAB to the IL-6 receptor. The HFR1 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 93, the sequence after HFR1 is referred to as SEQ ID NO: 114, the HCDR1 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 95, the sequence after HCDR1 is referred to as SEQ ID NO: 115 and the LCDR1 sequence of TOCILIZUMAB is referred to as SEQ ID NO: 87, the sequence after the side of LCDR1 is shown in SEQ ID NO: 116, the LCDR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: 107, and the sequence after side of LCDR2 is shown in SEQ ID NO:
[Figure 9]
9 is a graph showing the neutralization activity of TOCILIZUMAB and H3pI / L73 in BaF / gp130.
[Fig. 10]
10 is a graph showing changes in plasma concentrations of TOCILIZUMAB and H3pI / L73 after intravenous administration in a crab monkey.
[Figure 11]
FIG. 11 is a graph showing changes in plasma concentrations of TOCILIZUMAB and H3pI / L73 after intravenous administration in human IL-6 receptor transgenic mice.
12]
12 is a diagram showing the results of evaluating the heterogeneity derived from the C-terminal by cation exchange chromatography of TOCILIZUMAB, TOCILIZUMAB delta K, and TOCILIZUMAB delta GK.
[Fig. 13]
Fig. 13 shows the results of evaluating the heterogeneity of TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2, and TOCILIZUMAB-SKSC derived from disulfide bonds by cation exchange chromatography.
[Fig. 14]
14 is a diagram showing the denaturation curves and the Tm values of each Fab domain measured by differential scanning calorimetry (DSC) of TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2, and TOCILIZUMAB-SKSC.
[Fig. 15]
FIG. 15 is a graph showing changes in plasma concentrations of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 after intravenous administration in human FcRn transgenic mice.
[Fig. 16]
16 is a graph showing the neutralization activity of TOCILIZUMAB, control and Fv5-M83 in BaF / gp130.
[Figure 17]
17 is a graph showing the neutralization activity of TOCILIZUMAB, Fv3-M73 and Fv4-M73 in BaF / gp130.
[Fig. 18]
FIG. 18 is a graph showing changes in plasma concentrations of TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 after intravenous administration in a crab monkey.
19]
FIG. 19 is a graph showing changes in CRP concentration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 after intravenous administration in a crab monkey.
20]
FIG. 20 is a graph showing the change of the unconjugated soluble IL-6 receptor ratio of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration in crab monkeys.
[Fig. 21]
Figure 21 is a graph showing differences in the formation of high molecular weight species (HMW) of antibody Fv4-M73 over time for different formulations (AD).
[Figure 22]
Figure 22 shows the difference in the formation of high molecular weight species of antibody Fv4-M73 at two points in time (at 25 degrees Celsius) due to the presence of different pH values, buffer type, NaCl concentration and arginine and absence (delta HMW: The amount of increase from the beginning).
[Fig. 23]
23 shows the difference in the formation of high molecular weight species (HMW) of antibody Fv4-M73 at one point in time (at 25 degrees Celsius) with different pH values, buffer type, NaCl concentration, and presence and absence of arginine Fig.
24]
Figure 24 shows the difference in the formation of high molecular weight species of antibody Fv4-M73 at three points in time (at 40 degrees Celsius) due to different pH values, buffer type, NaCl concentration, and presence and absence of arginine (at delta HMW: The amount of increase from the beginning).
[Fig. 25]
Figure 25 shows the difference in the formation of high molecular weight species (HMW) of antibody Fv4-M73 at one point in time (at 40 degrees Celsius) due to different pH values, buffer type, NaCl concentration, and presence and absence of arginine Fig.
[Fig. 26]
Figure 26 shows the difference in the formation of low molecular weight species of antibody Fv4-M73 at three points in time (at 40 degrees Celsius) due to the presence of different pH values, buffer type, NaCl concentration and arginine and absence (delta LMW: In Fig.
[Fig. 27]
Figure 27 is a diagram showing the results of anion exchange chromatography performed with a solution of the antibody Fv4-M73 stored under different pH values.
[Fig. 28]
Figure 28 is a diagram showing the results of anion exchange chromatography performed with a solution of antibody Fv4-M73 stored under three different pH values, different concentrations of NaCl, arginine and two different buffering agents.
29]
Figure 29 shows the difference in the formation of high molecular weight species of antibody Fv4-M73 (delta HMW: increase from baseline) versus two different cryo / thaw cycles, different concentrations of NaCl, arginine and two different buffers Graph.
30]
Figure 30 shows the difference (delta HMW: increase from the beginning) in the formation of high molecular weight species of antibody Fv4-M73 at three different points in time (at 40 degrees Celsius and 25 degrees Celsius) and four different formulations (EH) Fig.
[Fig. 31]
Figure 31 is a graph showing the difference (delta HMW: increase from baseline) in the formation of high molecular weight species of antibody Fv4-M73 for two different cryo / thaw cycles and four different formulations (EH).
[Fig. 32]
Figure 32 is a graph showing differences (delta HMW: increase from baseline) in the formation of high molecular weight species of antibody Fv4-M73 for the six different formulations at 5 degrees Celsius at 3 and 6 months.
[Figure 33]
33 is a graph showing the difference (delta HMW: increase from the beginning) in the formation of high molecular weight species of antibody Fv4-M73 for 6 different preparations at 3 and 6 months at -20 degrees Celsius.

The present invention provides pharmaceutical formulations comprising at least one polypeptide selected from the following (a) to (f):

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: A polypeptide comprising CDR3 having a sequence,

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: A polypeptide comprising CDR3 having a sequence,

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: A polypeptide comprising CDR3 having a sequence,

(d) CDR1 having the sequence of SEQ ID NO: 10 (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and CDR3 having the sequence of SEQ ID NO: 12 Lt; / RTI >

(e) CDR1 having the sequence of SEQ ID NO: 13 (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR3 having the sequence of SEQ ID NO: 15 Lt; / RTI >

(f) CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and CDR3 having the sequence of SEQ ID NO: 18 Lt; / RTI >

Polypeptides and antibodies of the invention may be formulated according to conventional methods (see, e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).

As used herein, "medicament," "medicament," and "medicinal composition" are intended to include a polypeptide and / or an antibody as an active ingredient (s), adapted to be administered directly or after reconstitution to an animal, &Quot; refers to a liquid or solid formulation. If desired, the agent may contain a pharmaceutically acceptable carrier and / or excipient. (E. G., Ascorbic acid, methionine), colorants, fragrances, preservatives, stabilizers, buffers, chelating agents (e. G. EDTA), suspension agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, and fodder.

Polypeptides and / or antibody-containing preparations according to the invention preferably do not contain HSA (human serum albumin), gelatin, and such proteins as stabilizers.

The polypeptides and / or antibody-containing preparations according to the invention preferably contain a polypeptide and / or antibody at a high concentration of at least 10 mg / ml, preferably at least 50 mg / ml, more preferably at least 80 mg / ml It is a liquid medicinal preparation. The concentration may be 10 to 240 mg / ml, may be 10 mg / ml, more preferably 50 mg / ml, even more preferably 100 to 200 mg / ml.

The liquid pharmaceutical preparations according to the present invention are preferably prepared by not performing the lyophilization step (s).

Buffering agents that can be used in the present invention are those which are capable of adjusting the pH within a desired range and are pharmaceutically acceptable. In the high concentration polypeptide and / or antibody-containing preparation according to the invention, the pH of the preparation is preferably 4.5 to 7, more preferably 5.5 to 6.6. Such buffers are known to those of ordinary skill in the art, examples of which include inorganic salts such as phosphate (sodium or potassium) and sodium bicarbonate; Organic acid salts such as citrate (sodium or potassium), sodium acetate and sodium succinate; And acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid and gluconic acid. In addition, Tris buffer, Good's buffer such as MES and MOPS, histidine (e.g., histidine hydrochloride) and glycine can also be used. In high concentration polypeptide and / or antibody-containing preparations according to the invention, the buffer is preferably a histidine or citrate buffer, wherein a histidine buffer is particularly preferred. The concentration of the buffer solution is generally 1 to 500 mM, preferably 5 to 100 mM, more preferably 10 to 20 mM. When a histidine buffer is used, the buffer solution preferably contains histidine at a concentration of 5 to 25 mM, more preferably 10 to 20 mM.

The preparations according to the invention additionally contain a surfactant. Typical examples of surfactants are inorganic surfactants, for example sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate; Glycerol fatty acid esters such as glycerol monocaprylate, glycerol monomyristate and glycerol monostearate; Polyglycerol fatty acid esters such as decaglyceryl monostearate, decassylseryl distearate and decaglyceryl monolinoleate; Polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan monostearate; Polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol tetra stearate and polyoxyethylene sorbitol tetraoleate; Polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glyceryl monostearate; Polyethylene glycol fatty acid esters such as polyethylene glycol distearate; Polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; Polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene cetyl ether; Polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; Polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil); Polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; Polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; Surfactants having an HLB of 6 to 18, such as polyoxyethylene fatty acid amides, e.g., polyoxyethylene octadecanamide; Anionic surfactants such as alkyl sulphates having C ₁₀ -C ₁₈ alkyl groups such as sodium cetyl sulphate, sodium lauryl sulphate and sodium oleyl sulphate; A polyoxyethylene alkyl ether sulfate having an average molar number of ethylene oxide units added such as sodium polyoxyethylene lauryl sulfate of 2 to 4 and an alkyl group of 10 to 18 carbon atoms; Lauryl alkyl sulfosuccinate salts having a C ₈ -C ₁₈ alkyl group, such as sodium sulfosuccinate; Natural surfactants such as lecithin and glycerophospholipids; Sphingoid lipids such as sphingomyelin; And sucrose esters of C ₁₂ -C ₁₈ fatty acids.

Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers, particularly preferably polysorbates 20, 21, 40, 60, 65, 80, 81 and 85 and pluronic type Surfactants, most preferably polysorbates 20 and 80, and Flonic F-68 (Poloxamer 188).

The content of the surfactant (s) added to the antibody preparation according to the present invention is generally 0.0001 to 10% (w / v), preferably 0.001 to 5%, more preferably 0.005 to 3%.

The preparation according to the present invention may further contain an acidic amino acid such as methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine and such amino acid as well as arginine as a stabilizer.

In the antibody-containing formulation or antibody-containing solution formulation according to the invention, the addition of carbohydrate (s) (e.g. sugar (s)) can inhibit the formation of dimers during refrigeration / thaw cycles. Sugars that can be used include non-reducing oligosaccharides, for example, non-reducing disaccharides such as sucrose and trehalose, or non-reducing trisaccharides such as raffinose, and particularly preferably non-reducing oligosaccharides. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

In an antibody-containing formulation or an antibody-containing solution formulation according to the present invention, the formation of a multimer and abatement product during prolonged storage may be inhibited by the addition of carbohydrate (s) (e.g., sugar (s)). Sugars which can be used include sugar alcohols such as mannitol and sorbitol; Non-reducing oligosaccharides include, for example, non-reducing disaccharides such as sucrose and trehalose, particularly preferred among non-reducing oligosaccharides, or non-reducing trisaccharides such as raffinose. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

The sugar should be added at a concentration of 0.1 to 500 mg / ml, preferably 10 to 300 mg / ml, more preferably 25 to 100 mg / ml.

In another aspect, the formulation according to the invention preferably consists essentially of the following ingredients

A) a modified anti-IL-6 receptor antibody;

B) cationic amino acids (e.g., arginine, histidine, and / or lysine);

C) Buffer (s) (e.g., histidine or citrate).

The carbohydrate (s) (e.g., sugars) and / or the surfactant (s) may also be included in the formulation, depending on the purpose.

Stabilizers may include methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, and such amino acids.

The term "substantially constituted" means herein that components other than those components which are usually added to the formulation are not included, and those components which are usually added to the formulation include the suspension compositions, solubilizers, isotonic agents, preservatives , Adsorption inhibitors, diluents, vehicle, pH-adjusting agents, softening agents, sulfur-containing agents and antioxidants.

Examples of such suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, rabic rubber, milled tragacanth, carboxymethylcellulose sodium and polyethylene sorbitan monolaurate.

Examples of the solubilizing agent include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol and castor oil fatty acid ethyl ester.

Examples of such isotonizing agents include sodium chloride, potassium chloride and calcium chloride.

Examples of such preservatives include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropylcellulose, methylcellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione and C ₁ And compounds having a sulfhydryl group such as a -C ₇ thioalkane.

Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole, alpha-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L- Chelating agents such as sodium bisulfite, sodium bisulfite, sodium bisulfite, triamylmorpholinic acid salt, propylmorpholinic acid salt, and sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

However, the preparations (medicament, pharmaceutical composition) according to the present invention which can be used for preventing or treating IL-6-related diseases including inflammatory diseases are not limited to the above and may suitably contain other conventional carriers . Particularly, there may be mentioned, for example, light anhydrous silicic acid, lactose, crystallized cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone , Gelatin, medium chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts. In preparing the injectable liquid solution, the anti-IL-6 receptor antibody is dissolved in an isotonic solution containing, for example, physiological saline, glucose or other adjuvant. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Suitable solubilizers may also be combined with, for example, alcohols (such as ethanol), polyalcohols (such as propylene glycol, PEG), and nonionic surfactants (polysorbate 80 and HCO-50).

If necessary, the polypeptide may be incorporated into microcapsules (microcapsules made of hydroxyethylcellulose, gelatin, poly (methylmethacrylate), etc.) or in a colloidal drug delivery system (liposomes, albumin microspheres, microemulsions, Nano-capsules, etc.) (see, e. G., Remigton ' s Pharmaceutical Science 16th edition, Oslo Ed. (1980)). Further, methods of making drugs as delayed-release agents are well known and they can be applied to polypeptides (Larger et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105, US Patent No. 3,773,919, European Patent Application (EP) No. 58,481, Sidman et al., Biopolymers (1983) 22: 547-56, EP No. 133,988). In addition, the liquid volume for subcutaneous injection can be increased by adding or mixing the hyaluronidase to the drug (see, for example, WO 2004/078140).

The pharmaceutical composition of the present invention can be administered either orally or parenterally, but is preferably administered parenterally. In particular, the composition is administered to a patient by injection or transdermal administration. Injections include, for example, intravenous, intramuscular, or total or local administration by subcutaneous injection. The composition can be injected locally at the site of treatment, particularly around the site by intramuscular injection. Transdermal dosage forms include, for example, ointments, gels, creams, feminine care products and patches which can be administered topically or wholly. In addition, the administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage can be selected, for example, from the range of 0.0001 mg to 100 mg of active ingredient per kg body weight for each administration. Alternatively, when the composition is administered to a human patient, for example, the active ingredient may be selected from the range of 0.001 to 1000 mg per kg of body weight for each patient. Single dose amounts preferably contain, for example, from about 0.01 to 50 mg / kg (body weight) of an antibody of the invention. However, the antibody of the present invention is not limited to the above content.

According to the present invention, as can be seen from the results of the examples described below, cationic amino acids combined with one or more cationic amino acids (arginine, histidine, and / or lysine, preferably arginine) The preparation can be made stable with long term storage or dimerization and demineralization of the antibody during freezing / thawing.

To determine the shelf life stability of high concentration antibody-containing preparations, the inventors studied the effects of various additives by performing size exclusion chromatography and anion exchange chromatography experiments. As a result, it was found that the solution of the high concentration antibody dissolved in the buffer solution containing the amino acid arginine had a lower dimer content than in the solution without additional arginine. These results indicate that arginine is effective as a stabilizer to inhibit antibody dimerization.

Thus, by adding arginine as a stabilizer, it is possible to provide a stable antibody preparation in which the dimerization of the antibody is reduced.

One embodiment of the present invention is a stable antibody-containing preparation characterized by containing an antibody and arginine in a buffer solution.

As the arginine used in the present invention, the arginine compound itself, derivatives thereof, and salts thereof may be used. L-arginine and salts thereof are preferable.

When the preparation according to the present invention contains arginine, the concentration of arginine is preferably 1 to 1500 mM, more preferably 50 to 1000 mM, still more preferably 50 to 200 mM.

The polypeptide of the present invention is not particularly limited, but is preferably an antigen binding substance having a binding activity to a human IL-6 receptor. Preferred examples of the antigen binding substance include a heavy chain variable region (VH) of an antibody, a light chain variable region (VL) of an antibody, a heavy chain of an antibody, a light chain of an antibody, and an antibody.

In the above-mentioned polypeptides (a) to (f), preferred examples of the polypeptides of (a) to (c) include heavy chain variable regions of the antibody and preferred examples of the polypeptides of (d) to (f) A light chain variable region of an antibody.

These variable regions can be used as part of an anti-human IL-6 receptor antibody. An anti-human IL-6 receptor antibody in which these variable regions are used has excellent binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and / or excellent physical properties. In the present invention, the improvement of excellent pharmacokinetics or pharmacokinetics means reduction of "Clearance (CL)", which is one of the pharmacokinetic parameters calculated from the concentration change in plasma when the antibody is administered in vivo, (AUC) ", "Mean Residence Time ", and" increase in plasma half-life (t1 / 2) ". In the present invention, the improvement of excellent physical properties or physical properties is not particularly limited, but means improvement of stability and reduction of heterogeneity.

The framework region (FR) of a human antibody linked to a CDR is selected such that the CDRs form a good antigen binding site. The FR used in the variable region of the present invention is not particularly limited, and any FR may be used, but a human-derived FR is preferably used. It is possible to use a human-derived FR having a natural sequence. Alternatively, substitutions, deletions, additions, and / or insertions of one or more amino acids may be introduced into the framework region having a native sequence, if necessary, so that the CDR forms an appropriate antigen binding site. For example, a mutant FR sequence having the desired properties can be selected by measuring and evaluating the binding activity of an antibody having FR substituted with an amino acid to an antigen (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

In addition, substitution, deletion, addition and / or insertion of one or more amino acids in the above-mentioned CDR sequence may be used. The CDR sequence after substitution, deletion, addition and / or insertion of one or more amino acids preferably has an activity equivalent to that of the CDR sequence before modification with respect to binding activity, neutralizing activity, stability, immunogenicity and / or pharmacokinetics. The number of amino acids to be substituted, deleted, added and / or inserted is not particularly limited, but is preferably 3 amino acids or less, more preferably 2 amino acids or less, and more preferably 1 amino acid to one CDR.

The method of substituting one or more amino acid residues with other amino acids of interest can be carried out by, for example, site-specific mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, -directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. , Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984). The gapped duplex DNA approach to oligonucleotide-directed mutation construction Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci 1985. Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, US A. 82, 488-492). The method can be used to replace the desired amino acid with another amino acid of interest in the antibody. Also, as amino acid substitution methods, amino acids can be obtained by obtaining appropriate frameworks and CDRs using library techniques such as framework shuffling (Mol Immunol. 2007 Apr; 44 (11): 3049-60) and CDR repair (US 2006/0122377) .

The present invention also provides a pharmaceutical preparation comprising at least one antibody selected from the following (a) to (i).

(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: (CDR1 of VL1), CDR2 having the sequence of SEQ ID NO: 11 (CDR2 of VL1), and a heavy chain variable region comprising CDR3 having the sequence of SEQ ID NO: 10 An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(b) CDR2 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 having the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: (CDR1 of VL3), CDR2 having the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR2 having the sequence of SEQ ID NO: 13 (VL3 of VL3), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(c) CDR2 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR2 of SEQ ID NO: CDR3 having the sequence of SEQ ID NO: 16 (CDR1 of VL5), CDR2 having the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a heavy chain variable region comprising the CDR3 having the sequence of SEQ ID NO: An antibody comprising a light chain variable region comprising a CDR3 having the sequence of SEQ ID NO: 3;

(d) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83) and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);

(g) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 29 (VL3); And

(i) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain variable region having the sequence of SEQ ID NO: 30 (VL5).

The antibodies described above may be used as anti-human IL-6 receptor antibodies with good binding activity, good pharmacokinetics, good safety, reduced immunogenicity, and / or good physical properties.

The framework regions of human antibodies linked to the CDRs of the present invention are selected so that the CDRs form good antigen binding sites. The FR used in the variable region of the present invention is not particularly limited, and any FR may be used, but a human-derived FR is preferably used. It is also possible to use a human-derived FR having a natural sequence. Alternatively, substitution, deletion, addition and / or insertion of one or more amino acids, etc., can be introduced into the framework region having a native sequence, if necessary, so that the CDR forms an appropriate antigen binding site. For example, a mutant FR sequence having the desired properties can be selected by measuring and evaluating the binding activity of an antibody having FR substituted with an amino acid to an antigen (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

On the other hand, the normal region used for the antibody in the present invention is not particularly limited, and any normal region may be used. Preferred normal regions used for the antibody in the present invention include, for example, normal regions derived from humans (IgG1, IgG2, IgG3, IgG4, C-kappa, C-lambda normal regions and the like). One or more amino acids may be substituted, deleted, added and / or inserted in a normal region derived from a human. The heavy chain constant region of human origin can be, for example, a normal region having the amino acid sequence of SEQ ID NO: 31 (the normal region of VH4-M73), a normal region having the amino acid sequence of SEQ ID NO: 32 (the normal region of VH3- Region, the normal region having the amino acid sequence of SEQ ID NO: 33 (the normal region of VH5-M83), while the preferred human-derived light chain constant region comprises the amino acid sequence of SEQ ID NO: 34 (VL1) A normal region having the amino acid sequence of SEQ ID NO: 35 (VL3), and a normal region having the amino acid sequence of SEQ ID NO: 36 (VL5).

In addition, one or more amino acids may be substituted, deleted, added, and / or inserted in the CDR sequences described above. The CDR sequence after substitution, deletion, addition and / or insertion of one or more amino acids preferably has an activity equivalent to that of the CDR sequence before modification with respect to binding activity, neutralizing activity, stability, immunogenicity and / or pharmacokinetics. The number of amino acids to be substituted, deleted, added and / or inserted is not particularly limited, but is preferably 3 amino acids or less, more preferably 2 amino acids or less, and more preferably 1 amino acid to one CDR.

Each variable region of the antibody can be used as part of a molecule that reacts with an anti-human IL-6 receptor. The variable region may also include substitution, deletion, addition and / or insertion of one or more amino acids (e.g., 5 amino acids or less, preferably 3 amino acids or less). The method of substituting one or more amino acid residues with another amino acid of interest includes, for example, the above-mentioned method.

The present invention also includes a polypeptide comprising the above-described variable region.

Each heavy and light chain of the antibody can be used as part of a molecule that reacts with an anti-human IL-6 receptor. The anti-human IL-6 receptor antibodies using these heavy chains and light chains have excellent binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and / or excellent physical properties.

Such heavy and light chains may include substitution, deletion, addition and / or insertion of one or more amino acids (e.g., 10 amino acids or less, preferably 5 amino acids or less, more preferably 3 amino acids or less). The method of substituting one or more amino acid residues with another amino acid of interest includes, for example, the above-mentioned method.

Substitution, deletion, addition and / or insertion of one or more amino acids can be done for the variable region, the normal region, or both.

The invention also encompasses polypeptides comprising the heavy and light chains described above.

In the present invention, the antibody is preferably a humanized antibody.

Humanized antibodies are also referred to as reshaped human antibodies. Such human antibodies are obtained by grafting complementarity-determining regions (CDRs) from non-human mammals to the CDRs of human antibodies. General gene recombination techniques for the production of such antibodies are also known (see European Patent Application Publication No. 125023, WO 96/02576).

Specifically, for example, a DNA sequence designed to link a CDR of interest with a framework region of interest (FR) comprises a number of oligonucleotides prepared so as to have a portion overlapping both terminal regions of CDR and FR, , And synthesized by the PCR method (see the method described in WO98 / 13388). The humanized antibody is obtained by linking the obtained DNA with a DNA encoding a human antibody normal region or a human antibody normal region recombinant, inserting the DNA into an expression vector, and introducing the vector into a host to produce the antibody (European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96/02576).

The framework regions of human antibodies linked to the CDRs are selected so that the CDRs form a good antigen binding site. If necessary, amino acid substitution, deletion, addition and / or insertion, etc., can be introduced into the framework region of the antibody variable region.

A human antibody normal region variant in which one or more amino acids are substituted, deleted, added and / or inserted in the human antibody normal region or the human antibody normal region can be used as a normal region of the humanized antibody.

For example, in the case of the H chain, C gamma 1, C gamma 2, C gamma 3, C gamma 4, C mu, C delta, C alpha 1, C alpha 2 and C epsilon, , And C lambda can be used. The amino acid sequence of C-kappa is shown in SEQ ID NO: 38, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of C-Gamma 1 is shown in SEQ ID NO: 40, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 39. The amino acid sequence of C-Gamma 2 is shown in SEQ ID NO: 42, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 41. The amino acid sequence of C-Gamma 4 is shown in SEQ ID NO: 44, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 43.

In addition, to improve antibody stability or antibody production stability, the human antibody C region can be modified. Any isotype human antibodies such as IgG, IgM, IgA, IgE or IgD can be used in antibody humanization, but IgG is preferably used in the present invention. IgG1, IgG2, IgG3, IgG4, etc. can be used as IgG.

In addition, amino acids in the variable regions (for example, CDR, FR) and the normal region of the humanized antibody can be deleted, added, inserted, and / or substituted with other amino acids. In the present invention, the antibody also includes humanized antibodies including amino acid substitutions and the like.

In the present invention, as long as the antibody has a binding activity to the IL-6 receptor and / or a neutralizing activity, it includes not only a bivalent antibody represented by IgG but also a polyvalent antibody represented by a monovalent antibody or IgM. The multivalent antibody of the present invention includes a multivalent antibody having the same antigen binding site, and a multivalent antibody in which all or a part of the antigen binding site is different. In the present invention, as long as the antibody binds to the IL-6 receptor protein, it is not limited to the whole molecule of the antibody but also includes a low-molecular-weight antibody and its water-containing plant.

The low-molecular-weight antibody is an antibody comprising an antibody fragment in which a part of a whole antibody (for example, whole IgG) is deficient and has an activity of binding to and / or neutralizing the IL-6 receptor, (For example, whole IgG or the like) is deficient. The low-molecular-weight antibody in the present invention is not particularly limited as long as it contains a part of the whole-length antibody. However, the low-molecular-weight antibody preferably includes VH or VL, and particularly preferably includes both VH and VL. Other preferred low-molecular-weight antibodies in the present invention include low-molecular-weight antibodies, including, for example, antibody CDRs. The low-molecular-weight antibody may comprise all or part of the six CDRs of the antibody.

In the present invention, the low-molecular-weight antibody preferably has a smaller molecular weight than the whole-length antibody. However, low-molecular-weight antibodies can form multimers, such as dimers, trimers, or tetramers, and thus their molecular weight is sometimes greater than the molecular weight of the full-length antibody.

In particular, antibody fragments include, for example, Fab, Fab ', F (ab') 2 and Fv. On the other hand, the low-molecular-weight antibody includes, for example, Fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody and sc (Fv) . Multimers (for example, dimers, trimers, tetramers and polymers) of these antibodies are also included in the low-molecular-weight antibody in the present invention.

Antibody fragments can be obtained, for example, by treating the antibody with an enzyme to generate antibody fragments. As the enzyme producing an antibody fragment, for example, papain, pepsin, or plasmin are known. Alternatively, a gene encoding these antibody fragments can be constructed, introduced into an expression vector, and then expressed in a suitable host cell (see, for example, Co, MS et al., J. Immunol. (1994) 152, Plockthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, E., et al., J. Biol., 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) Methods in Enzymology (1989) 121, 663-669, Bird, RE et al., TIBTECH (1991) 9, 132-137) .

The digestive enzyme cleaves a specific position of the antibody fragment to give an antibody fragment of the specific structure shown below. Gene engineering techniques can be applied to the enzymatically obtained antibody fragments to delete any portion of the antibody.

The antibody fragments obtained by using the digestive enzyme are as follows.

Papain digestion: F (ab) 2 or Fab

Pepsin digestion: F (ab ') 2 or Fab'

Plasmin digestion: Facb

The low-molecular-weight antibody in the present invention includes an antibody fragment in which an arbitrary region has been deleted as long as they have binding activity and / or neutralizing activity to the IL-6 receptor.

"Diabody" refers to a bivalent antibody fragment constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) EP404,097, WO93 / 11161, etc.). A diabody is a dimer composed of two polypeptide chains. In each polypeptide chain making up dimers, VL and VH are generally linked by a linker in the same chain. Generally, the linker in the diabody is sufficiently short that VL and VH can not bind to each other. Specifically, the number of amino acid residues constituting the linker is, for example, about 5 residues. Thus, VL and VH encoded on the same polypeptide chain will not form single chain variable region fragments and will form dimers with another single chain variable region fragment. As a result, the diabody has two antigen binding sites.

ScFv antibodies are single chain polypeptides produced by combining VH and VL with linkers and the like (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883, Plueckthun "The Pharmacology of Monoclonal Antibodies "Vol.113, Resenburg and Moore, Springer Verlag, New York, pp. 269-315 (1994)). The H chain V region and the L chain V region of scFv can be derived from any of the antibodies described herein. The peptide linker for linking the V regions is not particularly limited. For example, any single chain peptide containing about 3 to 25 residues may be used as the linker. Concretely, it is possible to use a peptide linker described later.

The V regions of both chains can be connected by PCR, for example, as described above. First, DNA encoding the entire amino acid sequence or one desired partial amino acid sequence of the DNA shown below is used as a template for connecting V regions by PCR.

A DNA sequence encoding the H chain or H chain V region of the antibody, and

DNA sequence encoding the L chain or L chain V region of the antibody.

The DNA encoding the V region of the H chain and the L chain is amplified by a PCR method using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified. Next, DNA encoding the peptide linker portion is prepared. DNA encoding a peptide linker can also be synthesized by PCR. A base sequence, which can be used to link the amplification products of the synthesized V region, is added to the 5 'terminal side of the primer to be used. Then, PCR is carried out using the respective DNAs in [H chain V region DNA] - [Peptide linker DNA] - [L chain V region DNA] and primers for assembly PCR.

The primer for assembly PCR contains a combination of a primer that anneals to the 5'end of the [H chain V region DNA] and a primer that anneals to the 3'end of the [L chain V region DNA]. That is, the primer for assembly PCR is a set of primers that can be used to amplify DNA encoding the full-length sequence of the scFv to be synthesized. On the other hand, a base sequence that can be used for linking the respective V region DNAs is added to the above [peptide linker DNA]. These DNAs are then ligated, and then the entire length of the scFv is generated as an amplification product using primers for assembly PCR. Once a DNA encoding scFv is produced, an expression vector containing these DNAs and a recombinant cell transformed with the expression vector can be obtained by a conventional method. In addition, scFv can be obtained by expressing the scFv-encoding DNA by culturing the resulting recombinant cell.

The order of VH and VL to be combined is not particularly limited, and they may be listed in any order. An example of the listing is listed below.

[VH] Linker [VL]

[VL] Linker [VH]

sc (Fv) 2 is a single chain low molecular weight antibody prepared by combining two VHs and two VLs using a linker or the like (Hudson et al, J Immunol. Methods 1999; 231: 177-189). Sc (Fv) 2 can be produced, for example, by connecting scFvs using a linker.

Preferably, two VHs and two VLs of the antibody are listed in the order of VH, VL, VH and VL ([VH] linker [VL] linker [VH] linker [VL]) from the N-terminal side of the single- The order of one, two VHs and two VLs is not limited to the above arrangement, but may be arranged in any order. An example of the listing is listed below.

[VL] Linker [VH] Linker [VH] Linker [VL]

[VH] Linker [VL] Linker [VL] Linker [VH]

[VH] Linker [VH] Linker [VL] Linker [VL]

[VL] Linker [VL] Linker [VH] Linker [VH]

[VL] Linker [VH] Linker [VL] Linker [VH]

The amino acid sequence of the low molecular antibody VH or VL may contain substitution, deletion, addition and / or insertion. In addition, when VH and VL are associated, some of them may be deficient or other polypeptides may be added as long as they have antigen binding activity. In addition, the variable region can be chimerized or humanized.

In the present invention, the linker that binds the variable region of the antibody may be any peptide linker that can be introduced by gene engineering, or a synthetic compound linker such as those disclosed in Protein Engineering, 9 (3), 299-305, 1996 You can use the linker.

A preferred linker in the present invention is a peptide linker. The length of the peptide linker is not particularly limited, and the length of the peptide linker can be suitably selected by those skilled in the art depending on the purpose. The typical length is 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

The amino acid sequences of the peptide linkers include the following sequences.

Ser

Gly · Ser

Gly · Gly · Ser

Ser · Gly · Gly

Gly Gly Gly Ser (SEQ ID NO: 45)

Ser. Gly. Gly. Gly (SEQ ID NO: 46)

Gly Gly Gly Gly Ser (SEQ ID NO: 47)

Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 48)

Gly Gly Gly Gly Gly Ser (SEQ ID NO: 49)

Ser, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 50)

Gly, Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 51)

Ser, Gly, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 52)

(Gly Gly Gly Gly Ser (SEQ ID NO: 47)) n

(Ser · Gly · Gly · Gly · Gly (SEQ ID NO: 48)) n

Here, n is an integer of 1 or more.

The amino acid sequence of the peptide linker can be suitably selected by those skilled in the art depending on the purpose. For example, n, which determines the length of the above peptide linker, is usually 1 to 5, preferably 1 to 3, more preferably 1 or 2.

Synthetic compound linkers (chemical cross-linking agents) are cross-linking agents commonly used for cross-linking peptides such as N-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis (sulfosuccinimidyl) (BS), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol Bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST), bis [2- (succinimidoxycarbonyloxy) Ethyl] sulfone (BSOCOES) and bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl] sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available.

Generally, three linkers are required to combine the four antibody variable regions. The plurality of linkers may be the same or different linkers.

Antibodies in the present invention also include antibodies to which one or more amino acid residues have been added to the amino acid sequence of the antibody. In the present invention, the upper body also includes a fusion protein in which the above-mentioned antibody is fused with another peptide or protein. The fusion protein can be produced by ligating a polynucleotide encoding an antibody and a polynucleotide encoding another peptide or polypeptide into a frame, introducing the DNA into an expression vector, and expressing the vector in a host. Techniques known to those skilled in the art can be used. Such peptides or polypeptides fused with the antibodies of the present invention can be prepared by known peptides such as FLAG (Hopp, TP et al., BioTechnology (6), 1204-1210), 6 (histidine) A fragment of p18HIV, a fragment of T7-tag, an HSV-tag, an E-tag, a fragment of SV40T antigen, an Ick tag, a lck tag, Alpha-tubulin, B-tag, and Protein C fragments. The polypeptides fused with the antibodies of the present invention include, for example, GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin normal region, beta-galactosidase, and MBP (maltose binding protein) . Polynucleotides encoding such peptides or polypeptides that are commercially available can be fused with polynucleotides encoding the antibodies described herein. The fusion polypeptide can be prepared by expressing the thus prepared fusion polynucleotide.

In addition, the antibody described in the present invention may be a conjugate antibody bound to various molecules such as a polymer material including polyethylene glycol (PEG) and hyaluronic acid, a radioactive substance, a fluorescent substance, a luminescent substance, an enzyme, and toxin. The conjugated antibody can be obtained by chemically modifying the obtained antibody. Methods of modifying antibodies have been established in the art (see, for example, US 5057313 and US 5156840). The "antibody" in the present invention also includes the conjugated antibody.

In addition, the antibody in the present invention also includes an antibody having a modified sugar chain.

In addition, the antibody used in the present invention may be a bispecific antibody. Bispecific antibodies refer to antibodies having variable regions that recognize different epitopes in the same antibody molecule. Bispecific antibodies in the present invention are bispecific antibodies that recognize different epitopes on the IL-6 receptor molecule, or bispecific antibodies in which one antigen-binding site recognizes the IL-6 receptor and the other antigen-binding site recognizes another Lt; / RTI > Examples of the antigen to which the other antigen binding site of the bispecific antibody comprising the antibody of the present invention recognizing the IL-6 receptor binds are IL-6, TNFalpha, TNFRl, TNFR2, CD80, CD86, CD28, CD20, CD19, IL-1alpha, IL-beta, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin alpha, , LIGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM- M-CSF, M-CSFR, IFN-alpha, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL, and APRILR.

Methods for making bispecific antibodies are known. Bispecific antibodies can be produced, for example, by combining two antibodies that recognize different antigens. The antibody to be bound may be a half molecule each having an H chain and an L chain or a ¼ molecule containing only an H chain. Alternatively, fusion cells producing bispecific antibodies can be produced by fusion of hybridomas producing different monoclonal antibodies. In addition, bispecific antibodies can be produced by genetic engineering techniques.

As described below, the antibody used in the present invention may differ in amino acid sequence, molecular weight, isoelectric point, oligosaccharide, and form depending on the cell or host used for producing the antibody or the purification method. However, as long as the resultant antibody is functionally equivalent to the antibody described in the present invention, it should be considered included in the present invention. For example, when the antibody is expressed in a prokaryotic cell, such as E. coli, the methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. Such antibodies are also included in the antibodies described herein.

Polypeptides such as anti-IL-6 receptor antibodies in the present invention can be produced by methods known to those skilled in the art.

An anti-IL-6 receptor antibody can be produced, for example, by genetic recombinant techniques known to those skilled in the art based on the sequence of the obtained anti-IL-6 receptor antibody. Specifically, an anti-IL-6 receptor antibody can be produced by constructing a polynucleotide encoding an antibody based on the sequence of an antibody recognizing an IL-6 receptor, introducing the polynucleotide into an expression vector, and expressing the polynucleotide in an appropriate host cell (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496 Methods Enzymol (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol (1986); Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178,497-515; Lamoyi, (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

Accordingly, the present invention provides a method of producing a polypeptide encoded by a gene encoding (i) a polypeptide of the invention, or (ii) a gene encoding a polypeptide of the invention, wherein the method comprises detecting a polypeptide encoding a polypeptide of the invention And culturing the host cell containing the vector into which the nucleotide has been introduced.

More specifically, the present invention provides a method for producing a polypeptide of the present invention, comprising the steps of:

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

(b) a polypeptide encoded by the gene is obtained.

Examples of such vectors include M13 family vectors, pUC family vectors, pBR322, pBluescript, and pCR-Script. Also, when the purpose is subcloning and truncation of cDNA, examples of vectors other than the above-mentioned vectors include pGEM-T, pDIRECT, and pT7. Expression vectors are particularly useful for producing the antibodies described herein. For example, when an expression vector is intended for expression in E. coli, the vector must have characteristics that allow its amplification in E. coli. In addition, when the host is Escherichia coli such as JM109, DH5alpha, HB101 or XL1-Blue, the vector may be a promoter that allows its efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544 (Better et al., Science (1988) 240, 1041-1043), T7 promoter, and the like. These vectors include pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system (quasiagen)", pEGFP, and pET (in this case, BL21 expressing T7 RNA polymerase in the host) .

In addition, the expression plasmid vector may contain a signal sequence for antibody secretion. As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al. J. Bacteriol. (1987) 169, 4379) can be used to produce periplasmic E. coli. The vector may be introduced into the host cell by, for example, calcium chloride method or electroporation.

In addition to the vector for E. coli, the vector for producing the antibody described in the present invention may be, for example, an expression vector derived from a mammal such as pcDNA3 (manufactured by Invitrogen), pEF-BOS (Nucleic Acids. Bac-BAC baculovairus expression system (Gibco BRL), pBacPAK8), plant-derived expression vectors (e. G., 18 (17), p5322), pEF, pCDM8) (E.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retrovirus (e.g., pZIPneo), yeast- "Pichia Expression Kit (Invitrogen)", pNV11, SP-Q01), and staphylococcal expression vectors (for example, pPL608, pKTH50).

When the expression plasmid vector is used for expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is necessary to use a promoter necessary for expression in the cell, such as the SV40 promoter (Mulligan et al., Nature (1979) 277 , 108, MMLV-LTR promoter, EF1 alpha promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), or CMV promoter. It is more preferable that the vector has a gene for selection of transformed cells (for example, a drug resistance gene which can be discriminated by a drug (neomycin, G418, etc.)). Vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Further, when the objective is to stably express the gene and amplify the copy number of the gene in the cell, the CHO cell defect in the nucleic acid synthesis pathway can be detected by a vector having the complementary DHFR gene (for example, pSV2 -dhfr ("Molecular Cloning 2nd edition" Cold Spring Harbor Laboratory Press, (1989))) and amplifying the vector using methotrexate (MTX) can be used. When the objective is transient expression of a gene, a method of transforming a vector having a replication origin of SV40 (such as pcD) with a COS cell having a gene expressing SV40 T antigen on a chromosome can be used. In addition, it is possible to use a starting point of a conjugate derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like. In order to amplify the gene copy number in the host cell system, the expression vector may contain an aminoglycoside transporase (APH) gene, a thymidine kinase (TK) gene, E. coli Xanthine guanine phosphoribosyltransferase (Ecogpt) Gene, a dihydrofolate reductase (dhfr) gene, and the like.

The producing antibody described in the present invention can be isolated from a host cell or extracellularly (such as a medium) and purified as a substantially pure and homogeneous antibody. The antibody may be separated or purified using conventional separation and purification methods for antibody purification, but is not limited thereto. For example, the antibody can be suitably selected from a combination of column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, And can be separated and purified.

Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (see Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be carried out using liquid chromatography, for example, HPLC and FPLC. Columns used for affinity chromatography include Protein A column and Protein G column. Examples of columns using Protein A include Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention also encompasses highly purified antibodies using such purification methods.

The IL-6 receptor binding activity of the resulting antibody can be measured by methods known to those skilled in the art. Methods for measuring the antigen binding activity of an antibody include, for example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) and fluorescent antibody method. For example, when an enzyme immunoassay is used, an antibody-containing sample such as a culture supernatant and a purified antibody of an antibody-producing cell is added to a plate coated with an antigen. The secondary antibody labeled with an enzyme such as alkaline phosphatase is added, and the plate is incubated. After washing, the antigen binding activity can be assessed by measuring the absorbance by adding an enzyme substrate such as p-nitrophenylphosphoric acid.

Medicinal composition

The present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide as an active ingredient. The pharmaceutical composition of the present invention can be used for IL-6-related diseases such as rheumatoid arthritis. Accordingly, the present invention also provides a therapeutic agent for a disease such as arthritic rheumatism, which comprises the above antibody as an active ingredient. Preferred examples of the underlying diseases include, but are not limited to, arthritic rheumatism, elderly idiopathic arthritis, elderly idiopathic arthritis, castra man, systemic erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma atherosclerosis, ankylosing spondylitis, ankylosing spondylitis, psoriatic arthritis, chronic obstructive pulmonary disease (e.g., lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, Still's disease, amyloidosis, multiple sclerosis, COPD), IgA nephropathy, degenerative arthropathy, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, -cell non-Hodgkin's, pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve infiltration, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, The present invention relates to a method for the treatment of diabetic retinopathy, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, and the like, which are selected from the group consisting of dermatitis, atopic dermatitis, mesothelioma, multiple myositis, dermatomyositis, Includes postoperative inflammation.

The expression "comprising an anti-IL-6 receptor antibody as an active ingredient" means that the anti-IL-6 receptor antibody is contained as at least one of the active ingredients. In addition, the pharmaceutical composition of the present invention may contain other active ingredients together with the above-mentioned polypeptide.

The pharmaceutical composition of the present invention may be used not only for therapeutic purposes but also for preventive purposes.

Amino acids contained in the amino acid sequence described in the present invention can be modified after translation. For example, modification of the N-terminal glutamine (Gln) residue to pyroglutamic acid (pGlu) residue by pyroglutamylation can be performed by a person skilled in the art Is a well known formula. As such, when the amino acid is modified after translation, it is naturally included in the amino acid sequence described in the present invention.

The oligosaccharide to be bound to the antibody according to the present invention may be of any structure. The 297th (EU numbering) oligosaccharide may be any oligosaccharide structure (preferably a fucosylated oligosaccharide) or the oligosaccharide may not be present at that position (for example, it produces antibodies in E. coli, (EU numbering)).

All prior art documents cited herein are incorporated herein by reference.

[ Example ]

Hereinafter, the present invention will be described with the help of examples, but it will be understood that the present invention is not limited to these examples.

[Example 1]

TOCILIZUMAB of IL Identification of variable region mutation sites that improve affinity to the receptor

To improve the affinity of TOCILIZUMAB (H chain WT-IgG1 / SEQ ID NO: 53, L chain WT-kappa / SEQ ID NO: 54) to the IL-6 receptor, libraries of mutated CDR sequences were prepared Respectively. Screening libraries of CDR mutations found mutations that improved the affinity for the IL-6 receptor. This variation is shown in Fig. The combination of these mutations provided high affinity TOCILIZUMAB such as RDC-23 (H chain RDC23H-IgG1 / SEQ ID NO: 55, L chain RDC-23L-kappa / SEQ ID NO: 56). The affinity for the soluble IL-6 receptor and the biological activity measured using BaF / gp130 were compared between RDC-23 and TOCILIZUMAB (see reference example).

The affinity measurement results are shown in Table 1. Biological activity using BaF / gp130 (final concentration of IL-6 was 30 ng / mL) is shown in FIG. The results showed that the affinity of RDC-23 was about 60 times higher than that of TOCILIZUMAB, and the activity expressed by 100% inhibitory concentration of BaF / gp130 was about 100 times higher than that of TOCILIZUMAB.

[Example 2]

TOCILIZUMAB of Isoelectric point  To improve pharmacokinetics due to degradation Modified  Sympathy

In order to improve the pharmacokinetics of TOCILIZUMAB, studies were conducted to identify mutation sites that would lower the isoelectric point of the variable region without significantly deteriorating the binding to the IL-6 receptor. Screening of the predicted variable region mutation sites based on the stereochemistry model of TOCILIZUMAB found mutations that would lower the isoelectric point of the variable region without significantly reducing binding to the IL-6 receptor. These are shown in Fig. The combination of these mutations provided TOCILIZUMAB with reduced isoelectric point, including, for example, H53 / L28 (H chain H53-IgG1 / SEQ ID NO: 57, L chain L28-kappa / SEQ ID NO: 58). The affinity for the soluble IL-6 receptor, the isoelectric point, the drug activity in the mouse, and the biological activity measured using BaF / gp130 were compared between H53 / L28 and TOCILIZUMAB (see Reference Example).

The affinity measurement results are shown in Table 2. The measurement result of the biological activity (the final concentration of IL-6 was 30 ng / mL) measured using BaF / gp130 is shown in FIG. The results showed that the affinity of H53 / L28 was about 6 times higher than that of TOCILIZUMAB, and the activity expressed by 100% inhibitory concentration of BaF / gp130 was improved several times.

The results of isoelectric point measurements by isoelectric focusing electrophoresis known to those skilled in the art show that the TOCILIZUMAB and H53 / L28 isoelectric points are about 9.3 and about 6.5 to 6.7, respectively. Therefore, the isoelectric point of H53 / L28 was about 2.7 lower than that of TOCILIZUMAB. In addition, the theoretical isoelectric point of the VH / VL variable region was calculated using GENETYX (GENETYX CORPORATION). The results show that the theoretical isoelectric points of TOCILIZUMAB and H53 / L28 are 9.20 and 4.52, respectively. Therefore, the theoretical isoelectric point of H53 / L28 decreased by about 4.7 compared to TOCILIZUMAB.

In order to evaluate the pharmacokinetics of the modified antibody H53 / L28 with reduced isoelectric point, the pharmacokinetics of TOCILIZUMAB and H53 / L28 in normal mice were compared. A single dose of TOCILIZUMAB or H53 / L28 was administered intravenously (IV) and subcutaneously (SC) at 1 mg / kg to mice (C57BL / 6J, Charles River Japan) Plasma concentration trends of TOCILIZUMAB and H53 / L28 after intravenous or subcutaneous administration are shown in Figures 5 and 6, respectively. The pharmacokinetic parameters (clearance (CL), half-life (T1 / 2)) obtained using WinNonlin (Pharsight) are shown in Table 3. After intravenous administration, plasma half-life (T1 / 2) of H53 / L28 was extended to about 1.3 times of TOCILIZUMAB, while the clearance was reduced about 1.7 times. After subcutaneous administration, T1 / 2 of H53 / L28 was extended to about twice that of TOCILIZUMAB, while the clearance was reduced by about 2.1 times. Therefore, it has been found that pharmacokinetics can be greatly improved by lowering the isoelectric point of TOCILIZUMAB by amino acid substitution.

[Example 3]

TOCILIZUMAB The immunogenicity of Deteriorate  Identification of mutation points

Identification of mutation sites that reduce immunogenicity risk by T-cell epitopes in variable regions

The T-cell epitope present in the variable region sequence of TOCILIZUMAB was analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, the L chain CDR2 was predicted to have a T-cell epitope binding to HLA (i.e., having a sequence with high immunogenicity risk). Thus, TEPITOPE analysis was performed to examine amino acid substitutions that would reduce the immunogenicity risk of L chain CDR2 without compromising stability, binding activity, or neutralizing activity.

As described below, the screening results showed that the threonine at L51 (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) of L chain CDR2 (SEQ ID NO: 59) of TOCILIZUMAB was replaced with glycine , Indicating that substitution of arginine in L53 with glutamic acid (SEQ ID NO: 60) can reduce immunogenicity risk without lowering stability, binding activity, or neutralizing activity.

TOCILIZUMAB L chain CDR2 (SEQ ID NO: 59)

T-cell epitope removal TOCILIZUMAB L chain CDR2 (SEQ ID NO: 60)

[Example 4]

TOCILIZUMAB Variable area Framework sequence  Immunogenicity by full humanization Lee Sc Abatement

In the process of humanization of TOCILIZUMAB, several mouse sequences remain in framework sequences to maintain binding activity (Cancer Res. 1993 Feb 15; 53 (4): 851-6). These sequences are H27, H28, H29 and H30 in the H chain FR1 of the variable region sequence of TOCILIZUMAB and H71 (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. Remaining mouse sequences are a potential source of increased immunogenicity risk. Thus, we examined whether the framework sequence could be fully humanized and further reduce the immunogenicity risk of TOCILIZUMAB.

The results show that the H chain FR1 (SEQ ID NO: 61) of TOCILIZUMAB was replaced with the humanized H chain FR1-A (SEQ ID NO: 62) shown below and the H chain FR3 (SEQ ID NO: 63) of TOCILIZUMAB was replaced with the humanized H chain By substitution with FR3 (SEQ ID NO: 64), it was shown that the entire framework of TOCILIZUMAB can be fully humanized without compromising stability, binding activity, and neutralizing activity.

TOCILIZUMAB H chain FR1 (SEQ ID NO: 61)

Humanized H chain FR1-A (SEQ ID NO: 62) (derived from Germline IMGT hVH4)

TOCILIZUMAB H chain FR3 (SEQ ID NO: 63)

Humanized H chain FR3 (SEQ ID NO: 64) (derived from Mol. Immunol. 2007, 44 (4): 412-422)

[Example 5]

TOCILIZUMAB of IL -6 receptor pH  Identification of Mutation Sites for Improved Pharmacokinetics by Dependent Binding

One of the ways to improve the pharmacokinetics of TOCILIZUMAB is to improve the molecule so that one molecule of TOCILIZUMAB binds and neutralizes multiple IL-6 receptors repeatedly. TOCILIZUMAB is bound to the transmembrane IL-6 receptor and then inserted into the intracellular endosomes by internalization while bound to the transmembrane IL-6 receptor and then transferred to the lysosome bound to the transmembrane IL-6 receptor At the same time, it is presumed to be decomposed by lysosomes. Specifically, one molecule of TOCILIZUMAB typically binds to one or two molecules of the membrane type IL-6 receptor (mono-or two-dimensional) and is degraded into lysosomes after interpolation. Therefore, one molecule of TOCILIZUMAB can bind and neutralize only one or two molecules of the membrane type IL-6 receptor.

Thus, the present inventors have found that if it is possible to produce TOCILIZUMAB that binds in a pH-dependent manner, where the binding of TOCILIZUMAB is maintained under neutral conditions, but significantly under acidic conditions, TOCILIZUMAB binding in a pH- Was dissociated from the transmembrane IL-6 receptor (antigen) in endosomes and bound to the FcRn present in endosomes, leading to return to plasma. Once in the plasma, TOCILIZUMAB, which binds in a pH-dependent manner, will again be able to bind to the membrane-type IL-6 receptor. By repeating such binding in plasma and dissociation in endosomes, it was thought that one molecule of TOCILIZUMAB could repeatedly bind and neutralize multiple molecules of IL-6 receptor. Thus, TOCILIZUMAB binding in a pH-dependent manner, as compared to TOCILIZUMAB, is presumed to have improved pharmacokinetics.

To dissociate TOCILIZUMAB from the IL-6 receptor under acidic conditions in the endosome, the binding should be significantly weaker than under neutral conditions. Since strong IL-6 receptor binding is required for neutralization at the cell surface, at a cell surface pH of pH 7.4, the antibody should bind to the IL-6 receptor either equally or more strongly than TOCILIZUMAB. The endosomal pH is generally reported to be 5.5 to 6.0 (Nat Rev Mol Cell Biol. 2004 Feb; 5 (2): 121-32). Thus, if TOCILIZUMAB binding in a pH-dependent manner is modified to bind weakly to the IL-6 receptor at pH 5.5 to 6.0, it is expected to dissociate from the IL-6 receptor under acidic conditions in the endosome. Specifically, if TOCILIZUMAB binding in a pH-dependent manner is enhanced to bind strongly to the IL-6 receptor at pH 7.4, the pH of the cell surface, TOCILIZUMAB can bind and neutralize multiple molecules of IL-6 receptor, Therefore, pharmacokinetics can be improved.

Possible methods to confer a pH dependence on the binding of TOCILIZUMAB to the IL-6 receptor are those in which the histidine residue has a pKa of about 6.0 to 6.5 and its proton dissociation state is neutral (pH 7.4) and acidic (pH 5.5 to pH 6.0 ) Conditions, the histidine residue is introduced into the variable region of TOCILIZUMAB. Thus, screening was performed to identify histidine introduction sites in the variable region based on the stereostructure model of TOCILIZUMAB. In addition, a library for screening was designed by randomly substituting selected variable region sequences of TOCILIZUMAB with histidine. The screening was performed using the binding to the IL-6 receptor at pH 7.4 and dissociation from the IL-6 receptor, or the decrease in affinity at pH 5.5 to 5.8 as an indicator.

As a result, the present inventors have found mutation sites conferring TOCILIZUMAB binding to the IL-6 receptor with pH dependence (binding at pH 7.4 and dissociation at pH 5.8). This is shown in FIG. In Figure 8, the substitution of tyrosine with histidine at H27 is not a CDR but a variation in the H chain FR1. However, Eur. J. Immunol. 1992. 22: 1719-1728, the sequence with histidine at H27 is the human sequence (SEQ ID NO: 65). Thus, the antibody can be fully humanized by using the next framework in combination with Example 4.

The humanized H chain FR1-B (SEQ ID NO: 65)

For example, a combination of mutations including H3pI / L73 (H chain H3pI-IgG1 / SEQ ID NO: 66, L chain L73-kappa / SEQ ID NO: 67) can provide TOCILIZUMAB with pH dependent binding properties. H3pI / L73 and TOCILIZUMAB showed affinity for soluble IL-6 receptor at pH 7.4, dissociation rate from membrane-type IL-6 receptor at pH 7.4 and pH 5.8, bioactivity using BaF / gp130, And human IL-6 receptor transgenic mice (see Reference Example).

Table 4 shows the affinity measurement results for the soluble IL-6 receptor at pH 7.4. Fig. 9 shows the results of measurement of biological activity (final concentration of IL-6 30 ng / mL) obtained using BaF / gp130. These results show that H3pI / L73 corresponds to TOCILIZUMAB in terms of affinity for soluble IL-6 receptor at pH 7.4 and activity against BaF / gp130.

The dissociation rates of TOCILIZUMAB or H3pI / L73 from the membrane-type IL-6 receptor at pH 7.4 and pH 5.8 are shown in Table 5. Compared with TOCILIZUMAB, the dissociation rate of H3pI / L73 at pH 5.8 was faster and the pH dependence of the dissociation rate from the membrane type IL-6 receptor improved about 2.6 fold.

A single dose of TOCILIZUMAB or H3pI / L73 was intravenously administered to the crab monkeys at 1 mg / kg and the plasma concentration trend was evaluated. FIG. 10 shows changes in plasma concentration after intravenous administration of TOCILIZUMAB or H3pI / L73. The results showed that the pharmacokinetics of H3pI / L73 in crab monkeys were significantly improved compared to TOCILIZUMAB.

A single dose of TOCILIZUMAB or H3pI / L73 was administered to human IL-6 receptor transgenic mice (hIL-6R tg mouse, Proc Natl Acad Sci US A. May 1995; 92 (11): 4862-6) at 25 mg / kg Were administered intravenously and the changes in plasma concentration were evaluated. Figure 11 shows changes in plasma concentration after intravenous administration of TOCILIZUMAB or H3pI / L73. The results showed that the pharmacokinetics of H3pI / L73 in human IL-6 receptor transgenic mice was significantly improved compared to TOCILIZUMAB.

H3pI / L73, a TOCILIZUMAB with pH-dependent binding properties, showed significantly improved pharmacokinetics in geese monkey and human IL-6 receptor transgenic mice compared to TOCILIZUMAB. This suggests that it is possible to bind and neutralize multiple IL-6 receptors in a single molecule by binding the antigen at pH 7.4 and dissociating from the antigen at pH 5.8. It was also conceived that further pharmacokinetics could be improved by conferring IL-6 receptor binding with a stronger pH dependence than H3pI / L73.

[Example 6]

TOCILIZUMAB Optimization of the normal region of

Reduction of heterogeneity of H chain C-terminal of TOCILIZUMAB

In the case of the heterogeneity of the H chain C-terminal sequence of the IgG antibody, amidation of the C-terminal carboxyl group by deletion of the lysine residue of the C-terminal amino acid and deletion of both glycine and lysine of the C-terminal 2 amino acid has been reported (Anal Biochem. 2007 Jan 1; 360 (1): 75-83.). Also in TOCILIZUMAB, the main component is a sequence in which the lysine of the C-terminal amino acid existing in the base sequence is deleted by the post-translational modification, the subcomponent in which the lysine remains and the subcomponent in which the C-terminal carboxyl group is amidated by both the glycine and lysine deletion Contributes to heterogeneity. It is not easy to manufacture them in large quantities as pharmaceuticals while maintaining the target substance / related substances related to the heterogeneity between the preparations, and it will be more expensive. If possible, it is desirable to be a single material and to have reduced heterogeneity when developing antibodies as pharmaceuticals. Therefore, when developing an antibody as a medicine, it is preferable that heterogeneity of the H chain C terminal is not present.

The C-terminal amino acid was modified to reduce the C-terminal amino acid heterogeneity. The results showed that C-terminal amino acid heterogeneity could be avoided by pre-cleaving lysine and glycine residues at the C-terminus of the H chain normal region of TOCILIZUMAB from the nucleotide sequence. TOCILIZUMAB, TOCILIZUMAB (TOCILIZUMAB delta K, H chain WT-IgG1 delta K / SEQ ID NO: 68, L chain WT-kappa / SEQ ID NO: 54) lacking the lysine residue at the C terminus and lysine and glycine residue at the C terminus The missing TOCILIZUMAB (TOCILIZUMAB delta GK, H chain WT-IgG1 delta GK / SEQ ID NO: 69, L chain WT-kappa / SEQ ID NO: 54) was evaluated for heterogeneity by cation exchange chromatography. L of MES / NaOH, 250 mmol / L of NaCl (pH of 6.1), and a mobile phase B of 25 mmol / L of MES / NaOH, 250 mmol / 6.1). Appropriate flow rates and gradients were used. The results of the evaluation by cation exchange chromatography are shown in Fig. The results showed that C-terminal amino acid heterogeneity could be reduced by pre-cleaving both lysine and glycine residues at the C-terminus of the H chain normal region from the nucleotide sequence. All C-terminal sequences of the human antibody normal regions IgG1, IgG2 and IgG4 contain lysine and glycine at positions 447 and 446 of the EU numbering (see, Sequences of proteins of immunological interest, NIH Publication No. 91-3242). Therefore, the methods of reducing the C-terminal amino acid heterogeneity found in this study are predicted to be applicable to the IgG2 and IgG4 normal regions, and their dog variants.

Reduction of heterogeneity derived from disulfide bond of IgG2 isotype of TOCILIZUMAB

The isotype of TOCILIZUMAB is IgG1. Since TOCILIZUMAB is a neutralizing antibody, in terms of immunogenicity and side effects, binding to the Fc gamma receptor may be undesirable. A possible way to decrease Fc gamma receptor binding is to change the isotype of IgG antibodies from IgGl to IgG2 or IgG4 (Ann Hematol. 1998 Jun; 76 (6): 231-48). From the viewpoint of binding to Fc gamma receptor I and pharmacokinetics, IgG2 was thought to be more preferable than IgG4 (Nat Biotechnol. 2007; 25 (12): 1369-72). On the other hand, when the antibody is developed as a medicine, the physical properties of the protein, particularly the uniformity and stability, are very important. IgG2 isotype has been reported to have a very high heterogeneity by disulfide bonds in the hinge region (J Biol Chem. 2008 Jun 6; 283 (23): 16206-15.). It is not easy and costly to manufacture them in large quantities as pharmaceuticals while maintaining the target substance / related substances related to heterogeneity derived from interspecific disulfide bonds. Thus, a single material is desired. Therefore, when an IgG2 isotype antibody is developed as a medicine, it is preferable to reduce the heterogeneity derived from the disulfide bond without lowering the stability.

For the purpose of reducing the heterogeneity of IgG2 isotype, various individual variants were examined. As a result, in the IgG2 normal region, the 131th (EU numbering) cysteine residue and the 133th (EU numbering) arginine residue in the CH1 domain of the H chain were substituted with serine and lysine, (SEQ ID NO: 70) in which cysteine of the first (EU numbering) was replaced with serine, the heterogeneity could be reduced without lowering the stability. IgG1 (H chain WT-IgG1 / SEQ ID NO: 53, L chain WT-kappa / SEQ ID NO: 54), TOCILIZUMAB-IgG2 : 54), and TOCILIZUMAB-SKSC (H chain WT-SKSC / SEQ ID NO: 70, L chain WT-kappa / SEQ ID NO: 54) were prepared and heterogeneity and stability were evaluated. Heterogeneity was assessed by cation exchange chromatography. ProPac WCX-10 (Dionex) column was used. Mobile phase A was 20 mM sodium acetate (pH 5.0) and mobile phase B was 20 mM sodium acetate, 1 M NaCl (pH 5.0). Appropriate flow rates and gradients were used. The results of the evaluation by cation exchange chromatography are shown in Fig. The stability was evaluated based on the heat denaturation intermediate temperature (Tm value) measured by differential scanning calorimetry (DSC) (VP-DSC, manufactured by Microcal). The results of DSC measurement at 20 mM sodium acetate, 150 mM NaCl, pH 6.0 and Tm values of the Fab domain are shown in FIG.

The results showed that the heterogeneity of TOCILIZUMAB-IgG2 was significantly increased compared to TOCILIZUMAB-IgG1, but heterogeneity could be greatly reduced by TOCILIZUMAB-SKSC. In addition, the DSC of TOCILIZUMAB-IgG2 compared to TOCILIZUMAB-IgG1 gave a low stability, a shoulder peak (Fab *) component with a low Tm at the thermodynamic peak of the Fab domain, presumably due to the heterologous component. However, in the case of TOCILIZUMAB-SKSC, the shoulder peak (low Tm) thought to be due to the hetero component was lost and the Tm value was about 94 占 폚, which was equivalent to the Fab domain of TOCILIZUMAB-IgG1 and TOCILIZUMB-IgG2. Thus, TOCILIZUMAB-SKSC was found to have high stability.

Identification of normal region mutations to improve pharmacokinetics of TOCILIZUMAB

As described above, from the IgG1 isotype of TOCILIZUMAB, the heterogeneity of the C-terminal is reduced, the binding property to the Fc gamma receptor is reduced, and the heterogeneity of the antibody having the IgG2 isotype normal region is reduced while maintaining high stability Can be achieved. Furthermore, it is preferred that said normal region also has better pharmacokinetics than IgGl, which is an isotype of TOCILIZUMAB.

Screening was performed to find a normal region with a plasma half-life superior to that of an antibody having an IgG1 isotype normal region, and TOCILIZUMAB-SKSC having a high degree of stability and a reduced heterogeneity with respect to an antibody having the aforementioned IgG2 isotype normal region To identify the mutation sites to improve the pharmacokinetics of the drug. As a result, we found WT-M58 (SEQ ID NO: 72 (amino acid sequence)), wherein the 137th (EU numbering) glutamic acid was replaced by glycine and the 138th serine was replaced by glycine , The 268th histidine was replaced with glutamine, the 355th arginine was replaced with glutamine, the 419th glutamine was replaced with glutamic acid, and the 446th glycine and the 447th glycine were substituted for the H- Of the lysine. In addition, for IgG1, WT-M44 (SEQ ID NO: 73 (amino acid sequence)) in which 434th asparagine was replaced with alanine was prepared. In addition, WT-M83 (SEQ ID NO: 74 (amino acid sequence)) was prepared by deletion of 446th glycine and 447th lysine from M44 in order to reduce the heterogeneity of the H chain C-terminal. Further, in WT-M58, WT-M73 (SEQ ID NO: 75 (amino acid sequence)) was prepared by substituting 434th asparagine with alanine.

L chain WT-kappa / SEQ ID NO: 54), TOCILIZUAMB-M44 (H chain WT-M44 / SEQ ID NO: 73, L chain WT-kappa / SEQ ID NO: : 54) and TOCILIZUMAB-M73 (H chain WT-M73 / SEQ ID NO: 75, L chain WT-kappa / SEQ ID NO: 54) were prepared and affinity to human FcRn and pharmacokinetics (See Reference Example as a method).

The binding of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 to human FcRn was assessed using Biacore. As shown in Table 6, the binding of TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 was about 2.7 times, 1.4 times and 3.8 times higher than TOCILIZUMAB-IgG1, respectively.

TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 were evaluated for their pharmacokinetics in human FcRn transgenic mice. The results are shown in Fig. As shown in Fig. 15, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 were found to show improved pharmacokinetics compared to TOCILIZUMAB-IgG1. The effect of improving pharmacokinetics was correlated with the ability to bind to human FcRn. In particular, the concentration of TOCILIZUMAB-M73 in plasma after 28 days was about 16 times that of TOCILIZUMAB-IgG1. Therefore, it was also estimated that the antibody having the normal region of M73 had significantly improved pharmacokinetics as compared to the antibody having the normal region of IgG1.

[Example 7]

PK / PD Improved full humanization IL -6 receptor antibody

TOCILIZUMAB variants were constructed by combining a plurality of mutations in the variable region and the normal region of TOCILIZUMAB found in the above embodiment. The full humanized IL-6 receptor antibody positively predicted from various screenings is Fv3-M73 (H chain VH4-M73 / SEQ ID NO: 25, L chain VL1-kappa / SEQ ID NO: 28), Fv4- SEQ ID NO: 26, L chain VL3-kappa / SEQ ID NO: 29) and Fv5-M83 (H chain VH5-M83 / SEQ ID NO: 27, L chain VL5-kappa / SEQ ID NO: 30).

The affinity of the prepared Fv3-M73, Fv4-M73, and Fv5-M83 to the IL-6 receptor was compared with TOCILIZUMAB (see Reference Example). The affinities of these antibodies to soluble IL-6 receptor at pH 7.4 are shown in Table 7. In addition, their BaF / gp130 neutralizing activity was compared with TOCILIZUMAB and control (VQ8F11-21 hIgG1 as described in Reference Example, known high affinity anti-IL-6 receptor antibody, US 2007/0280945) Reference). The results obtained by measuring the biological activity of these antibodies using BaF / gp130 are shown in Fig. 16 (TOCILIZUMAB, control and Fv5-M83 with a final concentration of 300 ng / mL of IL-6) ng / mL, TOCILIZUMAB, Fv3-M73 and Fv4-M73). As shown in Table 7, Fv3-M73 and Fv4-M73 had about 2 to 3 times higher affinity than TOCILIZUMAB, while Fv5-M83 showed about 100 times higher affinity than TOCILIZUMAB (Fv5 It was difficult to measure the affinity of -M83, and therefore it was difficult to measure the affinity of Fv5-IgG1 (H chain VH5-IgG1 / SEQ ID NO: 76, L chain VL5-kappa / SEQ ID NO: Mars was measured and the normal region is generally thought to have no effect on affinity). In addition, as shown in Fig. 17, Fv3-M73 and Fv4-M73 show slightly stronger activity compared to TOCILIZUMAB. As shown in Fig. 16, Fv5-M83 has a very strong activity of 100 times or more as compared with TOCILIZUMAB in terms of 50% inhibitory concentration. Fv5-M83 also showed about 10-fold higher neutralizing activity than the control (known high affinity anti-IL-6 receptor antibody) in terms of 50% inhibitory concentration.

The dissociation rates of TOCILIZUMAB, Fv3-M73, and Fv4-M73 from the membrane-type IL-6 receptor were measured at pH 7.4 and pH 5.8. The pH dependence of the dissociation rate of Fv3-M73 and Fv4-M73 from the membrane-type IL-6 receptor was 11 and 10 times higher than that of TOCILIZUMAB, respectively, as shown by the results shown in Table 8 . A significant improvement in the pH dependence of the dissociation rate compared to H3pI / L73 described in Example 5 suggested that the pharmacokinetics of Fv3-M73 and Fv4-M73 would be significantly improved compared to H3pI / L73.

The isoelectric points of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 were measured by isoelectric point electrophoresis using methods known to those skilled in the art. The results were approximately 9.3 for TOCILIZUMAB, 8.4 to 8.5 for control, 5.7 to 5.8 for Fv3-M73, 5.6 to 5.7 for Fv4-M73 and 5.4 to 5.5 for Fv5-M83. Thus, each antibody had significantly reduced isoelectric point compared to TOCILIZUMAB and control. In addition, the theoretical isoelectric point of the variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION). The results showed that the theoretical isoelectric point was 9.20 for TOCILIZUMAB, 7.79 for control, 5.49 for Fv3-M73, 5.01 for Fv4-M73 and 4.27 for Fv5-M83. Each antibody had significantly reduced isoelectric point compared to TOCILIZUMAB and control. The pharmacokinetics of Fv3-M73, Fv4-M73 and Fv5-M83 were thought to be improved as compared to TOCILIZUMAB and the control, as it is shown in Example 2 that the pharmacokinetics are improved by lowering the isoelectric point.

T-cell epitopes in the variable region sequences of TOCILIZUMAB, Fv3-M73, Fv4-M73 and Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, as shown in Example 3, TOCILIZUMAB was predicted to have a T-cell epitope capable of binding algebra to HLA. In contrast, the number of sequences predicted to bind to the T-cell epitope in Fv3-M73, Fv4-M73 and Fv5-M83 was greatly reduced. In addition, the framework of Fv3-M73, Fv4-M73 or Fv5-M83 does not have any mouse sequences and is therefore fully humanized. This suggests that immunogenicity risk may be significantly reduced in Fv3-M73, Fv4-M73 and Fv5-M83 compared to TOCILIZUMAB.

[Example 8]

Full humanization IL -6 receptor antibody monkey PK / PD  exam

TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 were administered intraperitoneally to the geese monkey at a dose of 1 mg / kg, and the plasma concentration trend was evaluated (see Reference Example). FIG. 18 shows changes in plasma concentration after intravenous administration of TOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83. The results showed that Fv3-M73, Fv4-M73, and Fv5-M83, respectively, showed significantly improved pharmacokinetics in TOCILIZUMAB and control compared to control. Among them, Fv3-M73 and Fv4-M73 showed significantly improved pharmacokinetics compared to TOCILIZUMAB.

We evaluated the efficacy of each antibody that neutralizes the murine monkey IL-6 receptor. Five micrograms / kg of the crab monkey IL-6 from day 6 to day 18 (from day 3 to day 10 for TOCILIZUMAB) were subcutaneously administered to the umbilicus, and after 24 hours, (See Reference Example for the method). The change in CRP concentration after administration of each antibody is shown in Fig. In order to evaluate the efficacy of each antibody neutralizing the soluble-type crab monkey IL-6 receptor, plasma concentrations of the unconjugated and soluble type crab monkey IL-6 receptor in crab monkeys were measured, The available IL-6 receptor ratios were calculated (see Reference Examples for methods). The change in IL-6 receptor ratio, which is unconjugated and soluble after administration of each antibody, is shown in Fig.

Fv3-M73, Fv4-M73, and Fv5-M83 neutralized the murine crab monkey IL-6 receptor in a more persistent manner compared to TOCILIZUMAB and control (known high affinity anti-IL-6 receptor antibodies) Over a long period of time. In addition, Fv3-M73, Fv4-M73, and Fv5-M83 neutralized soluble CSC IL-6 receptor in a more persistent manner compared to TOCILIZUMAB and control, respectively, and the unconjugated and available type of crab monkey IL-6 And inhibited the increase of the receptor over a long period of time. This finding shows that both Fv3-M73, Fv4-M73 and Fv5-M83 are superior to TOCILIZUMAB and the control in sustaining the neutralization of the solid and soluble IL-6 receptor. Of these, Fv3-M73 and Fv4-M73 were very good at sustaining neutralization. Fv5-M83, on the other hand, suppressed more CRP and non-binding and soluble type of crab monkey IL-6 receptor than Fv3-M73 and Fv4-M73. Thus, Fv5-M83 was thought to be more potent than Fv3-M73, Fv4-M73 and control (known high affinity anti-IL-6 receptor antibodies) in neutralizing the membrane-type and soluble IL-6 receptor. Thus, the results in vivo of the crab monkeys show that the stronger affinity of Fv5-M83 for the IL-6 receptor and the stronger affinity of Fv5-M83 for the BaF / gp130 assay system Activity.

This finding suggests that Fv3-M73 and Fv4-M73 are superior to TOCILIZUMAB and the control in that they continue to function as anti-IL-6 receptor neutralizing antibodies, thus greatly reducing the dose and frequency of administration do. In addition, Fv5-M83 was found to be excellent in terms of the strength of action as an anti-IL-6 receptor neutralizing antibody as well as to maintain its activity. Thus, Fv3-M73, Fv4-M73 and Fv5-M83 are predicted to be useful as IL-6 antagonist drugs.

[Example 9]

Fv4 - M73 Stable for Buffer  Choosing the type

Fv4 - M73 is manufactured as described above. Fv4-M73 consists of the artificial normal region M73 for improved heterogeneity, stability, safety and pharmacokinetics as described in Examples 6-7, and the stability profile of Fv4-M73 (such as the most stable buffer species) May be different from an antibody consisting of a native constant region. Antibodies consisting of the native IgG1 normal region are generally reported to be most stable under histidine-acetate buffer (WO / 2006/0044908). Therefore, the effect of the type of buffer on Fv4-M73 was examined.

Fv4-M73 was formulated to a final concentration of 37 mg / mL in four different buffers with pH 6.0 (as described in Table 9). Samples were analyzed by size exclusion chromatography and anion exchange chromatography with UV detection over two months under storage conditions at < RTI ID = 0.0 > 40 C. < / RTI > The percentage of agglomerates formed in the formulation is plotted over time and is shown in FIG. The percentage increase in the aggregate is an indication of the reduced stability of the antibody. Therefore, the percentage increase has been used as an index to compare the stabilizing effects of different buffers.

As shown in Figure 21, the formulation with histidine-HCl buffer (formulation A) and citrate buffer (formulation D) was the most stable and the formulation with acetate buffer (formulation C) was the most unstable. The histidine-acetate buffer (formulation B) was slightly more unstable than the histidine-HCl buffer, presumably due to the destabilizing effect of the acetate buffer.

Thus, Fv4-M73 with an artificial steady region was found to be most stable in histidine-HCl buffer and citrate buffer, not in the histidine-acetate buffer reported to be the most stable buffer for the native IgG1 antibody.

Way

Size Exclusion Chromatography (SEC): Size exclusion chromatography was performed to screen the antibody preparation for the presence of antibody aggregates and fragments. The sample was injected into a size exclusion G3000 SWXL column (TOSOH). The mobile phase was 50 mM sodium phosphate, 300 mM sodium chloride (pH 7.0) equally moving at a flow rate of 0.5 mL / min. The eluted protein was detected by UV absorption at 200 nM. The relative content of all protein species detected was reported as the area percent of the product peaks relative to the total area of all other detection peaks. Peaks eluting earlier than antibody monomer peaks were recorded in fractional percentiles while peaks eluting earlier than antibody monomer peaks were recorded in aggregate percentiles.

Sample preparation: Samples were diluted to 0.3 to 2 mg / mL as mobile phase and 15 microliters were injected into the column.

Anion Exchange Chromatography: Anion exchange chromatography was performed to analyze the antibody formulation for the presence of heterogeneity, particularly for the presence of deamidation (the material eluting as an acidic species after demineralization with the main peak). The sample was injected at 40 占 폚 onto a DEAE-NPR column (TOSOH). The mobile phase was A) 10 mM Tris-HCl (pH 7.5), B) 10 mM Tris-HCl, 500 mM sodium chloride (pH 7.5) and migrated to a gradient of 100% mobile phase A and after 30 minutes 70% % Mobile phase B, after 1 minute, to 100% mobile phase B, which was maintained for 4 minutes and the column was washed at a flow rate of 1. 0 mL / min. The eluted protein was detected by UV absorption at 280 nM.

Sample preparation: The sample was diluted to 0.05 to 0.33 mg / mL with mobile phase and injected into the column with a volume of 100 microliters.

[Example 10]

100 mg / mL in Fv4 - M73 For the stability of pH , NaCl , And arginine- HCl Effect of

Fv4-M73 was formulated at a final shade of 100 mg / mL in different buffer preparations (as described in Table 10) and stored at 25 < 0 > C and 40 < 0 & ≪ / RTI > for 30 minutes at room temperature and thawing for 30 minutes) were analyzed by size exclusion chromatography and anion exchange chromatography with UV detection over 2 months. The percentage of agglomerates present in the formulation and the percentage of agglomerates increased from initial values after storage at 25 [deg.] C and 40 [deg.] C 2/4/8 weeks are shown in Figures 22-25. An increase in the content of the agglomerates is an indication of reduced stability.

It is assumed that the low molecular weight species (LMW) described in Figure 26 is the result of direct hydrolysis of the peptide bond at weak points such as the hinge region under acidic or basic conditions. This is common for monoclonal antibodies, especially in liquid formulations at high temperatures. The acidic species eluted between about 22.5 and 26 minutes, increased under basic conditions and described in FIG. 27, are reported to be related to the charge heterogeneity of monoclonal antibodies by incubating the antibody under elevated temperature and basic pH and then observing more acidic species As a result of nonenzymatic deamidation of the asparagine residue (general formula of the antibody). Given the results of Figures 22-26, Fv4-M73 was found to be stable in the fluid in association with flocculation and abatement at a pH of about 5.5 to 6.3. Optimal stability was observed at a pH of about 5.5 to 6.3 with respect to the main peak to total peak area ratio.

Within the scope of this study, the addition of 100 mM NaCl (20 mM buffer, 150 mM NaCl) to a buffer containing 20 mM buffer and 50 mM NaCl was followed by the addition of 20 mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl), leading to an equally stable antibody solution, suggesting that NaCl has no stabilizing effect. On the other hand, the addition of 100 mM arginine to a buffer containing 20 mM buffer and 50 mM NaCl showed a significant stabilizing effect of the antibody solution in relation to the agglutinates, suggesting that arginine-HCl has a significant stabilizing effect. With respect to the buffer, the solution stability was slightly better to use the histidine-HCl buffer than the citrate buffer.

With respect to the freeze-thaw study ("FT" represents the freezing / thawing circulation water) as shown in Figure 29, the significant content of aggregates was observed at lower pH or lower salt concentration in the formulation. Addition of 100 mM arginine (20 mM buffer, 50 mM NaCl, 100 mM arginine-HCl) to a buffer containing 20 mM buffer and 50 mM NaCl was performed by adding 100 mM NaCl (20 mM buffer, 150 mM NaCl ), Which suggests that arginine-HCl has a significant stabilizing effect on freezing-thawing. Optimal stability contained 100 mM arginine-HCl and was observed at a pH range of about 5.0-6.6.

[Example 11]

100 mg / mL in Fv4 - M73 To the stability of Per party  And arginine- HCl Effect of

Fv4-M73 was formulated at a final concentration of 100 mg / mL in different buffer formulations (as described in Table 11) and the samples were stored at 25 < 0 > C and 40 < 0 > C and also subjected to a certain number of freeze- And analyzed by size exclusion chromatography and anion exchange chromatography with UV detection over 2 months. The percentage of agglomerates contained in the increased formulation from the initial value is plotted over time and is shown in Figures 30 and 31. The content of agglomerates increased by percent is an indication of reduced stability.

As shown in Figures 30 and 31, Formulations F and G provided lower stability than Formulation H, which is less stable than arginine-HCl in liquid phase in which sucrose and trehalose were stored at 25 占 폚 and 40 占 폚 , And in freeze-thaw, formulations F and G provided comparable or higher stability to formulation H, suggesting that sucrose and trehalose exhibit a significant stability effect in freeze-thaw.

[Example 12]

200 mg / mL in Fv4 - M73 Stability; pH , Arginine- HCl  And Trehalose  effect

Fv4-M73 was formulated at a final concentration of 200 mg / mL in six different preparations as described in Table 12. < tb > < TABLE >

Samples were incubated at 5 < 0 > C and -20 < 0 > C for 3 and 6 months, respectively. Initial samples, 3-month samples, and 6-month samples were analyzed by size exclusion chromatography with UV detection as described in Example 9. The percentage of agglomerates increased from the initial value at 5 째 C for the formulation after 3 months and 6 months is shown in Fig. 3 months and 6 months The percentage of agglomerates increased from the initial value at -20 ° C for the formulation is shown in FIG.

32, Fv4-M73 was apparently more stable at the lower pH (most stable at pH 5.5 and the least stable at pH 6.5), maintained at a higher arginine concentration (Most stable at 150 mM arginine-HCl and the least stable at 50 mM arginine-HCl). The addition of 50 mM trehalose showed some stabilizing effect on Fv4-M73 in the liquid phase stored at 5 < 0 > C. The preferred liquid formulation for Fv4-M73 should contain at least 100 mM arginine-HCl along with a histidine buffer at pH 5.5 to 6.0, and additional stabilizers if necessary .

As shown in Figure 33, in the frozen state stored at -20 占 폚, Fv4-M73 tends to be more stable at higher pH and is apparently more stable at higher arginine concentrations (most stable at 150 mM arginine-HCl 50 mM arginine-HCl). The addition of 50 mM trehalose showed a significant stabilizing effect on Fv4-M73 in the frozen state stored at -20 < 0 > C. The bulk material of the antibody is often stored in a frozen state at -20 캜 to -70 캜. When considering the cost of storage and transportation in a frozen state, storage at -20 ° C is preferable. A preferred bulk material preparation for storage of Fv4-M73 at -20 [deg.] C should contain at least 100 mM arginine-HCl with histidine buffer (pH 5.5-6.5) and sugar (such as trehalose).

[Reference Example]

Preparation of recombinant soluble human IL-6 receptor

Soluble Formulation of Human IL-6 Receptor Antigen The human IL-6 receptor was prepared as follows. J. Biochem. (Yamasaki et al., Science 1988; 241: 825-828 (GenBank # X12830), which contains an amino acid sequence from the first to the 344th amino acid sequence on the N-terminal side reported in Japanese Patent Application Laid- ) In a normal (constant) manner. The soluble human IL-6 receptor was purified by affinity chromatography using gel column chromatography and three-column chromatography: Blue Sepharose 6 FF column chromatography, a specific antibody against SR344, and gel filtration column chromatography, And purified from the culture supernatant of CHO cells. The fraction eluted as the main peak was used as the final purified sample.

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

Based on the published Rhesus monkey IL-6 receptor gene sequence (Birney et al, Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556-61) Respectively. A DNA fragment encoding the full length of the human monkey IL-6 receptor gene was prepared using the PCR method using the primer and the cDNA prepared from the crab monkey pancreas as a template. The resulting DNA fragment was inserted into a mammalian cell expression vector, and a stable CHO expression strain (cyno. SIL-6R-producing CHO cell) was prepared using this vector. cyno. The culture of sIL-6R producing CHO cells was purified with a HisTrap column (GE Healthcare Bioscience) and then concentrated using Amicon Ultra-15 Ultracel-10k (Millipore). A final purified sample of soluble goat's monkey IL-6 receptor (hereinafter cIL-6R) was obtained through further purification on a Superdex 200 pg 16/60 gel filtration column (GE Healthcare Bioscience).

Preparation of Recombinant Crab Monkey IL-6 (cIL-6)

The crab monkey IL-6 was prepared by the following procedure. A nucleotide sequence encoding 212 amino acids registered under SWISSPROT Accession No. P79341 was prepared and cloned into a mammalian cell expression vector. The resulting vector was introduced into CHO cells to produce a stable expression cell line (cyno. IL-6 producing CHO cell). cyno. The culture of IL-6 producing CHO cells was purified using SP-Sepharose / FF column (GE Healthcare Bioscience) and then concentrated with Amicon Ultra-15 Ultracel-5k (Millipore). The final purification of crab monkey IL-6 (hereinafter referred to as cIL-6) was carried out by further purification on Superdex 75 pg 26/60 gel filtration column (GE Healthcare Bioscience) and concentration by Amicon Ultra-15 Ultracel-5k (Millipore) A sample was obtained.

Production of high affinity anti-IL-6 receptor antibody

To express the known high affinity anti-IL-6 receptor antibody, VQ8F11-21 hIgG1, a vector for mammalian cell expression was constructed. VQ8F11-21 hIgG1 is described in US 2007/0280945 A1 (US 2007/0280945 A1, H chain amino acid sequence: SEQ ID NO: 77, L chain amino acid sequence: SEQ ID NO: 78). The antibody variable region was prepared by PCR (assembly PCR) using a combination of synthetic oligo DNA and IgG1 was used for the normal region. The antibody variable region and the normal region were ligated by assembly PCR method and inserted into a mammalian expression vector to construct expression vectors of the desired H chain and L chain. The nucleotide sequence of the obtained expression vector was determined by a method known to a person skilled in the art. Expression and purification of high affinity anti-IL-6 receptor antibody (hereinafter referred to as "control") was performed using the expression vector prepared above by the method described in Example 1.

Production, expression and purification of TOCILIZUMAB mutants

The mutants of TOCILIZUMAB were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the method described in the attached manual. The resulting plasmid fragment was inserted into a mammalian cell expression vector to produce the desired H chain and L chain expression vectors. The nucleotide sequence of the obtained expression vector was determined by a method known to a person skilled in the art. Antibodies were expressed by the following method. HEK293H cell line (Invitrogen) derived from human fetal kidney cancer was suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen). The cells were seeded with 10 mL per dish of adhesive cell dish (diameter 10 cm, CORNING) at a cell density of 5 to 6 x 10 ⁵ cells / mL and cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ) The whole day was incubated overnight. The medium was aspirated off and 6.9 mL of CHO-S-SFM-II (Invitrogen) medium was added. The prepared plasmid was introduced into the cells by lipofection. The resulting culture supernatant was recovered and centrifuged (about 2000 g for 5 minutes at room temperature) to remove the cells and sterilized by filtration through a 0.22 micrometer filter MILLEX (R) -GV (Millipore) to obtain a culture supernatant. using the ^{rProtein A Sepharose TM Fast Flow (Amersham} Biosciences) and purified antibodies from the culture supernatant obtained by the technique known in the art. In order to measure the purified antibody concentration, the absorbance at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the value obtained using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

Establishment of human gp130-expressing BaF3 cell line

In order to obtain a cell line proliferating in an IL-6-dependent manner, a BaF3 cell line expressing human gp130 was established by the following procedure.

Full length human gp130 cDNA (Hibi et al., Cell 1990; 63: 1149-1157 (GenBank # NM_002184)) was amplified by PCR and cloned into the expression vector pCOS2Zeo to construct pCOS2Zeo / gp130. pCOS2Zeo is an expression vector constructed by removing the DHFR gene expression site from pCHOI (Hirata et al., FEBS Letter 1994; 356: 244-248) and inserting the Zeocin resistance gene. The full length human IL-6R cDNA was amplified by PCR and cloned with pcDNA3.1 (+) (Invitrogen) to construct hIL-6R / pcDNA3.1 (+).

10 micrograms of pCOS2Zeo / gp130 were mixed in BaF3 cells (0.8 × 10 ⁷ cells) suspended in PBS and pulsed with 0.33 kV and 950 microFD using Gene Pulser (Bio-Rad). BaF3 cells having the genes introduced by electroporation were suspended in RPMI1640 medium (Invitrogen) containing 0.2 ng / mL mouse interleukin-3 (Peprotech) and 10% Fetal Bovine Serum (FBS, HyClone) The cells were cultured and selected by adding RPMI 1640 medium containing 100 ng / mL human interleukin-6 (R & D systems), 100 ng / mL human interleukin-6 soluble receptor (R & D systems) and 10% FBS, (Hereinafter referred to as " BaF3 / gp130 "). This BaF / gp130 proliferates in the presence of human interleukin-6 (R & D systems) and soluble human IL-6 receptor and thus evaluates the proliferation inhibitory activity (or IL-6 receptor neutralizing activity) Lt; / RTI >

Evaluation of biological activity by human gp130-expressing BaF3 cells (BaF / gp130)

IL-6 / IL-6 receptor-dependent growth of BaF3 / gp130 was used to evaluate IL-6 receptor neutralizing activity. After washing three times in RPMI 1640 medium containing 10% FBS, BaF3 / gp130 cells were incubated with human interleukin-6 (TORAY) at a concentration of 600 ng / mL to 60 ng / mL at 5 x 10 ⁴ cells / ng / mL to 30 ng / mL), an appropriate amount of soluble human IL-6 receptor and 10% FBS. The cell suspension was divided into 96 well plates (CORNING) (50 microliters per well). Next, the purified antibody was diluted with RPMI1640 containing 10% FBS and added to each well (50 microliters / well). The cells were cultured for 3 days at 37 ° C under 5% CO ₂ . WST-8 reagent (Cell Counting Kit-8, Tojin Chemical Research Co., Ltd.) was diluted 2-fold with PBS. Immediately after adding 20 microliters of the reagent to each well, the absorbance at 450 nm (reference wavelength 620 nm) was measured using a SUNRISE CLASSIC (TECAN). After incubation for 2 hours, the absorbance at 450 nm (reference wavelength 620 nm) was measured again. The neutralizing activity of IL-6 receptor was assessed using the absorbance change for 2 hours as an indicator.

Evaluation of Binding to Soluble Human IL-6 Receptor by Biacore

The kinetic analysis of the antigen-antibody reaction was performed using Biacore T100 (GE Healthcare). An appropriate amount of protein A or protein A / G or anti-IgG (gamma -chain specific) F (ab ') ₂ is immobilized on the sensor chip by an amine coupling method and the desired antibody is bound on the chip at pH 7.4 , human IL-6 receptor was measured by flowing soluble IL-6 receptor prepared at various concentrations at pH 7.4 as an analyte on a chip. All measurements were performed at 37 ° C. The kinetic parameters K _a (1 / M s) and the dissociation rate constant K _d (1 / s), which are kinetic parameters, were calculated from the sensorgrams obtained from the measurements. Next, K _D (M) was calculated based on the above rate constant. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare).

Evaluation of pH-dependent dissociation of membrane-bound IL-6 receptor by Biacore

The antigen-antibody reaction with the membrane-type IL-6 receptor at pH 5.8 and pH 7.4 was observed using Biacore T100 (GE Healthcare). Binding to the membrane type IL-6 receptor was evaluated by evaluating the binding to the soluble human IL-6 receptor immobilized on the sensor chip. SR344 was biotinylated by methods known to those skilled in the art. Based on the affinity of streptavidin and biotin, a biotinylated human IL-6 receptor was immobilized on a sensor chip via streptavidin. All measurements were carried out at 37 ° C. The mobile phase buffer was 10 mM MES (pH 5.8), 150 mM NaCl, and 0.05% Tween 20. Clones expressing pH-dependent binding were coupled with soluble human IL-6 receptors by injection under conditions of pH 7.4 (10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 in buffer of the injected sample). Next, pH dependent dissociation of each clone was observed at pH 5.8, the mobile phase pH. The dissociation rate constant (K _d (1 / s)) at pH 5.8 was calculated by fitting only the dissociation phase at pH 5.8 using Biacore T100 Evaluation Software (GE Healthcare). The sample concentration was 0.25 micrograms / mL. Binding was performed in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween20 and dissociation was performed in 10 mM MES (pH 5.8), 150 mM NaCl, and 0.05% Tween20. Similarly, dissociation rate constants (K _d (1 / s)) at pH 7.4 were calculated by fitting only the dissociation phase at pH 7.4 using Biacore T100 Evaluation Software (GE Healthcare). The sample concentration was 0.5 micrograms / mL. Binding was performed in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween20 and dissociation was performed in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween20.

Evaluation of binding to human FcRn

FcRn is a complex of FcRn and beta 2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J. Exp. Med. 180 (6), 2377-2381 (1994)). As a template, a DNA fragment encoding the gene of the gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) and the prepared primer. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular region (Met1-Leu290) containing a signal region was amplified by PCR and inserted into an animal cell expression vector (human FcRn amino acid sequence / SEQ ID NO: : 79). Likewise, an oligo DNA primer was prepared based on the public beta 2-microglobulin gene sequence (Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002)). A DNA fragment encoding the gene of the gene was prepared by PCR using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template and the primer thus prepared. Using the DNA fragment obtained as a template, a DNA fragment encoding the beta 2-microglobulin total length (Met1-Met119) including the signal region was amplified by PCR and inserted into a mammalian cell expression vector (human beta 2- microglobulin amino acid sequence / SEQ ID NO: 80).

The soluble human FcRn was expressed by the following procedure. Plasmids prepared for human FcRn and beta 2-microglobulin were introduced into human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) using lipofection method using 10% FBS (Invitrogen). The resulting culture supernatant was recovered and purified by the method described in (J Immunol. 2002 Nov 1; 169 (9): 5171-80) using IgG Sepharose 6 Fast Flow (Amersham Bioscience) And further purified by HP (GE Healthcare).

Measurement of antibody plasma concentration in mice

Antibody concentrations in mouse plasma were determined by ELISA according to methods known to those skilled in the art.

PK / PD test to measure antibody concentration, CRP concentration, unconjugated and soluble IL-6 receptor in plasma in monkey

The concentration in the crab monkey plasma was measured by an ELISA method according to a method known to a person skilled in the art.

The CRP concentration was measured with an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.) using a Sears CRP (Kanto Chemical Co., Ltd.).

The unconjugated and soluble type of crabgrass monkey IL-6 receptor in the crab monkey plasma was measured by the procedure described below. A total of 30 microliters of plasma from a crab monkey were added to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 micrometer filter cup (Millipore) to remove all IgG type antibodies IL-6 Receptor Antibody and Anti-Human IL-6 Receptor Antibody-Soluble Type Crab Monkey IL-6 Receptor Complex) were adsorbed to Protein A. The solution in the cup was then spun down with a high speed centrifuge and the pass solution was withdrawn. The pass solution does not contain the anti-human IL-6 receptor antibody-soluble form of the goat's monocyte IL-6 receptor complex bound to protein A. Therefore, it is possible to measure the concentration of IL-6 receptor that is unconjugated and soluble by measuring the soluble IgE receptor IL-6 receptor concentration in the protein A pathway solution. Soluble human crab monkey IL-6 receptor concentration was measured using a method known to those skilled in the art for measuring the concentration of soluble human IL-6 receptor. The soluble human crab monkey IL-6 receptor (cIL-6R) prepared as described above was used as a standard. The IL-6 receptor ratio, which is non-binding and soluble, was calculated by the following equation.

                         SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA   <120> PHARMACEUTICAL FORMULATION CONTAINING IMPROVED ANTIBODY MOLECULES <130> C1-A0905P <150> JP 2009-069095 <151> 2009-03-19 <160> 117 <170> PatentIn version 3.4 <210> 1 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 1 His Asp His Ala Trp Ser 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 2 Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Gly 1 5 10 15 <210> 3 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 3 Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 4 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 4 His Asp His Ala Trp Ser 1 5 <210> 5 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 5 Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Gln Gly 1 5 10 15 <210> 6 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 6 Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 7 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 7 Asp Asp His Ala Val Ser 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 8 Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Asp 1 5 10 15 <210> 9 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 9 Leu Leu Ala Arg Ala Thr Ala Met Asp Val 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 10 Gln Ala Ser Arg Asp Ile Ser Ser His Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 11 Tyr Gly Ser His Leu Leu Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 12 Gly Gln Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 13 Gln Ala Ser Thr Asp Ile Ser Ser His Leu Asn 1 5 10 <210> 14 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 14 Tyr Gly Ser His Leu Leu Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 15 Gly Gln Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 16 Gln Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 17 Tyr Gly Ser Glu Leu Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 18 Gly Gln Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 19 <211> 119 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 19 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 Ser 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 Thr 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         115 <210> 20 <211> 119 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 20 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 Ser 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         115 <210> 21 <211> 119 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 21 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 His Ala Val 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 Thr Leu     50 55 60 Gln Asp 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 Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 22 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 22 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg 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 Leu Leu Ser Gly Val Ser 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             100 105 <210> 23 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 23 Asp Ile Gln Met Thr Gln Ser 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 Leu Leu Ser Gly Val Ser 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             100 105 <210> 24 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 24 Asp Ile Gln Met Thr Gln Ser 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 Glu Ser Gly Val Ser 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             100 105 <210> 25 <211> 443 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 25 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 Ser 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 Thr 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 Ser 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 Glu 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> 26 <211> 443 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 26 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 Ser 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 Ser 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 Glu 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> 27 <211> 447 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 27 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 His Ala Val 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 Thr Leu     50 55 60 Gln Asp 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 Leu Leu Ala Arg Ala Thr Ala Met Asp Val 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 Ser 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 Ser 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 Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 <210> 28 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 28 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg 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 Leu Leu Ser Gly Val Ser 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> 29 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 29 Asp Ile Gln Met Thr Gln Ser 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 Leu Leu Ser Gly Val Ser 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> 30 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 30 Asp Ile Gln Met Thr Gln Ser 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 Glu Ser Gly Val Ser 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> 31 <211> 324 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 31 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 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> 32 <211> 324 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 32 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 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> 33 <211> 328 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 33 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 Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro                 325 <210> 34 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 34 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 35 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 35 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 36 <211> 107 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 36 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 37 <211> 327 <212> DNA <213> Homo sapiens <400> 37 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttgataa 327 <210> 38 <211> 107 <212> PRT <213> Homo sapiens <400> 38 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 39 <211> 990 <212> DNA <213> Homo sapiens <400> 39 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 40 <211> 330 <212> PRT <213> Homo sapiens <400> 40 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 Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 41 <211> 984 <212> DNA <213> Homo sapiens <400> 41 gctagcacca agggcccatc ggtcttcccc ctggcgccct cctccaagag cacctccgag 60 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300 aaatcttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540 gtggtcagcg tcctcaccgt cgtgcaccag gactggctga acggcaagga gtacaagtgc 600 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 960 tccctgtctc cgggtaaatg ataa 984 <210> 42 <211> 326 <212> PRT <213> Homo sapiens <400> 42 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 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 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 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 Gly Lys                 325 <210> 43 <211> 995 <212> DNA <213> Homo sapiens <400> 43 gctagcacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300 aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 360 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960 ctctccctgt ctctgggtta atgataagcg gccgc 995 <210> 44 <211> 326 <212> PRT <213> Homo sapiens <400> 44 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 Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly                 325 <210> 45 <211> 4 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 45 Gly Gly Gly Ser One <210> 46 <211> 4 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 46 Ser Gly Gly Gly One <210> 47 <211> 5 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 47 Gly Gly Gly Gly Ser 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 48 Ser Gly Gly Gly Gly 1 5 <210> 49 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 49 Gly Gly Gly Gly Gly Ser 1 5 <210> 50 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 50 Ser Gly Gly Gly Gly Gly 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 51 Gly Gly Gly Gly Gly Gly Ser 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 52 Ser Gly Gly Gly 1 5 <210> 53 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Ser 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> 54 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 54 Asp Ile Gln Met Thr Gln Ser 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 Ser 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> 55 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 55 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Val Leu Ala Arg Ile 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 Ser 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 Ser 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> 56 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 56 Asp Ile Gln Met Thr Gln Ser 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 Ser 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 Gly Gln Gly Asn Arg 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> 57 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 57 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 His 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 Ser 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 Ser 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> 58 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 58 Asp Ile Gln Met Thr Gln Ser 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 Ser 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> 59 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 59 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 60 Tyr Gly Ser Glu Leu His Ser 1 5 <210> 61 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 61 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ser Ser Gly Tyr Ser Ile Thr             20 25 30 <210> 62 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 62 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             20 25 30 <210> 63 <211> 32 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 63 Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg             20 25 30 <210> 64 <211> 32 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 64 Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg             20 25 30 <210> 65 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 65 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             20 25 30 <210> 66 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 66 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 Ser Ser His Asp             20 25 30 His Ala His 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 Ser 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 Ser 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> 67 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 67 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln His 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 His Ser Gly Val Ser 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> 68 <211> 448 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 68 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Ser 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 <210> 69 <211> 447 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 69 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Ser 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> 70 <211> 445 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 70 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Glu Ser 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 Ser 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 His 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 Glu 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 Arg 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 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 71 <211> 445 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 71 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Ser 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 Cys 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 His 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 Glu 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 Arg 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 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 72 <211> 443 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 72 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Glu 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 Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 <210> 73 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Ser 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 Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly         435 440 445 Lys      <210> 74 <211> 447 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 74 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Ser 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 Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 <210> 75 <211> 443 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 75 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser 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 Ser 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 Glu 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> 76 <211> 449 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 76 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 His Ala Val 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 Thr Leu     50 55 60 Gln Asp 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 Leu Leu Ala Arg Ala Thr Ala Met Asp Val 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 Ser 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 Ser 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> 77 <211> 446 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 77 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Phe Thr Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Lys Gly Arg Asp Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr     210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser     290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 78 <211> 214 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 78 Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu 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> 79 <211> 267 <212> PRT <213> Homo sapiens <400> 79 Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 1 5 10 15 Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro             20 25 30 Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys         35 40 45 Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu     50 55 60 Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80 Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys                 85 90 95 Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu             100 105 110 Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly         115 120 125 Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln     130 135 140 Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160 His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp                 165 170 175 Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly             180 185 190 Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu         195 200 205 Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly     210 215 220 Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240 Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His                 245 250 255 Ala Gly Leu Ala Gln Pro             260 265 <210> 80 <211> 99 <212> PRT <213> Homo sapiens <400> 80 Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu 1 5 10 15 Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro             20 25 30 Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys         35 40 45 Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu     50 55 60 Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80 Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp                 85 90 95 Arg Asp Met              <210> 81 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 81 Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 82 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 82 Phe Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 83 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 83 Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 84 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 84 Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 85 Leu Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 86 Ser Leu Ala Arg Ala Thr Ala Met Asp Tyr 1 5 10 <210> 87 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 87 Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 88 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 88 Arg Ala Ser Thr Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 89 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 89 Arg Ala Ser Arg Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 90 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 90 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 91 Gly Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 92 Gln Gln Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 93 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 93 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ser Ser Gly Tyr Ser Ile Thr             20 25 30 <210> 94 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 94 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             20 25 30 <210> 95 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 95 Ser Asp His Ala Trp Ser 1 5 <210> 96 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 96 Asp Asp His Ala Trp Ser 1 5 <210> 97 <211> 14 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 97 Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly 1 5 10 <210> 98 <211> 14 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 98 Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile Gly 1 5 10 <210> 99 <211> 16 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 99 Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Gln Asp 1 5 10 15 <210> 100 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 100 Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser 1 5 10 <210> 101 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 101 Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 102 <211> 23 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 102 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys             20 <210> 103 <211> 23 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 103 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys             20 <210> 104 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 104 Gln Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 105 <211> 15 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 105 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 106 <211> 15 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 106 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile Tyr 1 5 10 15 <210> 107 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 107 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 108 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 108 Tyr Thr Ser Glu Leu Glu Ser 1 5 <210> 109 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 109 Tyr Thr Ser Arg Leu Leu Ser 1 5 <210> 110 <211> 32 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 110 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys             20 25 30 <210> 111 <211> 32 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 111 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys             20 25 30 <210> 112 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 112 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 113 <211> 10 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 113 Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 1 5 10 <210> 114 <211> 30 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 114 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly His Ser Ile Thr             20 25 30 <210> 115 <211> 6 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 115 His Asp His Ala Trp Ser 1 5 <210> 116 <211> 11 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 116 Arg Ala Ser Gln Asp Ile Ser Ser His Leu Asn 1 5 10 <210> 117 <211> 7 <212> PRT <213> Artificial <220> An artificially synthesized polypeptide sequence <400> 117 Tyr Thr Ser His Leu His Ser 1 5

Claims (21)

(SLE), lupus nephritis, Crohn's disease, chronic myelogenous leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, Aplastic anemia, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), IgA nephropathy, deformed arthropathy, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney Cancer, B-cell non-Hodgkin's, pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve infiltration, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, Sex dermatitis , Atopic dermatitis, mesothelioma, multiple myositis, dermatomyositis, uveitis, whole uveitis, middle uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.
(Iv) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73), and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);
(V) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73), and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);
(Vi) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83), and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1); And
(Viii) An antibody comprising a heavy chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5). 3. The stable pharmaceutical formulation according to claim 1 comprising at least one buffer selected from histidine and citrate buffer. 3. The stable pharmaceutical preparation according to claim 1 or 2, comprising at least one cationic amino acid. 4. The pharmaceutical composition according to any one of claims 1 to 3, which comprises 1-500 mM of at least one buffer selected from histidine and citrate buffer, 1-1500 mM of at least one cationic amino acid, 1-200 mg / mL of antibody, and 1- 400 mM. &Lt; / RTI &gt; The formulation according to claim 3 or 4, wherein the cationic amino acid is arginine. 5. The preparation according to claim 4, wherein the carbohydrate is sucrose or trehalose. 7. The formulation according to any one of claims 1 to 6, further comprising a surfactant. 8. The preparation according to any one of claims 1 to 7, wherein the preparation contains at least one selected from a polypeptide and an antibody in an amount of 10 mg / ml or more. 9. The preparation according to any one of claims 1 to 8, wherein the preparation contains at least one selected from the group consisting of a polypeptide and an antibody in a content of 50 mg / ml or more. 10. The preparation according to any one of claims 1 to 9, wherein the preparation contains at least 80 mg / ml of at least one selected from a polypeptide and an antibody. 11. The preparation according to any one of claims 1 to 10, which contains at least one selected from the group consisting of a polypeptide and an antibody in a content of not more than 240 mg / ml. 12. The formulation according to any one of claims 1 to 11 having a pH in the range of 4.5 to 7.0. 13. The formulation of claim 12 having a pH in the range of 5.5 to 6.6. 14. The formulation according to any one of claims 1 to 13, wherein the formulation is a liquid. 15. The formulation according to claim 14, which has not been applied to freeze drying during preparation of the formulation. 16. The formulation according to any one of claims 1 to 15, wherein at least one dimerization selected from a polypeptide and an antibody molecule is reduced. 17. The formulation according to any one of claims 1 to 16, wherein at least one dimerization selected from a polypeptide and an antibody molecule is inhibited. 18. The formulation according to any one of claims 1 to 17 for subcutaneous administration. A method for stabilizing a solution containing an antibody selected from at least one of the following (iv) to (vii), comprising adding at least one cationic amino acid.
(Iv) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);
(V) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);
(Vi) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83), and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1); And
(Viii) An antibody comprising a heavy chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5). A method for stabilizing an antibody during a cooling / thaw cycle of a solution containing an antibody selected from one or more of the following (iv) to (vii), comprising adding one or more cationic amino acids.
(Iv) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 20 (the variable region of VH3-M73) and a light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL3);
(V) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 (the variable region of VH4-M73) and a light chain variable region having the sequence of SEQ ID NO: 22 (variable region of VL1);
(Vi) an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 21 (the variable region of VH5-M83), and a light chain variable region having the sequence of SEQ ID NO: 24 (variable region of VL5);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain having the sequence of SEQ ID NO: 29 (VL3);
(Viii) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain having the sequence of SEQ ID NO: 28 (VL1); And
(Viii) An antibody comprising a heavy chain having the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain having the sequence of SEQ ID NO: 30 (VL5). delete

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