201100100 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種含有多胜肽及/或抗體之配方，特別 是關於一種含有高濃度之經修飾的抗IL6受體抗體的穩定 液體配方。 【先前技術】 近年來’已有各種抗體配方被開發出來且實用化。多 種配方用於靜脈内注射。由於每次投予的抗體量大（約 l〇〇mg~200mg) ’且皮下注射的體積通常受限，因此，設計 用於皮下注射用的含抗體配方需要增加液體中的抗體濃 度。 含有咼濃度抗體的溶液中會發生不欲的崩解，此崩解 包括了形成不溶及/或可溶的凝集物。此等不溶及可溶的凝 集物由於與抗體分子結合，而可能形成於液體狀態中。當 配方液體長時間儲存，抗體分子的生物活性可能由於天I 醢胺（asparglne)殘基的去醯胺化（deamidati〇n)而喪失或 降低。冷凍及解凍循環也會形成崩解及凝集的抗體分子。 已有許多針對提供穩定化配方的方案被提出，其中， 即使：該配方長時間貯存，有效成分的喪失會降低。此種 配方係藉由將-有效成分與各種添加劑溶解於緩衝溶液中 而獲％•目A對於提供抑制長時間儲存中二聚體化及去酿 化、及穩定及適合皮下投予的含高濃度抗體之配方仍有需 求。 抗體由於在血漿中高度穩定且副作用少，作為醫藥品 201100100 ^ 受到注重。其中，已有多種IgG型抗體醫藥品可在市場上 取得，目前許多抗體醫藥品正在開發當中（Janice Μ Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz， Monoclonal antibody successes in the clinic, Nature Biotechnology 23, 1073-1078(2005) and Pavlou AK, Belsey MJ. , The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59(3)·· 389-96)。IL-6為一種細胞激素，涉及多種自體免 ® 疫疾病、發炎性疾病、惡性腫瘤等（Nishimoto N，Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2 (11 ) : 61 9-26 ) ° TOC ILIZUMAB 為一種人型化抗IL-6受體IgGl抗體，專一性地連接於IL-6 受體。TOC ILIZUMAB會中和IL-6的生物活性，因此被認為 可作為IL - 6相關疾病性的治療劑，例如：類風濕性關節炎 (W0 92/19759， W0 96/11020， WO96/12503，及 Maini RN， q Taylor PC, Szechinski J, Pavelka K, Broil 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)。在日本，TOCILIZUMAB 已被證實作為卡斯托曼病（Cast 1 eman di sease)及類風濕 5 201100100 性關節炎的治療劑（NishimotoN，KanakuraY, AozasaK， Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, NishiokaK, Hara M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S, Kodaraa F, Nakahara H, Hagihara K， Yosh i zak i K, K i sh i motο T. Human i zed anti-inter1eukin-6 receptor antibody treatment of multicentric Castleman disease. Blood. 2005 Oct 15;106(8):2627-32)。 人型化抗體’例如TOCILIZUMAB為第一代抗體藥品。 為了改良第一代抗體藥品的效力、便利性及成本，目前正 開發第二代抗體藥品。多種可應用於第二代抗體藥品的技 術正在開發。已有報導增強效應子（e f f e c t 〇 r )功能、抗原 結合能力、藥動學、及穩定性的技術，以及減少免疫原性 風險的技術。作為增強藥物效力或減低劑量的方法，已報 導藉由IgG抗體的Fc區域的胺基酸取代，增強抗體依存性 細胞媒介的細胞毒性活性（ADCC活性）、或補體依存性細胞 毒性活性（CDC活性）的技術（Kim SJ, Park Y，Hong HJ.， Antibody engineering for the development of therapeutic antibodies. , Mol Cells. 2005 Aug 31 ; 20( 1 ):17-29. Review)。再者，親和性成熟化（Affinity maturation)據報告作為用於增強抗原結合能力或抗原中 和能力的技術（Rothe A，H〇sse RJ，Powei· be. Ribosome display for improved biotherapeutic molecules.201100100 VI. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a formulation containing a multi-peptide and/or antibody, and more particularly to a stable liquid formulation containing a high concentration of a modified anti-IL6 receptor antibody. [Prior Art] In recent years, various antibody formulations have been developed and put into practical use. A variety of formulations are used for intravenous injection. Since the amount of antibody administered per administration is large (about 1 mg to 200 mg) and the volume of subcutaneous injection is usually limited, the antibody-containing formulation designed for subcutaneous injection needs to increase the concentration of the antibody in the liquid. Undesirable disintegration occurs in solutions containing hydrazine concentrations, which include the formation of insoluble and/or soluble agglomerates. These insoluble and soluble agglomerates may form in a liquid state due to binding to antibody molecules. When the formulation liquid is stored for a long period of time, the biological activity of the antibody molecule may be lost or reduced due to deamidatination of the asparglne residue. The freezing and thawing cycles also form disintegrating and agglutinating antibody molecules. A number of proposals have been made for providing a stabilizing formulation in which, even if the formulation is stored for a long period of time, the loss of active ingredient is reduced. This formulation is obtained by dissolving the active ingredient and various additives in a buffer solution to provide a high concentration for inhibiting dimerization and de-growth in long-term storage, and for stable and suitable subcutaneous administration. There is still a need for a formulation of concentrated antibodies. The antibody is highly stable in plasma and has few side effects, and is considered as a pharmaceutical product. Among them, a variety of IgG-type antibody drugs are available on the market, and many antibody drugs are currently under development (Janice Μ Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnology 23, 1073-1078 (2005) and Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59(3)·· 389-96). IL-6 is a cytokine involved in a variety of autoimmune diseases, inflammatory diseases, malignant tumors, etc. (Nishimoto N, Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2 (11) : 61 9-26 ) ° TOC ILIZUMAB is a humanized anti-IL-6 receptor IgG1 antibody that is specifically linked to the IL-6 receptor. TOC ILIZUMAB neutralizes the biological activity of IL-6 and is therefore considered to be a therapeutic agent for IL-6-related diseases, such as rheumatoid arthritis (W0 92/19759, W0 96/11020, WO 96/12503, and Maini RN, q Taylor PC, Szechinski J, Pavelka K, Broil 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). In Japan, TOCILIZUMAB has been confirmed as a therapeutic agent for Cast 1 eman di sease and rheumatoid 5 201100100 arthritis (Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, NishiokaK, Hara M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S, Kodaraa F, Nakahara H, Hagihara K, Yosh i zak i K, K i sh i motο T. Human i Zed anti-inter1eukin-6 receptor antibody treatment of multicentric Castleman disease. Blood. 2005 Oct 15;106(8):2627-32). Humanized antibodies such as TOCILIZUMAB are first generation antibody drugs. In order to improve the efficacy, convenience and cost of first-generation antibody drugs, second-generation antibody drugs are currently being developed. A variety of techniques are available for the development of second-generation antibody drugs. Techniques for enhancing the effector (e f f e c t 〇 r ) function, antigen binding ability, pharmacokinetics, and stability, and techniques for reducing the risk of immunogenicity have been reported. As a method for enhancing drug potency or reducing dose, it has been reported to enhance antibody-dependent cellular cytotoxic activity (ADCC activity) or complement-dependent cytotoxic activity (CDC activity) by amino acid substitution of the Fc region of an IgG antibody. (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review). Furthermore, Affinity maturation has been reported as a technique for enhancing antigen binding ability or antigen neutralization ability (Rothe A, H〇sse RJ, Powei·be. Ribosome display for improved biotherapeutic molecules.
Expert Opin Biol Ther. 2006 Feb; 6(2):1 77-87)。此技 201100100 術能藉由導入胺基酸突變於變異區等之互補決定（CDR) 區，使增強抗原結合活性。此抗原結合能力之增強，改良 體外生物學活性或減少劑量，且因此改良體内的效力 (Rajpal A, Beyaz N, Haber L, Cappucci11i G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries·, Proc Natl Acad Sci USA. 2005 Jun 14; 1 02(24):8466-71. Epub 2005 Jun 6)。現在，Motavizumab(由親和性成熟化生產） 正在進行臨床試驗，期待其比起第一代抗RSV抗體醫藥 品，Palivizumab 有更優越的效果（WuH，PfarrDS, 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 ❹ an(1 Lower Respiratory Tract· J Mol Biol. 2007，368, 652-665)。已有人報告具有親和性約為〇 〇5nM的一種抗 IL-6受體抗體（即，較TOCILIZUMAB具有更大的親和性）（w〇 2007/143168)。然而，並未有人報告敘述具有高於〇. 〇5nM 之親和性的人類抗體、人型化抗體或嵌合抗體。 目前的抗體醫藥品面臨的一個問題在於，伴隨著投予 極大量的蛋白質所帶來的高生產成本。比如， TOCILIZUMAB，一人型化抗IL_6受體IgG1抗體，以靜脈内 注射據估計劑量為約8mg/kg/月（Maini RN，Tayl〇『pc， 7 201100100Expert Opin Biol Ther. 2006 Feb; 6(2): 1 77-87). This technique 201100100 enhances antigen binding activity by introducing an amino acid mutation to the complementarity determining (CDR) region of the variant region or the like. This enhanced antigen binding capacity improves in vitro biological activity or reduces dosage, and thus improves efficacy in vivo (Rajpal A, Beyaz N, Haber L, Cappucci 11i G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R A general method for greatly improving the affinity of antibodies by using combinatorial libraries·, Proc Natl Acad Sci USA. 2005 Jun 14; 1 02(24): 8466-71. Epub 2005 Jun 6). Now, Motavizumab (produced by affinity maturation) is undergoing clinical trials, and it is expected to have superior effects compared to the first generation of anti-RSV antibody drugs, Palivizumab (WuH, PfarrDS, 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 ❹ an (1 Lower Respiratory Tract· J Mol Biol. 2007,368, 652-665 An anti-IL-6 receptor antibody having an affinity of about 5 nM has been reported (i.e., has greater affinity than TOCILIZUMAB) (w〇2007/143168). However, no report has been reported. A human antibody, a humanized antibody, or a chimeric antibody that is higher than 〇. 〇 5 nM. A problem faced by current antibody pharmaceuticals is the high production cost associated with the administration of extremely large amounts of protein. , TOCILIZUMAB, a humanized anti-IL_6 receptor IgG1 antibody, administered intravenously at an estimated dose of about 8 mg/kg/month (Maini RN, Tayl〇『pc, 7 201100100
Szechinski J, Pavelka K, Broil 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. Toe i1i zumab, In European patients with rheumatoid arthritis who had an incomplete response to methotrexate., Arthritis Rheum. 2006 Sep; 54(9):281 7-29)。於慢性自體免疫疾 病’較佳的投予形式為皮下配方。一般而言，皮下配方需 要為高濃度配方。從穩定性等的角度，I 型抗體配方的 極限’一般為約 l〇〇mg/inl (Shire SJ，Shahrokh Z，Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93(6):1390-402)。可藉由增加抗體在血漿中的半衰期以延 長其藥效、賦予此抗體高穩定性’提供能以皮下投予、長 間隔投予的低成本、便利的第二代抗體醫藥品，藉此減少 投予的蛋白質量。Szechinski J, Pavelka K, Broil 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. Toe i1i zumab, In European patients with rheumatoid arthritis who had an incomplete response to methotrexate., Arthritis Rheum. 2006 Sep; 54(9):281 7-29). A preferred form of administration for chronic autoimmune disease is a subcutaneous formulation. In general, subcutaneous formulations require a high concentration formulation. From the standpoint of stability and the like, the limit of the type I antibody formulation is generally about 10 mg/inl (Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93(6): 1390-402). By increasing the half-life of the antibody in plasma to prolong its efficacy and confer high stability to the antibody, it provides a low-cost, convenient second-generation antibody drug that can be administered subcutaneously or at long intervals, thereby reducing The amount of protein administered.
FcRn與抗體藥動學密切相關。關於同型抗體的血漿中 半衰期差異，已知IgGl及lgG2比起IgG3及IgG4具有優 越的血製中半衰期（Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25 (12):1369-72)。作為更進一步改良具有優越血漿半衰期 的I gG 1及I gG2抗體的方法’已知有以恆定區的胺基酸置 換’增強對 FcRn 結合的方法（Hinton PR, Xi〇ng jm，J〇hlfs 201100100FcRn is closely related to antibody pharmacokinetics. Regarding the difference in plasma half-life of isotype antibodies, it is known that IgG1 and lgG2 have superior mid-life half-life compared to IgG3 and IgG4 (Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25 (12): 1369-72 ). As a method of further improving the I gG 1 and I gG2 antibodies having superior plasma half-life, it is known to replace the amino acid with a constant region to enhance the binding of FcRn (Hinton PR, Xi〇ng jm, J〇hlfs 201100100).
MG, Tang MT, Keller S, Tsurshita N,m An engineered human IgGl anti body with longer serum half-life., JMG, Tang MT, Keller S, Tsurshita N, m An engineered human IgGl anti body with longer serum half-life., J
Immunol. 2006 Jan 1; 176(1 ):346-56 and Ghetie V，Popov S, Borvak J, RaduC, 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)。從免疫原性的觀點，相較於恆定區， 將可變區的胺基酸取代，可進一步改良血漿半衰期（W〇 2007/114319)。然而’至今為止，尚沒有報告經由改變可 變區來改良IL-6受體抗體的血漿半衰期之方法。 另一項在開發生物醫藥品時遭遇的重要問題為免疫原 性。一般而言，小鼠抗體的免疫原性，藉由抗體人型化會 減低。據推測’免疫原性風險可藉由使用生殖細胞系 (germl ine)框架序列作為抗體人型化中的模板而進一步減 低（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)。然而，即便是人類抗TNF抗體， Adalimumab，仍顯示高頻率（13%〜17%)的免疫原性，而且在 顯現免疫原性的病患中，治療效果被發現下降（Bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wolbink GJ. Clinical response to ada1i mumab： The relationship with anti-adalimumab antibodies and serum adalirauraab 9 201100100 concentration in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; [Epub ahead of print] and Bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B. Immunogeneci ty, efficacy and adverse events of adalimuraab in RA patients. Rheumatol Int. 2007 Jan; 27(3):269-74)。即便是人類抗體的CDR，也可能存在T細 胞抗原決定位，且在CDR的T細胞抗原決定位為免疫原性 的可能原因。已知預測T細胞抗原決定位的電腦模擬（i n 8土11(20)及體内（111¥1¥0)方法（¥311¥&116 1，6&115 6111&115¥， Parren PW, Stas P, Lasters I. Immunogenicity screening in protein drug development. Expert Opin Biol Ther. 2007 Mar; 7(3):405-18 and Jones TD, Philips 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 Cl domain of factor VIII. J Thromb Haemost. 2005 May; 3 ( 5 ) : 9 91 -1 0 0 0 )。據推測’免疫原性風險可藉由使用此等 方法移除T細胞抗原決定位而減低（Chirino AJ，Ary ML， Msrshsll SA. Minimizing th6 immunogenicity of protein therapeutics. Drug Discov Today. 2004 Jan 15; 9(2):82-90)。 人型化抗IL-6受體igGl抗體’ TOCILIZUMAB，為人型 化小鼠抗體PM1而得到的IgGl抗體。CDR的移植係使用人 類NEW及RE I序列分別作為Η及L鏈的模板框架而進行。 10 201100100 然而’為了維持活性’有5個小鼠序列胺基酸保留在框架 中作為必要胺基酸（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)。先前並無報告將在人型化抗體 TOC ILIZUMAB框架中的剩餘小鼠序列全部人型化而不減低 活性者。再者，T0CILIZUMAB的CDR序列為小鼠序列，因 η 此’如同Adal lmumab，在CDR可能具有Τ細胞抗原決定位， 而可能會有潛在的免疫原性風險。於T0CILIZUMAB的臨床 試驗中，有效劑量8mg/kg中未檢測到抗T0CILIZUMAB抗 體，但是，在2mg/kg及4mg/kg的劑量有觀察到（w〇 20 04/09 62 73)。此顯示關於T0CILIZUMAB之免疫原性仍有 改良的餘地。然而，並無藉由胺基酸置換來減低 TOCILIZUMAB免疫原性風險的報告。 ❹ T0CILIZUMAB同型物（i s〇type)為IgGl。此同型物的差 異’係指恆定區序列的不同。由於恆定區序列被推測對於 效應子功能、藥物動力、物理性質等有強烈的影響，因此， 在開發抗體醫藥品時，選擇恆定區序列十分重要（Salfeld JG. Isotype selection in antibody engineering. NatImmunol. 2006 Jan 1; 176(1):346-56 and Ghetie V, Popov S, Borvak J, RaduC, 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). From the viewpoint of immunogenicity, the substitution of the amino acid of the variable region compared to the constant region can further improve the plasma half-life (W〇 2007/114319). However, to date, no method for improving the plasma half-life of IL-6 receptor antibodies by altering the variable region has been reported. Another important issue encountered in the development of biopharmaceuticals is immunogenicity. In general, the immunogenicity of mouse antibodies is reduced by the humanization of antibodies. It is speculated that the risk of immunogenicity can be further reduced by using the germin ine framework sequence as a template for antibody humanization (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). However, even human anti-TNF antibodies, Adalimumab, showed high frequency (13% to 17%) immunogenicity, and the therapeutic effect was found to be reduced in patients with immunogenicity (Bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wolbink GJ. Clinical response to ada1i mumab: The relationship with anti-adalimumab antibodies and serum adalirauraab 9 201100100 concentration in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; [Epub ahead of print] and Bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B. Immunogeneci ty, efficacy and adverse events of adalimuraab in RA patients. Rheumatol Int. 2007 Jan; 27(3) :269-74). Even the CDRs of human antibodies may have T cell epitopes and a possible cause of immunogenicity in the T cell epitope of the CDRs. Computer simulation of predicting T cell epitopes (in 8 soil 11 (20) and in vivo (111 ¥ 1 ¥0) methods (¥311¥&116 1,6&115 6111&115¥, Parren PW, Stas P, Lasters I. Immunogenicity screening in protein drug development. Expert Opin Biol Ther. 2007 Mar; 7(3):405-18 and Jones TD, Philips 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 Cl domain of factor VIII. J Thromb Haemost. 2005 May; 3 ( 5 ) : 9 91 -1 0 0 0 ). Presumed to be 'immunogenic risk Can be reduced by using these methods to remove T cell epitopes (Chirino AJ, Ary ML, Msrshsll SA. Minimizing th6 immunogenicity of protein therapeutics. Drug Discov Today. 2004 Jan 15; 9(2): 82-90) The humanized anti-IL-6 receptor igG1 antibody 'TOCILIZUMAB, an IgG1 antibody obtained by humanized mouse antibody PM1. The CDR grafting uses human NEW and RE I sequences as template frameworks for Η and L chains, respectively. 10 201100100 However, there are 5 in order to maintain activity. The mouse sequence amino acid remains in the framework as an essential amino acid (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). There have been no previous reports of humanization of the remaining mouse sequences in the humanized antibody TOC ILIZUMAB framework without reducing the activity. Furthermore, the CDR sequence of TOCIILZUMAB is a mouse sequence, and since η is like Adal lmumab, it may have a sputum cell epitope in the CDR, and there may be a potential immunogenic risk. In the clinical trial of T0CILIZUMAB, no anti-T0CILIZUMAB antibody was detected at an effective dose of 8 mg/kg, but was observed at doses of 2 mg/kg and 4 mg/kg (w〇 20 04/09 62 73). This shows that there is still room for improvement regarding the immunogenicity of TOCILIZUMAB. However, there is no report of reducing the risk of TOCILIZUMAB immunogenicity by amino acid replacement. ❹ T0CILIZUMAB isoform (i s〇type) is IgGl. The difference in this isoform refers to the difference in the sequence of the constant region. Since the constant region sequence is presumed to have a strong influence on effector function, drug kinetics, physical properties, etc., it is important to select a constant region sequence when developing antibody drugs (Salfeld JG. Isotype selection in antibody engineering. Nat)
Biotechnol· 2007 Dec; 25( 1 2):1 369-72)。近年來，抗體 醫藥品的安全性變得很重要。抗體Fc部分與Fc 7受體的 交互作用（效應子功能），可能會在TGN1412第j期臨床試Biotechnol· 2007 Dec; 25(1 2): 1 369-72). In recent years, the safety of antibody drugs has become important. The interaction of the Fc portion of the antibody with the Fc 7 receptor (effector function) may be in the phase J clinical trial of TGN1412
驗中造成嚴重的負效果（Strand V，Kimberly R，IsuesJD 11 201100100Serious negative effects in the test (Strand V, Kimberly R, IsuesJD 11 201100100
Biologic therapies in rheumatology： lessons learned future directions. Nat Rev Drug Discov. 2007 Jan; 6 ( 1 ). 7 5 9 2)針對5又计為中和抗原生物活性的抗體醫藥品 而言，對於效應子功能例如ADCC重要的結合至Fc 7受 體’並不需要。從負效果的觀點來看，結合至Fc T受體 甚至是不宜的。減少結合至Fcr受體的方法，係將igG 抗體的同型物從igG1改變為IgG2或IgG4 (Gessner je，Biologic therapies in rheumatology: lessons learned future directions. Nat Rev Drug Discov. 2007 Jan; 6 ( 1 ). 7 5 9 2) For antibody pharmaceuticals that are considered to be neutralizing antigen bioactivity, for effector functions such as The important binding of ADCC to the Fc 7 receptor is not required. From a negative effect point of view, binding to the Fc T receptor is even unfavorable. The method of reducing binding to the Fcr receptor is to change the isotype of the igG antibody from igG1 to IgG2 or IgG4 (Gessner je,
Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1 998 Jun; 76(6):231-48)。從藥 物動力學及Fct受體結合的觀點，IgG2比IgG4更適合 (Salfeld JG. Isotyper selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25(12):1369-72)。1'0(：1112_八6為一況-6受體的中和抗 體，其同型物為IgGl。因此，從潛在的不利效果的觀點，Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1 998 Jun; 76(6): 231-248). IgG2 is more suitable than IgG4 from the viewpoints of pharmacokinetics and Fct receptor binding (Salfeld JG. Isotyper selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25(12): 1369-72). 1'0 (:1112_8) is a neutralizing antibody of the -6 receptor, and its isoform is IgGl. Therefore, from the viewpoint of potential adverse effects,
IgG2可能為較佳的同型物，因為不需要如ADCC的效應子 功能。 同時’當開發抗體醫藥品時，蛋白質的物化性質，尤 其同質性（homogeneity)及穩定性非常關鍵。已知igG2同 型物具有由鼓鏈區的雙硫鍵衍生出的顯著異質性（DiHon TM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, Plant M, Henkle B, Li Y, Deechongkit S, Varnum B, Wypych J, Balland A, Bondarenko PV. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008 Jun 12 201100100 ' 6;283(23):16206—1 5)。欲在生產之間維持衍生自雙硫鍵的 目標物質/相關物質關聯的異質性，大規模製造為醫藥品並 不容易且可能更花費成本。因此，希望儘可能為單一物質。 再者’針對抗體之Η鏈C端序列的異質性，已有刪除c端 胺基酸離胺酸殘基，以及藉由刪除兩c端甘胺酸及離胺酸 之胺基酸的醯胺化C端羧基之報導（J〇hns〇n KA，IgG2 may be a preferred isoform because effector functions such as ADCC are not required. At the same time, when developing antibody pharmaceuticals, the physicochemical properties of proteins, especially their homogeneity and stability, are critical. The igG2 isoform is known to have significant heterogeneity derived from the disulfide bond of the drum chain region (DiHonTM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, Plant M, Henkle B, Li Y, Deechongkit S , Varnum B, Wypych J, Balland A, Bondarenko PV. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008 Jun 12 201100100 '6;283(23):16206-1 5). To maintain the heterogeneity associated with the target substance/related substance derived from the disulfide bond between productions, mass production into a pharmaceutical product is not easy and may be more costly. Therefore, it is desirable to be as single substance as possible. Furthermore, 'the heterogeneity of the C-terminal sequence of the Η chain of the antibody has been deleted, and the c-terminal amino acid lysine residue has been deleted, and the guanamine which removes the two c-terminal glycine and the amino acid of the lysine Report on the C-terminal carboxyl group (J〇hns〇n KA,
Paisley-Flango K, Tangarone BS, Porter TJ, R〇Use JC.Paisley-Flango K, Tangarone BS, Porter TJ, R〇Use JC.
Cation exchange-HPLC and mass spectrometry reveal f) C-terminal amidation of an IgGl heavy chain. Anal 81沉以!11.2007 以111;360( 1 ):75-83)。於開發12(52同型 物抗體為醫藥品時，較佳為減低此種異質性且維持高穩定 性。為了生產便利的、穩定的、高濃度、皮下投予的配方， 較佳為不僅穩定性高，而且血漿中半衰期優於T〇CIUZUMAB 之同型物IgGl者。然而，並無人報導具有IgG2同型物恆 定區之抗體的橫定區序列，與具有IgG1同型物恆定區之抗 Q 體相比，具有降低的異質性、高穩定性及優越的血漿半衰 期。 【發明内容】 本發明之一目的在於提供一種醫藥配方（以下也可稱 為「藥劑」或「醫藥組合物」，包含優於人型化抗^^受 體UG1抗體T0CILIZUMAB的第二代分子，藉由改變 T0CILIZUMAB的可變區及恆定區的胺基酸序列，以增強抗 原中和能力以及改良藥物動力，使得能以較低投予頻率而 展現延長的治療效果，且改良免疫原性、安全性以及物理 13 201100100 化學性質（穩定性及同質性）。 再者’本發明之配方提供含有高濃度之多胜肽及/或抗 體之配方’在長時間保存或多次冷凌/解束循環期間，該多 胜肽及/或抗體的二聚體化及去醯胺化受到抑制，及該配方 為穩疋且適於用在皮下投予。 已有密集的研究以解決上述問題，且已發現可藉由添 加胺基酸精胺酸或其鹽於溶液中作為穩定劑，可得到穩 定、含高濃度多胜肽/含抗體之配方。 …以下將詳述本發明。研究著重在獲得一種醫藥配方， 以第二代分子觀點來看為穩定的，第二代分子優於第一代 人型化抗1L —6受體1gG1抗體T0CILIZUMAB。關於所關注 的抗體的結果’本案發明人等發現到：於而UZUMAB的可 變區的多* CDR冑變’會改良對抗原的結合能力（親和 性）。本案發明人等因此藉由使用此等突變的組合，而成功 良親和14。本案發明人等亦藉由導入降低可變區序列 之等電點的修飾，而成功地改善藥物動力學。本案發明人 等亦藉由使仔對於IL-6受體抗原的結合為依存性，而 成功地改良藥物動力學’使得單一抗體分子可以中和多倍 的抗原。再者，他們藉由將殘留在t〇cilizumab框架中的 小既來源的序列完全地人型化’而且減少於電腦模擬預測 到的在可變區中的T細胞抗原決定位胜狀的數目’成功地 降低免疫原性風險。^ 再者’本案發明人等尚成功地發現針 對T0CILIZUMAB之值定區的新穎恒定區序列，相較於 IgG卜其對於Fe r受體之結合減低，可改良安全性 14 201100100 •s 比起IgGl ’藥物動力學改善’且減少因為igG2的鉸鏈區 雙硫鍵造成的異質性，及因為Η鏈C端造成的異質性，而 不減損穩定性。本案發明人等成功地藉由適當地組合CDR、 可變區及恆定區中的此等胺基酸序列改變，製造優於 T0CILIZUMAB的第二代分子。 本發明關於一種醫藥配方，包含一人型化抗IL-6受體 IgG抗體，其具有優越的抗原（il-6受體）結合能力、優越 的藥物動力學、優越的安全性以及物理性質（穩定性及同質 性）’且進一步’能藉由改變人型化抗IL-6受體IgGl抗體 TOC ILIZUMAB的可變區及恆定區的胺基酸序列，而減少免 疫原性；並關於用於製造此等醫藥組合物的方法。更詳言 之，本發明提供如下： (1) 一種醫藥組合物，包含至少一種選自以下的多胜 肽： (a) —多胜肽，包含CDR1、CDR2、CDR3，該CDR1包含 q 序列識別號1 (VH4-M73之CDR1)、該CDR2包含序列識別 號2 (VH4-M73之CDR2)，及該CDR3包含序列識別號3 (VH4-M73 之 CDR3); (b) —多胜肽，包含CDR1、CDR2、CDR3，該CDR1包含 序列識別號4 (VH3-M73之CDR1)、該CDR2包含序列識別 號5 (VH3-M73之CDR2)，及該CDR3包含序列識別號6 (VH3-M73 之 CDR3); (c) 一多胜肽，包含CDR1、CDR2、CDR3 ’該CDR1包含 序列識別號7 (VH5-M83之CDR1)、該CDR2包含序列識別 15 201100100 號8 (VH5-M83之CDR2)，及該CDR3包含序列識別號9 (VH5-M83 之 CDR3); (d) —多胜肽，包含CDR1、CDR2、CDR3，該CDR1包含 序列識別號10 (VL1之CDR1)、該CDR2包含序列識別號11 (VL1之CDR2)，及該CDR3包含序列識別號12 (VL1之CDR3); (e) —多胜肽，包含CDR1、CDR2、CDR3，該CDR1包含 序列識別號13 (VL3之CDR1)、該CDR2包含序列識別號14 (VL3之CDR2)，及該CDR3包含序列識別號15 (VL3之CDR3); (f) 一多胜肽，包含CDR卜CDR2、CDR3，該CDR1包含 序列識別號16 (VL5之CDR1)、該CDR2包含序列識別號Π (VL5之CDR2)，及該CDR3包含序列識別號18 (VL5之CDR3)。 (2) —種醫藥配方，包含至少一種選自以下的抗體： (a) —抗體，包含一重鏈可變區及一輕鏈可變區’該重 鏈可變區包含：包含序列識別號1 (VH4-M73之CDR1)之 CDR1、包含序列識別號2 (VH4-M73之CDR2)之CDR2，及包 含序列識別號3 (VH4-M73之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號1 〇 (VL1之CDR1)之CDR1、包含序列 識別號11 (VL1之CDR2)之CDR2,及包含序列識別號12 (VL1 之 CDR3)之 CDR3; (b) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鍵可變區包含：包含序列識別號4 (VH3-M73之CDR1)之 CDR1、包含序列識別號5 (之CDR2)之CDR2 ’及包 含序列識別號6 (VH3-M73之CDR3)之CDR3 ;該輕鏈可變區 包含：包含序列識別號1 3 (VL3之CDR1)之CDR1、包含序列 16 201100100 " 識別號14 (VL3之CDR2)之CDR2,及包含序列識別號15 (VL3 之 CDR3)之 CDR3;及 (c) 一抗體，包含一重鍵可變區及一輕鍵可變區，該重 鏈可變區包含：包含序列識別號7 (VH5-M83之CDR1)之 CDR1、包含序列識別號8 (VH5-M83之CDR2)之CDR2，及包 含序列識別號9 (VH5-M83之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號16 (VL5之CDR1)之CDR1、包含序列 識別號17 (VL5之CDR2)之CDR2,及包含序列識別號18 (VL5 〇 之 CDR3)之 CDR3; (d) —抗體，包含一重鍵可變區及一輕鍵可變區，該 重鏈可變區包含：序列識別號19 (VH4-M73之可變區）；該輕 鏈可變區包含：序列識別號22 (VL1之可變區）； (e) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號20 (VH3-M73之可變區）；該輕 鏈可變區包含：序列識別號23 (VL3之可變區）； 〇 (〇 —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號21 (VH5-M83之可變區）；該輕 鏈可變區包含：序列識別號24 (VL5之可變區）； (g) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號25 (VH4-M73);該輕鏈包含：序列識別號28 (VL1); (h) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號26 (VH3-M73);該輕鏈包含：序列識別號29 (VUh (i) 一抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號27 (VH5-M83);該輕鏈包含：序列識別號30 (VL5)。 201100100 (3) 如（1)或（2)之穩定的醫藥配方，包含一組胺酸及/ 或檸檬酸鹽緩衝液。 (4) 如（1)〜（3)中任一項之穩定的醫藥配方，包含至少 一陽離子性胺基酸。 (5) 如（1)〜（4)中任一項之穩定的醫藥配方，包含 l〜50 0mM組胺酸及/或檸檬酸鹽緩衝液、W5〇〇mM的至少一 陽離子性胺基酸、1〜200mg/mL的抗體，及卜400mM的碳水 化合物。 (6) 如（4)或（5)之配方，其中該陽離子性胺基酸為精胺 酸。 (7) 如（5)或（6)之配方，其中，該碳水化合物為蔗糖或 海藻糖（trehalose)。 (8) 如（1)至（7)中任一項之配方，更包含一界面活性 劑。 (9) 如（1)至（8)中任一項之配方，包含多胜肽及/或抗 體之量為至少10 mg/ml。 (10) 如（1)至（9)中任一項之配方，包含多胜肽及/或抗 體之量為至少50mg/ml。 (11) 如（1)至（10)中任一項之配方，包含多胜肽及/或 抗體之量為至少80mg/ml。 (12) 如（1)至（11)中任一項之配方，包含多胜肽及/或 抗體之量為少於或等於240mg/ml。 (13) 如（1)至（12)中任一項之配方，其中，PH介於 4. 5〜7. 0。 18 201100100 (14) 如（13)之配方，其中，pH介於5.5〜6.6。 (15) 如（1)至（14)中任一項之配方，其中，該配方為液 體。 (1 6)如（1 5)之配方，其中，在該配方製配期間，未經 冷凍乾燥。 (17) 如（1)至（16)中任一項之配方，其中，該多胜肽及 /或抗體分子的二聚體化減少。 (18) 如（1)至（17)中任一項之配方，其中，該多胜肽及 〇 /或抗體分子的二聚體化受抑制。 (19) 如（1)至（18)中任一項之配方，係供皮下投予。 (20) —種穩定含有該抗體之溶液的方法，包含添加至 少一陽離子性胺基酸，其中，該抗體為選自以下的至少一 種抗體： (a) —抗體，包含一重鏈可變區及一輕鏈可變區，該重 鏈可變區包含：包含序列識別號1 (VH4-M73之CDR1)之 Q CDR1、包含序列識別號2 (VH4-M73之CDR2)之CDR2，及包 含序列識別號3 (VH4-M73之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號10 (VL1之CDR1)之CDR1、包含序列 識別號11 (VL1之CDR2)之CDR2,及包含序列識別號12 (VL1 之 CDR3)之 CDR3; (b) —抗體，包含一重鍵可變區及一輕鍵可變區，該 重鏈可變區包含：包含序列識別號4 (VH3-M73之CDR1)之 CDR1、包含序列識別號5 (VH3-M7 3之CDR2)之CDR2，及包 含序列識別號6 (VH3-M73之CDR3)之CDR3;該輕鏈可變區 19 201100100 包含：包含序列識別號13 (VL3之CDRl )之CDRl、包含序列 識別號14 (VL3之CDR2)之CDR2,及包含序列識別號15 (VL3 之 CDR3)之 GDR3; (c) 一抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：包含序列識別號7 (VH5-M83之CDR1)之 CDR1、包含序列識別號8 (VH5-M83之CDR2)之CDR2，及包 含序列識別號9 (VH5-M83之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號16 (VL5之CDR1)之CDR1、包含序列 識別號17 (VL5之CDR2)之CDR2,及包含序列識別號18 (VL5 之 CDR3)之 CDR3; (d) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號19 (VH4-M73之可變區）；該輕 鏈可變區包含：序列識別號22(VL1之可變區）； (e) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號20 (VH3-M73之可變區）；該輕 鏠可變區包含：序列識別號23 (VL3之可變區）； (f) 一抗體’包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號21 (VH5-M83之可變區）；該輕 鍵可變區包含：序列識別號24 (VL5之可變區）； (8) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號25 (VH4-M73);該輕鏈包含：序列識別號28 (VL1); (h) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號26 (VH3-M73);該輕鏈包含：序列識別號29 (VL3); (i) 一抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 20 201100100 ' 識別號27 (VH5-M83);該輕鏈包含：序列識別號30 (VL5)。 (21) —種穩定抗體的方法，該抗體於一冷凍/解;!東循 環之溶液中，包含添加至少一陽離子性胺基酸，其中該抗 體選自以下至少一種抗體： (a) —抗體，包含一重鍵可變區及一輕鍵可變區，該重 鏈可變區包含：包含序列識別號1 (VH4-M73之CDR1)之 CDR1、包含序列識別號2 (VH4-M73之CDR2)之CDR2，及包 含序列識別號3 (VH4-M73之CDR3)之CDR3;該輕鏈可變區 ® 包含：包含序列識別號10 (VL1之CDR1)之CDR1、包含序列 識別號11 (VL1之CDR2)之CDR2’及包含序列識別號12 (VL1 之 CDR3)之 CDR3; (b) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：包含序列識別號4 (VH3-M73之CDR1)之 CDR1、包含序列識別號5 (VH3-M73之CDR2)之CDR2，及包 含序列識別號6 (VH3-M73之CDR3)之CDR3;該輕鏈可變區 ^ 包含：包含序列識別號13 (VL3之CDR1)之CDR1、包含序列 識別號14 (VL3之CDR2)之CDR2,及包含序列識別號15 (VL3 之 CDR3)之 CDR3; (c) 一抗體，包含一重鏈可變區及一輕鏈可變區，該 重鍵可變區包含：包含序列識別號7 (VH5-M83之CDR1)之 CDR1、包含序列識別號8 (VH5-M83之CDR2)之CDR2，及包 含序列識別號9 (VH5-M83之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號16 (VL5之CDR1)之CDR1、包含序列 識別號Π (VL5之CDR2)之CDR2,及包含序列識別號18 (VL5 21 201100100 之 CDR3)之 CDR3; (d) —抗體’包含一重鍵可變區及一.輕鍵可變區，該 重鏈可變區包含：序列識別號19 (VH4-M73之可變區）；該輕 鏈可變區包含：序列識別號22 (VL1之可變區）； (e) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號20 (VH3-M73之可變區）.該輕 鏈可變區包含：序列識別號23 (VL3之可變區）； (f) 一抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號21 (VH5-M83之可變區）；該輕 鏈可變區包含：序列識別號24 (VL5之可變區）； (g) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號25 (VH4-M73);該輕鏈包含：序列識別號28 (vu). (h) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號26 (VH3-M73);該輕鏈包含：序列識別號29 (VL3). 及 (i) 一抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號27 (VH5-M83);該輕鏈包含：序列識別號3〇 (VL5)。 上述人型化抗IL-6受體IgG抗體已有增強的效力及改 良的藥物動力學；因此，能以較少的投予頻率發揮延長的、▲ 療效果。 【實施方式】 本發明提供一種醫藥配方’包含至少一種選自以 * 多胜肽： (a)—多胜肽’包含CDR1、CDR2及CDR3，該CDR1包 22 201100100 含序列識別號1 (VH4-M73之CDRl)、該CDR2包含序列識 別號2 (VH4-M73之CDR2)，該CDR3包含序列識別號3 (VH4-M73 之 CDR3); (b) —多胜肽，包含CDRl、CDR2及CDR3，該CDR1包 含序列識別號4 (VH3-M73之CDR1)、該CDR2包含序列識 別號5 (VH3-M73之CDR2)，該CDR3包含序列識別號6 (VH3-M73 之 CDR3); (c) 一多胜肽，包含CDRl、CDR2及CDR3，該CDR1包 〇 含序列識別號7 (VH5-M83之CDR1) '該CDR2包含序列識 別號8 (VH5-M83之CDR2)，該CDR3包含序列識別號9 (VH5-M83 之 CDR3); (d) —多胜肽，包含CDRl、CDR2及CDR3，該CDR1包 含序列識別號10 (VL1之CDR1)，該CDR2包含序列識別號 11 (VL1之CDR2)，該CDR3包含序列識別號12(VL1之CDR3); (e) —多胜肽，包含CDRl、CDR2及CDR3，該CDR1包 q 含序列識別號13 (VL3之CDR1)，該CDR2包含序列識別號 14 (VL3之CDR2)，該CDR3包含序列識別號15(VL3之CDR3); (f) 一多胜肽，包含CDRl、CDR2及CDR3，該CDR1包 含序列識別號16 (VL5之CDR1)，該CDR2包含序列識別號 17 (VL5之CDR2)，該CDR3包含序列識別號18(VL5之CDR3)。 本發明之多胜肽及抗體可依照習知方法配方（參見例 如 Remington’ s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) ° 本發明中使用的「醫藥配方」、「藥劑」，及「醫藥 23 201100100 ’·物」係各3有多胜肽及/或抗體作為有效成分的液體 或固體配方’其係製備為適用於直接或是復原後投予動 物例如人類。右有需要，此配方可含有醫藥上可接受之 載體及/或添加物。例如，洗務劑（例如H 、 luronic)賦形劑、抗氧化劑（例如，抗壞血酸、甲硫胺 )著色劑風味劑、保存劑、安定劑、緩衝劑、螯合劑 (例如EDTA)、懸浮劑、等張劑、黏結劑、崩散劑、潤滑劑、 流動促進劑’及矯味劑。 本發明之含有多胜肽及/或抗體之配方，較佳為不含 HAS(人類血清白蛋白）、明膠、及此等蛋白質作為安定劑。 本發明之3有多胜肽及/或抗體之配方，較佳為一液體 4藥配方其包含咼濃度的多胜肽及/或抗體，濃度不低於 10mg/mL ’較佳為不低於5〇mg/mL，更佳為不低於8〇呢/‘。 展度介於10〜240mg/mL，可為i〇mg/mL，較佳為50mg/mL , 更佳為 100~200mg/mL。 本發明之液體醫藥配方，較佳為不經過冷凍乾燥步驟 製造。 可用於本發明之緩衝藥劑，為可調整pH於所欲範圍， 且為醫藥上可接受者。本發明之含高濃度多胜肽及/或抗體 之配方，配方之pH較佳為4 5〜7，更佳為5 5〜6· 6。此等 緩衝劑為熟習該技術領域之人士所知者，例如包括無機鹽 例如磷酸鹽（鈉鹽或鉀鹽），及碳酸氫鈉；有機酸鹽例如檸檬 酸鹽（鈉鹽或鉀鹽），乙酸鈉及琥珀酸鈉；及酸例如磷酸、碳 酸、檸檬酸、琥珀酸、蘋果酸，及葡萄糖酸。 24 201100100 再者’也可使用Tris缓衝液、Good’ s緩衝液，例 如：MES及mops、組胺酸（例如組胺酸鹽酸鹽），及甘胺酸。 依照、本發明之含有高濃度多胜肽及/或抗體之配方，此緩衝 液較佳為一組胺酸或檸檬酸鹽緩衝液，更佳為組胺酸緩衝 液。該緩衝溶液之濃度，一般而言為1至5〇〇mM，較佳為5 至l〇〇mM，更佳為至2〇mM。當使用組胺酸緩衝液時該 ❹ 緩衝溶液所含組胺酸濃度，較佳為5至25ιηΜ，更佳為1〇 至 2 0 m Μ。 ~ 本發明之配方可進一步包含界面活性劑。典型之界面 活性劑之例子，包括：非離子性界面活性劑，例如山梨㈣ 酐脂肪酸酿，例如山梨糖醇酐單癸酸醋、山梨糖醇肝單月 32及山梨糖料單棕搁酸酯；甘油脂肪酸s旨例如甘油 早:酸知、甘油單肉豆襄酸醋、及甘油單钱酸醋；多聚甘 油月曰肪酸酯例如十甘油單硬脂酸、+ # 及+廿e 、 早硬月曰酸酉曰、十甘油二硬脂酸酯、 /早亞油酸酯；聚氧乙婦 〇 ψ m ^ 米雜和酐脂肪酸酯，例如 聚氧乙烯山梨糖醇酐單月 ”如 油酸醋、聚氧乙婦山梨糖醇肝單硬腊酸〆：糖㈣早 糖醇酐單棕櫚酸酯、 θ聚乳乙烯山梨 氧乙烯山梨糖醇酐= 酐二油酸酯、及聚 酯，例如聚氧乙烯山幽& 乳乙烯山梨糖醇脂肪酸 久乳g席山梨糖醇四硬脂 糖醇四油酸酯.聚梟 " 及聚氧乙烯山梨 曰’取乳乙烯甘油脂酸 單硬脂酸酯；聚乙二醇 θ，列如聚氧乙烯甘油 取卜 月θ肪酸酯’例如聚Γ _ π 1氧乙烯烷基醚，例， 取乙—醇二硬脂酸酯； ^# 眾氣乙稀月桂鍵.甲 烷基醚，例如聚氧乙 ，聚氧乙烯聚氧丙烯 坪1我丙二醇鍵、乎 • 灰虱乙稀聚氧丙烯 25 201100100 =及聚氧乙稀聚氧丙稀物;聚氧乙烤院基苯鍵，例 t乳乙烯壬基苯越;聚氧乙烯硬化旣麻子油，例 乙烯篦麻子油，及聚氣乙，嫌石#^ u乳乙烯硬化藏麻子油（聚氧乙烯氫化 麻子油聚氧乙烯蜂蠟衍生物 端it 7 例如聚虱乙烯山梨糖醇蜂 壤1氧乙稀平毛脂衍生物，例如:聚氧乙稀羊毛脂;则為 6至18之界面活性劑，例如聚氧乙烯脂肪酸醯胺，例如聚 氧乙烯十八酿胺;陰離子界面活性劑，例如具有“院基 之烧基硫酸鹽，例如_基硫酸鈉、月桂基硫酸鈉，及、、由 基硫酸鈉；聚氧乙浠院基㈣酸鹽，其中，加成之氧乙稀單 70的平均莫耳數為2至4,且院基之碳原子數為10至18, 例如聚氧乙稀月桂基硫酸納；具冑基之燒基確基 琥㈣鹽，例如月桂基確基琥轴酸鈉;天然界面活性劑例如 卵磷脂’及甘油磷酸脂;抱合磷脂質，例如鞘磷脂;及C12-C18 脂肪酸之蔗糖醋。此等界面活性劑可各別添加到本發明之 配方或可將兩種以上的此等界面活性劑組合添加。 較佳的界面活性劑為聚氧乙烯山梨糖醇酐脂肪酸酯及 聚氧乙烯聚氧丙烯烷基醚，更佳為聚山梨糖醇酯20、21、 40、60、65、80、81及85’卩111]"〇1^形式的界面活性劑， 且最佳為聚山梨糖醇酯20及80，及Pluronic F-68(P〇l〇xamer 188)。 添加到本發明之抗體配方的界面活性劑量，一般為 0.000 1 至 10%(w/v)，較佳為 0.001 至 5%，更佳為 〇 〇〇5 至3%。 本發明之配方，可更包含一酸性胺基酸，例如精胺酸， 26 201100100 以及曱硫胺酸、甘胺酸、丙胺 ^ 本丙胺酸、色胺酸、鲜 胺酸、蘇胺酸、天冬醯胺、谷 4 定劑。 酿胺、及此等胺基酸作為安 依照本發明之含抗體之紀太十Α t配方或含抗體之溶液配方中， 可藉由添加碳水化合物（例如糖聽方中 η ^ ^ ^ ^ μ )抑制在冷凍/解凍循環 期間形成一聚體。可使用 項衣 匕括非還原性之寡糖，仏丨 非還原性二糖例如蔗糖及海 插；她s ^ ^ u 糖或非還原性三糖例如 Ο 棉子糖，更佳為非還原性裳 還m ㈣還輕募糖為非 定職一糖，更佳為蔗糖及海藻糖。 依照本發明之含抗體之配方或含 可藉由添加碳水化合物( 〇谷液配方中， 〇、☆ U如糖類）抑制長時間俘在湘f 成多聚體以及分解產物B間保存期間形 露醇以及山㈣.及非^ 類包括糖醇，例如甘 例如奔糖，以及海墓、、性券糖，例如’非還原性二糖， 糖以及海溱糖，或非還 其中，較佳者A #、—，例如棉子糖， ❹ 還原性-糖，# ϋ ^的非還原性寡糖為非 注—糖，更佳者為蔗糖及海藻糖。 糖類可添加 更佳為 25〜100fl]g/社。 300mg/inL，又 於另—. 成分組成’依照本發明之配方較佳為實質上由以下 A)經修飾的抗iL_6受體抗體； 胺醆)广陽離子性胺基酸(例如精胺酸、組胺酸，及/或離 )緩衝劑（例如，組胺酸或檸檬酸鹽） 27 201100100 依用途，可在本配方中包含碳水化合物(例如糖類)及/ 或界面活性劑。 、甘胺酸、丙胺酸、苯 、天冬醯胺、谷醯胺、 作為安定劑，可包含：甲硫胺酸 丙胺酸、色胺酸、絲胺酸、蘇胺酸 及此專胺基酸。Cation exchange-HPLC and mass spectrometry reveal f) C-terminal amidation of an IgGl heavy chain. Anal 81 sinks! 11.2007 to 111; 360(1): 75-83). In the development of 12 (52 isotype antibody is a pharmaceutical, it is preferable to reduce such heterogeneity and maintain high stability. In order to produce a convenient, stable, high concentration, subcutaneous administration formulation, it is preferred to be not only stable. High, and the half-life in plasma is better than that of T〇CIUZUMAB isoform IgGl. However, no one has reported the sequence of the region of the antibody with the constant region of the IgG2 isoform, compared to the anti-Q body with the constant region of the IgG1 isoform, Having reduced heterogeneity, high stability, and superior plasma half-life. SUMMARY OF THE INVENTION One object of the present invention is to provide a pharmaceutical formulation (hereinafter also referred to as "pharmaceutical" or "pharmaceutical composition", which is superior to human type. The second generation molecule of the anti-receptor UG1 antibody T0CILIZUMAB can enhance the antigen neutralization ability and improve the drug kinetics by changing the amino acid sequence of the variable region and the constant region of TOCILIZUMAB, enabling lower administration Frequency to exhibit prolonged therapeutic effects, and improved immunogenicity, safety, and physics 13 201100100 chemical properties (stability and homogeneity). For formulations containing high concentrations of peptides and/or antibodies 'dimerization and deamidation of the peptides and/or antibodies are inhibited during prolonged storage or multiple cold/unbundling cycles And the formulation is stable and suitable for subcutaneous administration. There have been intensive studies to solve the above problems, and it has been found that by adding an amino acid squalic acid or a salt thereof as a stabilizer in a solution, A stable, high concentration multi-peptide/antibody-containing formulation is obtained. The invention will be described in detail below. The research focuses on obtaining a pharmaceutical formulation which is stable from the second generation molecular point of view, and the second generation molecule is superior to the first A generation of humanized anti-1L-6 receptor 1gG1 antibody T0CILIZUMAB. Results on antibodies of interest 'The inventors of the present invention found that the multi* CDR transformation of the variable region of UZUMAB will improve the binding of antigens. Ability (affinity). The inventors of the present invention succeeded in affinity 14 by using a combination of such mutations. The inventors of the present invention also succeeded in improving by introducing a modification that reduces the isoelectric point of the variable region sequence. Pharmacokinetics. Ming and others have also succeeded in improving pharmacokinetics by making the babies dependent on the binding of IL-6 receptor antigens, so that single antibody molecules can neutralize multiple antigens. Furthermore, they will remain in The small source sequence in the t〇cilizumab framework is completely humanized 'and reduces the number of T cell epitopes in the variable region predicted by computer simulations' to successfully reduce the risk of immunogenicity. ^ Furthermore, the inventors of the present invention have succeeded in finding a novel constant region sequence directed to the value region of TOCILIZUMAB, which has improved safety compared to IgG, which reduces the binding of the Fe receptor. 14 201100100 •s compared to IgGl ' The pharmacokinetics improve 'and reduce the heterogeneity due to the disulfide bond in the hinge region of igG2, and the heterogeneity due to the C-terminus of the Η chain without detracting from stability. The inventors of the present invention succeeded in producing a second generation molecule superior to TOCILIZUMAB by appropriately combining these amino acid sequence changes in the CDR, variable region and constant region. The present invention relates to a pharmaceutical formulation comprising a humanized anti-IL-6 receptor IgG antibody having superior antigen (il-6 receptor) binding ability, superior pharmacokinetics, superior safety and physical properties (stable And homogeneity) 'and further' can reduce immunogenicity by altering the amino acid sequence of the variable and constant regions of the humanized anti-IL-6 receptor IgG1 antibody TOC ILIZUMAB; Methods of such pharmaceutical compositions. More specifically, the present invention provides the following: (1) A pharmaceutical composition comprising at least one polypeptide selected from the group consisting of: (a) a multi-peptide comprising CDR1, CDR2, CDR3, wherein the CDR1 comprises q sequence recognition No. 1 (CDR1 of VH4-M73), the CDR2 comprises SEQ ID NO: 2 (CDR2 of VH4-M73), and the CDR3 comprises SEQ ID NO: 3 (CDR3 of VH4-M73); (b) - multi-peptide, including CDR1, CDR2, CDR3 comprising SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising SEQ ID NO: 5 (CDR2 of VH3-M73), and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73) (c) a multi-peptide comprising CDR1, CDR2, CDR3 ' CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising sequence recognition 15 201100100 No. 8 (CDR2 of VH5-M83), and The CDR3 comprises SEQ ID NO: 9 (CDR3 of VH5-M83); (d) - a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising SEQ ID NO: 11 (CDR2 of VL1), and the CDR3 comprises SEQ ID NO: 12 (CDR3 of VL1); (e) - a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 13 (VL3) CDR1), the CDR2 comprises SEQ ID NO: 14 (CDR2 of VL3), and the CDR3 comprises SEQ ID NO: 15 (CDR3 of VL3); (f) a multi-peptide comprising CDR CDR2, CDR3 comprising sequence recognition No. 16 (CDR1 of VL5), the CDR2 comprises the sequence identifier Π (CDR2 of VL5), and the CDR3 comprises SEQ ID NO: 18 (CDR3 of VL5). (2) A pharmaceutical formulation comprising at least one antibody selected from the group consisting of: (a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: a sequence identifier number comprising: CDR1 of CDR1 (CDR1 of VH4-M73) comprising CDR2 of SEQ ID NO: 2 (CDR2 of VH4-M73), and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 of SEQ ID NO: 1 (CDR1 of VL1), CDR2 comprising SEQ ID NO: 11 (CDR2 of VL1), and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1); (b) - Antibody comprising a heavy chain a variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2' comprising SEQ ID NO: 5 (including CDR2) and comprising a sequence identifier CDR3 of SEQ ID NO: 6 (CDR3 of VL3); And a CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3); and (c) an antibody comprising a heavy bond variable region and a light bond variable region, the heavy chain variable region package Included: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); The chain variable region comprises: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5), CDR2 comprising SEQ ID NO: 17 (CDR2 of VL5), and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5 ;); (d) - an antibody comprising a heavy bond variable region and a light bond variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: a sequence identifier 22 (variable region of VL1); (e) - an antibody comprising a heavy chain variable region and a light chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73) The light chain variable region comprises: SEQ ID NO: 23 (variable region of VL3); 〇 (〇-antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: a sequence Identification No. 21 (variable region of VH5-M83); the light chain variable region comprises: SEQ ID NO: 24 (variable region of VL5); (g) - an antibody comprising a heavy chain and a light chain, The strand comprises: SEQ ID NO: 25 (VH4-M73); the light chain comprises: SEQ ID NO: 28 (VL1); (h) - an antibody comprising a heavy chain and a light chain comprising: SEQ ID NO: 26 ( VH3-M73); the light chain comprises: SEQ ID NO: 29 (VUh (i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprises: Sequence ID 30 (VL5). 201100100 (3) A stable pharmaceutical formulation as in (1) or (2) containing a group of amine acids and/or citrate buffers. (4) A stable pharmaceutical formulation according to any one of (1) to (3) comprising at least one cationic amino acid. (5) A stable pharmaceutical formulation according to any one of (1) to (4) comprising at least one cationic amino acid of 1 to 50 mM histidine and/or citrate buffer, W5 mM 1, 1 to 200 mg/mL of antibody, and 400 mM of carbohydrates. (6) The formulation of (4) or (5) wherein the cationic amino acid is arginine. (7) The formulation of (5) or (6), wherein the carbohydrate is sucrose or trehalose. (8) The formulation according to any one of (1) to (7), further comprising an surfactant. (9) The formulation according to any one of (1) to (8), wherein the amount of the peptide and/or the antibody is at least 10 mg/ml. (10) The formulation according to any one of (1) to (9), wherein the amount of the peptide and/or the antibody is at least 50 mg/ml. (11) The formulation according to any one of (1) to (10), wherein the amount of the multi-peptide and/or antibody is at least 80 mg/ml. (12) The formulation according to any one of (1) to (11), wherein the amount of the peptide and/or the antibody is less than or equal to 240 mg/ml. (1) The formula of any one of (1) to (12), wherein the pH is between 4. 5 and 7. 0. 18 201100100 (14) The formula of (13), wherein the pH is between 5.5 and 6.6. (15) The formulation of any one of (1) to (14), wherein the formulation is a liquid. (16) A formulation as in (1) wherein the formulation is not freeze-dried during the formulation. (17) The formulation according to any one of (1) to (16) wherein dimerization of the multi-peptide and/or antibody molecule is reduced. (18) The formulation according to any one of (1) to (17) wherein the dimerization of the multipeptide and the antibody molecule is inhibited. (19) The formulation according to any one of (1) to (18) for subcutaneous administration. (20) A method for stabilizing a solution containing the antibody, comprising adding at least one cationic amino acid, wherein the antibody is at least one antibody selected from the group consisting of: (a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: a Q CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73), a CDR2 comprising SEQ ID NO: 2 (CDR2 of VH4-M73), and comprising sequence recognition CDR3 of No. 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising SEQ ID NO: 11 (CDR2 of VL1), and sequence recognition CDR3 of No. 12 (CDR3 of VL1); (b) - an antibody comprising a heavy bond variable region and a light bond variable region comprising: SEQ ID NO: 4 (CDR1 of VH3-M73) CDR1, CDR2 comprising SEQ ID NO: 5 (CDR2 of VH3-M7 3), and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region 19 201100100 comprises: SEQ ID NO: 13 CDR1 of CDR1 of VL3, CDR2 comprising SEQ ID NO: 14 (CDR2 of VL3), and GDR3 comprising SEQ ID NO: 15 (CDR3 of VL3); c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83) comprising SEQ ID NO: 8 (VH5- CDR2 of CDR2) of M83, and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5), comprising SEQ ID NO: 17 ( CDR2 of CDR2) of VL5, and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5); (d) - an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: a sequence Identification No. 19 (variable region of VH4-M73); the light chain variable region comprises: SEQ ID NO: 22 (variable region of VL1); (e) - an antibody comprising a heavy chain variable region and a light chain a variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73); the lick variable region comprising: SEQ ID NO: 23 (variable region of VL3); (f) an antibody 'Contains a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); the light bond variable region comprising: SEQ ID NO: 2 4 (variable region of VL5); (8) - an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: SEQ ID NO: 28 (VL1) (h) - an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3); (i) an antibody comprising A heavy chain and a light chain comprising: sequence 20 201100100 'identification number 27 (VH5-M83); the light chain comprises: sequence identification number 30 (VL5). (21) A method for stabilizing an antibody, comprising: adding at least one cationic amino acid to a solution of a frozen/dissolved solution; wherein the antibody is selected from at least one of the following antibodies: (a) - an antibody , comprising a heavy bond variable region and a light bond variable region comprising: CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73) comprising SEQ ID NO: 2 (CDR2 of VH4-M73) CDR2, and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region® comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1) comprising SEQ ID NO: 11 (CDR1 of VL1) CDR2' and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1); (b) - an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: comprising a sequence identifier CDR1 of 4 (CDR1 of VH3-M73) comprising CDR2 of SEQ ID NO: 5 (CDR2 of VH3-M73), and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region ^ comprises : CDR1 comprising SEQ ID NO: 13 (CDR1 of VL3), CDR2 comprising SEQ ID NO: 14 (CDR2 of VL3), and CD containing SEQ ID NO: 15 (VL3) CDR3 of R3); (c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising the SEQ ID NO: 7 (CDR1 of VH5-M83), comprising the sequence CDR2 of SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5) CDR2 comprising the sequence identifier Π (CDR2 of VL5), and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5 21 201100100); (d) - antibody 'containing a heavy bond variable region and a light bond variable region, The heavy chain variable region comprises: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprises: SEQ ID NO: 22 (variable region of VL1); (e) - antibody, comprising one heavy a chain variable region and a light chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73). The light chain variable region comprises: SEQ ID NO: 23 (f) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); change Including: SEQ ID NO: 24 (variable region of VL5); (g) - an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: sequence recognition No. 28 (vu). (h) - an antibody comprising a heavy chain and a light chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3). i) An antibody comprising a heavy chain and a light chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprising: SEQ ID NO: 3 VL5. The above-described humanized anti-IL-6 receptor IgG antibody has enhanced potency and improved pharmacokinetics; therefore, it is possible to exert an extended therapeutic effect with a small frequency of administration. [Embodiment] The present invention provides a pharmaceutical formulation comprising at least one selected from the group consisting of: * multi-peptide: (a)-polypeptide comprising CDR1, CDR2 and CDR3, the CDR1 package 22 201100100 containing sequence identification number 1 (VH4- CDR1 of M73, the CDR2 comprises SEQ ID NO: 2 (CDR2 of VH4-M73) comprising SEQ ID NO: 3 (CDR3 of VH4-M73); (b) - a multi-peptide comprising CDR1, CDR2 and CDR3, The CDR1 comprises SEQ ID NO: 4 (CDR1 of VH3-M73), the CDR2 comprises SEQ ID NO: 5 (CDR2 of VH3-M73), and the CDR3 comprises SEQ ID NO: 6 (CDR3 of VH3-M73); The peptide comprises CDR1, CDR2 and CDR3, and the CDR1 comprises SEQ ID NO: 7 (CDR1 of VH5-M83) 'The CDR2 comprises SEQ ID NO: 8 (CDR2 of VH5-M83), and the CDR3 comprises SEQ ID NO: 9 ( CDR3) of VH5-M83; (d) - a multi-peptide comprising CDR1, CDR2 and CDR3, the CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1) comprising SEQ ID NO: 11 (CDR2 of VL1), CDR3 SEQ ID NO: 12 (CDR3 of VL1); (e) - Polypeptide comprising CDR1, CDR2 and CDR3, comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: SEQ ID NO: 14 (CDR2 of VL3), the CDR3 comprises SEQ ID NO: 15 (CDR3 of VL3); (f) a multi-peptide comprising CDR1, CDR2 and CDR3, the CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5), The CDR2 comprises SEQ ID NO: 17 (CDR2 of VL5) comprising SEQ ID NO: 18 (CDR3 of VL5). The multi-peptide and antibody of the present invention can be formulated according to a conventional method (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). "Pharmaceutical formula" and "pharmaceutical" used in the present invention, and "Pharmaceutical 23 201100100 'An object" is a liquid or solid formulation in which each of the three peptides and/or antibodies is used as an active ingredient, which is prepared for administration to an animal such as a human, either directly or after recovery. Where desired, the formulation may contain a pharmaceutically acceptable carrier and/or additive. For example, detergent (eg, H, luronic) excipients, antioxidants (eg, ascorbic acid, methyl thiamine) colorant flavors, preservatives, stabilizers, buffers, chelating agents (eg, EDTA), suspending agents, Isotonic agents, binders, disintegrating agents, lubricants, flow promoters, and flavoring agents. The multipeptide peptide and/or antibody containing formulation of the present invention preferably contains no HAS (human serum albumin), gelatin, and the like as a stabilizer. The present invention has a multi-peptide and/or antibody formulation, preferably a liquid 4 drug formulation comprising a ruthenium concentration of a peptide and/or an antibody at a concentration of not less than 10 mg/mL, preferably not less than 5 〇 mg / mL, more preferably no less than 8 / / '. The spread is between 10 and 240 mg/mL, and may be i〇mg/mL, preferably 50 mg/mL, more preferably 100 to 200 mg/mL. The liquid pharmaceutical formulation of the present invention is preferably manufactured without a freeze drying step. Buffering agents useful in the present invention are those which are pH adjustable to the desired range and which are pharmaceutically acceptable. In the formulation of the present invention containing a high concentration of multi-peptide and/or antibody, the pH of the formulation is preferably from 4 5 to 7, more preferably from 5 5 to 6. 6 . Such buffering agents are known to those skilled in the art and include, for example, inorganic salts such as phosphates (sodium or potassium salts), 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. 24 201100100 Further, Tris buffer, Good's buffer, such as MES and mops, histidine (such as histidine hydrochloride), and glycine can also be used. According to the formulation of the present invention containing a high concentration of multi-peptide and/or antibody, the buffer is preferably a group of amine acid or citrate buffer, more preferably a histidine buffer. The concentration of the buffer solution is generally from 1 to 5 mM, preferably from 5 to 1 mM, more preferably to 2 mM. When the histidine buffer is used, the concentration of histidine in the buffer solution is preferably from 5 to 25 ηηΜ, more preferably from 1 至 to 20 m Μ. ~ The formulation of the invention may further comprise a surfactant. Examples of typical surfactants include: nonionic surfactants such as sorbitol (iv) anhydride fatty acid, such as sorbitan monoacetate, sorbitol liver single 32 and sorbose mono-palmitate Glycerol fatty acid s, for example, glycerol early: acid, glycerin monomyristate, and glycerol monobasic acid vinegar; polyglycerol lauric acid ester such as decaglyceryl monostearic acid, + # and +廿e, Early hard month bismuth citrate, decaglyceryl distearate, / early linoleate; polyoxymethylene sulfonate m ^ m and anhydride fatty acid esters, such as polyoxyethylene sorbitan single month Such as oleic acid vinegar, polyoxyethylene sorbitol liver mono-barium glutamate: sugar (four) early sugar anhydride monopalmitate, θ poly-vinyl sorbitol oxyethylene sorbitan = anhydride dioleate, and poly Ester, such as polyoxyethylene mountain secure & styrene sorbitan fatty acid long-term milk g sorbitol tetrastearyl alcohol tetraoleate. Poly 枭 " and polyoxyethylene sorbet 'milk ethylene glycolic acid Monostearate; polyethylene glycol θ, such as polyoxyethylene glycerol, θ θ 肪 fatty acid ', such as poly Γ π 1 Ethylene alkyl ether, for example, taking ethyl alcohol distearate; ^# Ethylene ether laurel bond. Methyl ether, such as polyoxyethylene, polyoxyethylene polyoxypropylene ping 1 I propylene glycol bond, ash虱 稀 聚 聚 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 Oil, and polygas B, suspected stone #^ u milk ethylene hardened Tibetan pomace oil (polyoxyethylene hydrogenated puff oil polyoxyethylene beeswax derivative end it 7 such as polyethylene sorbitol bee soil 1 oxyethylene flat fat Derivatives, for example: polyoxyethylene lanolin; then 6 to 18 surfactants, such as polyoxyethylene fatty acid decylamines, such as polyoxyethylene octadecylamine; anionic surfactants, for example, having a "hospital basis" a sulphur-based sulphate, such as sodium sulphate, sodium lauryl sulfate, and sodium sulphate; polyoxyethylene ketone (tetra) acid salt, wherein the average molar number of the added oxyethylene sulphate 70 is 2 to 4, and the number of carbon atoms in the yard is 10 to 18, such as polyoxyethylene sodium lauryl sulfate; Base succinate (iv) salts, such as sodium lauryl succinate; natural surfactants such as lecithin' and glycerol phosphate; conjugated phospholipids, such as sphingomyelin; and C12-C18 fatty acids, sucrose vinegar. The formulations of the present invention may be separately added or two or more of these surfactants may be added in combination. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl groups. The ether, more preferably a polysorbate 20, 21, 40, 60, 65, 80, 81 and 85' 卩 111] " 〇 1 ^ form of surfactant, and preferably polysorbate 20 And 80, and Pluronic F-68 (P〇l〇xamer 188). The surfactant dose to be added to the antibody formulation of the present invention is generally from 0.000 1 to 10% (w/v), preferably from 0.001 to 5%, more preferably from 〇〇 5 to 3%. The formulation of the present invention may further comprise an acidic amino acid, such as arginine, 26 201100100 and guanidine thioglycolic acid, glycine acid, propylamine, a pro-alanine, tryptophan, alanine, threonine, day. Winter amide, Valley 4 fixative. The enriched amine, and the amino acid, can be used as a solution containing the antibody according to the present invention, or by adding a carbohydrate (for example, η ^ ^ ^ ^ μ in the sugar listener) ) inhibiting the formation of a polymer during the freeze/thaw cycle. Can be used to include non-reducing oligosaccharides, non-reducing disaccharides such as sucrose and seaweed; her s ^ ^ u sugar or non-reducing trisaccharides such as ruthenium raffinose, more preferably non-reducing Sang also m (four) also lightly raise sugar for non-scheduled sugar, more preferably sucrose and trehalose. The antibody-containing formulation or the inclusion thereof according to the present invention can be inhibited by adding a carbohydrate (in the formula of glutinous rice liquid, ☆, ☆ U such as a saccharide) to inhibit prolonged capture during storage between the poly-polymer and the decomposition product B. Alcohols and mountains (4). and non-categores include sugar alcohols, such as sugar, such as rum, and sea tombs, sugar, such as 'non-reducing disaccharides, sugar and jelly, or not, preferably. A #, -, for example, raffinose, 还原 reducing-sugar, # ϋ ^ non-reducing oligosaccharides are non-note-sugars, more preferably sucrose and trehalose. Sugar can be added more preferably 25~100fl]g/she. 300 mg/inL, yet another component composition 'The formulation according to the invention is preferably an anti-iL_6 receptor antibody modified substantially as follows: A) a broad cationic amino acid (eg arginine, Histamine, and/or a buffer (eg, histidine or citrate) 27 201100100 Depending on the application, carbohydrates (eg, sugars) and/or surfactants may be included in the formulation. , glycine acid, alanine, benzene, aspartame, situramine, as a stabilizer, may include: methionine alanine, tryptophan, serine, threonine and the amino acid .
此處使用之「實質上$ Λ- Τ- A 只負上組成」’意指不包含通常添加在 配方中的成分以外的成公，褅者 + J風刀通常添加在配方中的成分係如 下述k擇的添加劑成分，比如：懸浮劑、溶解化劑、等張 劑、保存劑、吸附抑制劑、稀釋劑 '載體、pH調整劑、鎮 靜劑、含硫之還原劑，及抗氧化劑。 W浮刈之例，包括甲基纖維素、聚山梨醇酯8 〇、羥基 乙基纖維素、阿拉伯膠（gumrabic)、粉末化黃耆膠、羧基 甲基纖維素鈉，及聚氧乙烯山梨糖醇酐單月桂酸酯。 /合解化劑之例，包括聚氧乙烯氫化篦麻子油、聚山梨 醇醋80、終驗醯胺、聚氧乙烯山梨糖醇酐單月桂酸酯、聚 乙二醇（macr〇g〇i)，及篦麻子油脂肪酸乙酯。 等張劑之例’包括氯化鈉、氯化鉀，及氯化药。 保存劑之例’包括對羥基苯甲酸甲酯、對羥基苯甲酸 乙酯、山梨酸、苯酚、甲酚，及氣甲酚。 吸附抑制劑之例子，包括：人類血清白蛋白、卵磷脂、 葡聚糖、氧乙烯—氧丙烯共聚物、羥基丙基纖維素、甲基纖 維素 I氧乙稀氫化篦麻子油，及聚乙二醇。 含硫之還原劑之例子，包括具有氫硫基之化合物，例 如：N-乙醯基半胱胺酸、N_乙醯基同半胱胺酸、硫辛酸 28 201100100 (thi oct i c acid)、硫二乙二醇、硫乙醇胺、硫甘油、硫山 梨醇、硫甘醇酸及其鹽’硫代硫酸納、谷胱甘肽，及Ci-h 抗氧化劑之例，包括：異抗壞血酸（eryth〇rbicAs used herein, "substantially $ Λ - Τ - A only constitutes a composition" means that it does not contain the ingredients that are usually added to the formula, and the components that are usually added to the formula are as follows: The additive components selected are, for example, a suspending agent, a dissolving agent, an isotonic agent, a preservative, an adsorption inhibitor, a diluent 'carrier, a pH adjuster, a sedative, a sulfur-containing reducing agent, and an antioxidant. Examples of W floating mites include methyl cellulose, polysorbate 8 oxime, hydroxyethyl cellulose, gumrabic, powdered xanthan gum, sodium carboxymethyl cellulose, and polyoxyethylene sorbose Alcoholic anhydride monolaurate. Examples of /resolving agents include polyoxyethylene hydrogenated castor oil, polysorbate 80, final decylamine, polyoxyethylene sorbitan monolaurate, polyethylene glycol (macr〇g〇i ), and castor oil fatty acid ethyl ester. Examples of isotonic agents include sodium chloride, potassium chloride, and chlorinated drugs. Examples of the preservative include 'methylparaben, ethylparaben, sorbic acid, phenol, cresol, and cresol. Examples of adsorption inhibitors include: human serum albumin, lecithin, dextran, oxyethylene-oxypropylene copolymer, hydroxypropylcellulose, methylcellulose oxyethylene hydrogenated castor oil, and polyethylene Glycol. Examples of the sulfur-containing reducing agent include a compound having a thiol group, for example, N-ethinyl cysteine, N-acetamido-cysteine, lipoic acid 28 201100100 (thi oct ic acid), Examples of thiodiethylene glycol, thioethanolamine, thioglycerol, sulphur sorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and Ci-h antioxidants, including: erythroin (eryth〇) Rbic
acid)、二丁基羥基甲苯、丁基化羥基苯曱醚（anis〇le)、 〇：_生育酚、生育酚乙酸酯、L-抗壞血酸及其鹽、L_抗壞血 醯基棕櫚酸酯、L-抗壞血醯基硬脂酸酯、亞硫酸氫鈉、亞 硫酸鈉、沒食子酸三戊酯、沒食子酸丙酯，及螯合劑例如 乙二胺四乙酸二鈉（EDTA)、焦磷酸鈉，及偏磷酸鈉。 然而，依本發明之配方（藥劑、醫藥組合物），可用於 預防或治療包括發炎疾病之IL_6相關之疾病，不限於上 述，且可適當包含其他習知的載體。詳言4，例如稍微去 水的石夕酸、乳糖、，结晶纖維素、甘露醇、殿粉、叛甲基纖 維素妈、m甲基纖維素鈉、㈣丙基纖維素、隸丙基甲 基纖維素、聚乙烯基乙縮駿二乙基胺基乙㈣、 。比洛酮、明膠、中鏈脂肪酸甘油g|、聚氧乙稀氫化歡麻子 油6〇、蔑糖、幾基甲基纖維素、玉米澱粉，及無機鹽。 其中也可含其他低分子量的多胜肽;蛋白質，例如血清 =白、明膠’及免疫球蛋白；及胺基酸。當製備用於 的水溶液時，將此簟始T T P > 等抗1L~6受體抗體溶解於例如含有生理 食鹽水、葡萄糖或其他佐劑 山_、n ㈣1之等張溶液。佐劑，包含例如:D_ 、二w -甘露糖、D-甘露醇，及氯化鈉。再者，可組人 適當的溶解劑，例如醇（ '' 及非雜；“…％專)、多讀(丙二醇、PEG等）， 非離子性界面活性劑（聚山梨醇醋80及⑽—5〇)。 29 201100100 視需要’可將此等多胜肽包裹在微膠囊内（由經基纖維 素、明膠、聚（甲基丙烯酸甲酯）等製作的微膠囊），或使進 入膠體藥物遞送系統（微脂體、白蛋白微球體、微乳劑、奈 米微粒、奈米膠囊等）（參見例如” Remingt〇n，S Pharmaceutical Science 16th edition” Oslo Ed· ( 1 980 ))。又，製備藥劑為持續釋放藥劑的方法為已知， 且可應用在此多胜肽（Langeretal., J· Biomed. Mater.Acid), dibutylhydroxytoluene, butylated hydroxyphenyl ether (anis〇le), hydrazine: tocopherol, tocopheryl acetate, L-ascorbic acid and its salt, L_ascorbic acid-based palmitic acid Ester, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium edetate (EDTA) , sodium pyrophosphate, and sodium metaphosphate. However, the formulation (agent, pharmaceutical composition) according to the present invention can be used for the prevention or treatment of a disease associated with IL_6 including an inflammatory disease, and is not limited to the above, and other conventional carriers can be appropriately included. 4, for example, slightly dehydrated water, lactose, crystalline cellulose, mannitol, temple powder, methylated cellulose, m-methyl cellulose sodium, (tetra) propyl cellulose, propyl propyl Cellulose, polyvinyl acetylene diethylaminoethyl (tetra), . Pilotonone, gelatin, medium chain fatty acid glycerin g|, polyoxyethylene hydrogenated euphorbia oil 6 〇, 蔑 sugar, benzyl cellulose, corn starch, and inorganic salts. It may also contain other low molecular weight polypeptides; proteins such as serum = white, gelatin' and immunoglobulins; and amino acids. When preparing an aqueous solution for use, the anti-1L-6 receptor antibody such as T T P > is dissolved in, for example, an isotonic solution containing physiological saline, glucose or other adjuvants, n (tetra) 1 . An adjuvant comprising, for example, D_, di-w-mannose, D-mannitol, and sodium chloride. Furthermore, suitable dissolving agents such as alcohols (''and non-hetero; "%%), multi-read (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80 and (10)- 5〇). 29 201100100 Depending on the need, these multi-peptides can be encapsulated in microcapsules (microcapsules made of cellulose, gelatin, poly(methyl methacrylate), etc.) or enter the colloidal drug. Delivery systems (lipids, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (see for example "Remingt〇n, S Pharmaceutical Science 16th edition" Oslo Ed. (1 980)). A method of administering a medicament as a sustained release agent is known and can be applied to this multi-peptide (Langer 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•’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 ( 1 983) 22:547-56; EP No. 1 33,988)。又， 用於皮下投予的液體體積可藉由添加或混合透明質酸酶至 一藥劑中而增加（例如見W02004/078140)。 本發明之醫藥組合物可藉由口服及非口服兩者投予， 但較佳為以非口服投予。詳言之，此等組合物係以注射或 經皮膚對於病患投予。注射包括，例如：利用靜脈内、肌二 内或皮下注射等的全身性及局部投予。此等组合物可在處 理部位或該部位周邊藉由尤其是肌肉内注射而局部注射地 經皮膚劑型’包括例如：軟膏劑、凝膠、乳霜 ' 〜相 敦糊劑 (poultice)及貼劑，其可局部或全身性投予。再 、 、 可’投予 方法可視病患的年紀及症狀適當選擇。投予劑量可從每a 投予每公斤體重例如0_ooolrog〜1〇〇ing有效成分的範 選擇。或者，當此等組合物係對於人類病患投予時，例 投予劑量可從每位病患每次投予每公斤/ 腹董例如 30 201100100 0.001mg~ 1 000mg有效成分。單一投予劑量較佳為包括例 如，本發.明抗體約〇.〇1〜50mg/kg體重。然而，本發明之抗 體之劑量不限於此等劑量。 如同可由下述實施例之結果所見，依照本發明，可得 到穩定的配方，其中，藉由添加一種以上陽離子性胺基酸 (精胺酸' 組胺酸，及/或離胺酸，更佳為精胺酸），或組合 陽離子性胺基酸及其他的胺基酸在此配方中，長時間保存 或冷凍/解凍期間，抗體之二聚體化及去醯胺化少。 為了評估此含有高濃度抗體之配方的保存期間穩定 性，本案發明人等藉由進行尺寸排除層析（sizeexclusi〇n chr〇matography )及陰離子交換層析試驗，研究各種添加劑 的效果。結果，發現到：於含有胺基酸精胺酸之緩衝溶液中 溶有高濃度抗體之溶液，二聚體之量低於沒有添加精胺酸 之溶液者。此等結果顯$，精胺酸作為安定劑可有效抑制 抗體二聚體化。Biopolymers (1 983) 22: 547-56; EP No. 1 33, 988). Further, the volume of liquid for subcutaneous administration can be increased by adding or mixing hyaluronidase into a medicament (see, for example, WO2004/078140). The pharmaceutical composition of the present invention can be administered by both oral and parenteral administration, but is preferably administered parenterally. In particular, such compositions are administered to a patient by injection or transdermally. Injection includes, for example, systemic and topical administration using intravenous, intramuscular or subcutaneous injection. Such compositions may be administered locally at the treatment site or around the site by intramuscular injection, especially by intramuscular injection, including, for example, ointments, gels, creams, and poultices and patches. It can be administered locally or systemically. The method of administration can be appropriately selected depending on the age and symptoms of the patient. The dose to be administered can be selected from the per-kg body weight per a, for example, 0 ooolrog~1〇〇ing active ingredient. Alternatively, when such compositions are administered to a human patient, the dosage may be administered from each patient per kg/abdomen, for example 30 201100100 0.001 mg to 1 000 mg of active ingredient per administration. The single administration dose preferably includes, for example, the present invention. The antibody is about 〜1 to 50 mg/kg body weight. However, the dose of the antibody of the present invention is not limited to such an equivalent dose. As can be seen from the results of the following examples, in accordance with the present invention, a stable formulation can be obtained in which, by adding more than one cationic amino acid (arginine 'histamine, and/or lysine, more preferably In the case of arginine, or a combination of a cationic amino acid and other amino acids, in this formulation, dimerization and deamidation of the antibody are less during prolonged storage or freezing/thawing. In order to evaluate the stability during storage of this high-concentration antibody-containing formulation, the inventors of the present invention studied the effects of various additives by performing size exclusion chromatography (sizeexclusi〇n chr〇matography) and anion exchange chromatography test. As a result, it was found that a solution in which a high concentration of the antibody was dissolved in a buffer solution containing amino acid arginine was used, and the amount of the dimer was lower than that in the case where no arginine was added. These results are significant, and arginine as a stabilizer can effectively inhibit antibody dimerization.
因此，藉由添加精胺酸作為安定劑，可提供一種穩定 的抗體配方，其中，減少抗體之二聚體化。 本發明之一具體實施例為，-種穩定的含有抗體的配 方’特徵為:於一緩衝的溶液中含有一抗體及精胺酸。 一用在本發明之精胺酸’可為任何精胺酸化合物本身、 其衍生物及其鹽，較佳為L —精胺酸及其鹽。 當依照本發明之配方含有精胺酸，精胺酸之濃度較佳 為卜l_mM，更佳為5(Μ〇〇_，又更佳為5〇 本發明之多胜肽不特別限定，然而，較佳為具有结人 31 201100100 到人類IL-6受體之活性的抗原結合物質，此等抗原結合物 質’較佳為包含例如：抗體重鏈可變區（VH)、抗體輕鏈可變 區（VL)'抗體重鏈、抗體輕鏈，及抗體。 上述（a)〜（f)之多胜肽中，（a)〜（c)之多胜肽為較佳之 抗體重鏈可變區之例子，而（dMf)之多胜肽為抗體輕鏈可 變區之例子。 此等可變區可使用作為一抗人類IL-6受體抗體之一 部分。使用如此之可變區的抗人類IL_6受體抗體具有優越 的結合活性、優異的藥物動力學、優異的安全性、減少的 免疫原性，及/或優越的物理化學性質。於本發明中，優異 的藥物動力學或藥物動力學之改良，係指以下任何一項： 「清除率（CL)」降低、「曲線下面積（AUC)」增加、「平均 殘留時間」增加，及”血漿半衰期（tl/2)，，增加，此等係 由备抗體投予到體Θ時’從血聚濃度之時間冑化而計算的 樂物動力學參數。纟此’優越的物理化學性質或改良的物 理化學性質，係指改良的穩定性、減少的異質性等，但不 7 灿一吧货' 避取使（ 开’成有利之抗原結合部位者。本發明巾，針對此等可變 而使用之FR不特別限定’可使用任意FR;然Therefore, by adding arginine as a stabilizer, a stable antibody formulation can be provided in which dimerization of the antibody is reduced. In one embodiment of the invention, a stable antibody-containing formulation is characterized by the inclusion of an antibody and arginine in a buffered solution. The arginine acid used in the present invention may be any arginine compound itself, a derivative thereof and a salt thereof, preferably L-arginine and a salt thereof. When the formulation according to the present invention contains arginine, the concentration of arginine is preferably 1 mM, more preferably 5 Μ〇〇, and still more preferably 5 多. The multipeptide of the present invention is not particularly limited, however, Preferably, it is an antigen-binding substance having the activity of human cell 31 201100100 to a human IL-6 receptor, and these antigen-binding substances preferably include, for example, an antibody heavy chain variable region (VH), an antibody light chain variable region. (VL) 'antibody heavy chain, antibody light chain, and antibody. Among the above multi-peptides (a) to (f), the multi-peptides of (a) to (c) are preferred antibody heavy chain variable regions. For example, the multi-peptide of (dMf) is an example of an antibody light chain variable region. These variable regions can be used as part of a primary antibody against human IL-6 receptor. Anti-human IL_6 using such a variable region The receptor antibody has superior binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and/or superior physicochemical properties. In the present invention, excellent pharmacokinetics or pharmacokinetics Improvement refers to any of the following: "Clearance rate (CL)" is reduced, "area under the curve (A UC) increase, "average residual time" increase, and "plasma half-life (tl/2), increase, which is calculated from the time when the antibody is administered to the body, and is calculated from the time of blood concentration. Physical kinetic parameters. ' This 'superior physical and chemical properties or improved physicochemical properties, refers to improved stability, reduced heterogeneity, etc., but not 7 can be a bar' to avoid (opening into a favorable The FR of the present invention, the FR used for such a variable is not particularly limited to 'any FR can be used;
:人類衍生的FR。可使用具有天然序列的人類衍生J :者，視需要，可將一個以上胺基酸取代、剛除、加成 或插入等導入到具有天然序列的框 ’ 足鉍L A 十 便侍此CDR形 的抗原結合部位。可利用例如測量及評估針對具有 201100100 ’ 基酸取代之FR之抗體對於一抗原之結合活性，而選擇具有 所望性質之突變FR序列（Sato, K. et al _，Cancer Res. (1993)53,851-856)。 又’可在上述CDR序列中將1個以上的胺基酸進行取 代、刪除、加成，及/或插入。較佳為，一個以上胺基酸進 行取代、刪除、加成及/或插入之後’一 CDR序列，就結合 活性、中和活性、穩定性、免疫原性，及/或藥物動力學， 與改變刖的CDR序列相比’具有同等活性。欲取代、刪除、 〇 加成及/或插入之胺基酸數目，不特別限定，然而，較佳為 3個胺基酸以下’更佳為為2個胺基酸以下，又更佳為每 個CDR ’ 1個胺基酸以下。 用於將一個以上胺基酸殘基取代為其他關注之胺基酸 的方法，例如：點突變（Hashimoto-Gotoh，T，Mizuno，T， Ogasahara, Y, and Nakagawa, Μ. (1995) An oligodeoxyribonucleotide-directed dual amber method 0 for s i te-d i rected mutagenesis. Gene 152. 271-275:Zo11er. MJ. and Smith. M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500;Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DAN approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12， 9441-9456; Kramer W, and Fritz HJ(1987) 33 201100100: Human derived FR. A human-derived J: having a natural sequence can be used, and more than one amino acid substitution, addition, addition or insertion can be introduced into a frame having a natural sequence as needed. Antigen binding site. For example, a mutant FR sequence having a desired property can be selected by measuring and evaluating the binding activity of an antibody having a FR of 201100100 'base acid substitution to an antigen (Sato, K. et al _, Cancer Res. (1993) 53, 851- 856). Further, one or more amino acids may be substituted, deleted, added, and/or inserted in the CDR sequence. Preferably, more than one amino acid undergoes a substitution, deletion, addition and/or insertion of a 'CDR sequence, which binds to activity, neutralizing activity, stability, immunogenicity, and/or pharmacokinetics, and changes The CDR sequences of sputum are equivalent in activity to '. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, however, it is preferably 3 or less of amino acids, more preferably 2 or less of amino acids, and even more preferably One CDR '1 amino acid below. A method for substituting one or more amino acid residues for other amino acids of interest, for example: point mutation (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, Μ. (1995) An oligodeoxyribonucleotide -directed dual amber method 0 for si te-d i rected mutagenesis. Gene 152. 271-275: Zo11er. MJ. and Smith. M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100 , 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DAN approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 9441-9456; Kramer W, and Fritz HJ (1987) 33 201100100
Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunke 1, TA ( 1 985) Rapid and efficient s i te-spec i f i c mutagenesis without phenotypic selection. Proc Natl Acad Sci U. S. A. 82，488-492)。此方法可用於將抗體中 之所望胺基酸以關注的其他胺基酸取代。再者，作為胺基 酸取代之方法，可利用基因庫技術例如.框架曳步 (framework shuffling) (Mol. Immunol. 2007 Apr; 44( 1 1 ) :3049-60)及 CDR 修復（US2006/0 1 22377)，進行胺基 酸取代以得到適當的框架及CDR。 本發明亦提供一種醫藥配方，包含選自以下至少一種 抗體： (a) —抗體，包含一重鏈可變區及一輕鏈可變區，該重 鏈可變區包含：包含序列識別號1 (VH4-M73之CDR1)之 CDR1、包含序列識別號2 (VH4-M73之CDR2)之CDR2，及包 含序列識別號3 (VH4-M73之CDR3)之CDR3 ;該輕鏈可變區 包含：包含序列識別號10 (VL1之CDR1)之CDR1、包含序列 識別號11 (VL1之CDR2)之CDR2,及包含序列識別號12 (VL1 之 CDR3)之 CDR3; (b) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：包含序列識別號4 (VH3-M73之CDR1)之 CDR1、包含序列識別號5 (VH3-M73之CDR2)之CDR2 ’及包 含序列識別號6 (VH3-M73之CDR3)之CDR3 ;該輕鏈可變區 包含：包含序列識別號1 3 (VL3之CDR1 )之CDR1、包含序列 201100100 ' 識別號14 (VL3之CDR2)之CDR2,及包含序列識別號15 (VL3 之 CDR3)之 CDR3; (c) 一抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：包含序列識別號7 (VH5-M83之CDR1)之 CDR1、包含序列識別號8 (VH5-M83之CDR2)之CDR2，及包 含序列識別號9 (VH5-M83之CDR3)之CDR3;該輕鏈可變區 包含：包含序列識別號16 (VL5之CDR1)之CDR1、包含序列 識別號17 (VL5之CDR2)之CDR2’及包含序列識別號18 (VL5 ^ 之 CDR3)之 CDR3; (d) —抗體，包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號19 (VH4-M73之可變區）；該輕 鏈可變區包含：序列識別號22 (VL1之可變區）； (e) —抗體’包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號20 (VH3-M73之可變區）；該輕 鏈可變區包含：序列識別號23 (VL3之可變區）； Q (f) 一抗體’包含一重鏈可變區及一輕鏈可變區，該 重鏈可變區包含：序列識別號21 (VH5-M83之可變區）；該輕 鏈可變區包含：序列識別號24 (VL5之可變區）； (S)—抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號25 (VH4-M73);該輕鏈包含：序列識別號28 (VL1); (h) —抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號26 (VH3-M73);該輕鏈包含：序列識別號29 (VL3); (i) 一抗體，包含一重鏈及一輕鏈，該重鏈包含：序列 識別號27 (VH5-M83);該輕鏈包含：序列識別號3〇 (VL5)。 35 201100100 以上所述抗體，可使用為具有優越結合活性'優里 藥物動力學、優異之容八w ^ 優吳之女全性、減低之免疫原性，及/或優越 的物理化學性質的抗人類IL-6受體抗體。 與本發明之CDR連結之人類抗體框架區係選取會使得 CDR形成有利之抗原结合部位者。本發明巾，針對此等可 變區而使用之FR不特別限^，可使用任意fr ;，然而，較佳 為使用人類衍生的FR。可使用具有天然、序列的人類衍生之 FR或者，視需要，可將一個以上胺基酸取代、刪除、加 成及/或插入等導入到具有天然序列的框架區，使得此 形成足夠的抗原結合部位。可利用例如測量及評估針對具 有胺基酸取代之FR之抗體對於一抗原之結合活性，而選擇 具有所望性質之突變FR序列（SatQ，K .㈡止，Cancer^ (1993) 53， 85卜856)。 同時’本發明之抗體使用之恆定區不特別限定，可使 用任意的恆定區。較佳的使用於本發明抗體的恆定區，包 括例如：人類衍生的恆定區（衍生自IgGl、IgG2、igG3、 I g(M、c /c、c又等的恆定區）。可在此人類衍生的恆定區 進行1個以上的胺基酸的取代、刪除、加成及/或插入。較 佳的人類衍生重鏈恆定區包括’例如：包含胺基酸序列識別 號31之怪定區（VH4_M73之恆定區）、包含胺基酸序列識別 號32之恆定區（VH3-M73之恆定區）、包含胺基酸序列識別 號33之值定區（VH5_M83之恆定區），而較佳之人類 4r 王之 輕鍵值定區包括，例如：包含胺基酸序列識別號34之恆定 區（VLl)、包含胺基酸序列識別號35之恆定區（VL3)、包含 36 201100100 • 胺基酸序列識別號36之恆定區（VL5)。 又’可在上述CDR序列中將i個以上的胺基酸進行取 代、刪除、加成，及/或插入。較佳為，一個以上胺基酸進 行取代、刪除、加成及/或插入之後’一 CDR序列，就結合 活性、中和活性、穩定性、免疫原性，及/或藥物動力學， 與改變前的CDR序列相比，具有同等活性。欲取代、刪除' 加成及/或插入之胺基酸數目，不特別限定，然而，較佳為 3個胺基酸以下，更佳為為2個胺基酸以下，又更佳為每 Ο 個CDR，1個胺基酸以下。 此等抗體的各可變區，可作為與抗人類IL_6受體反應 的分子的一部分。此等恆定區尚可包含丨個以上胺基酸（例 如，5個或以下胺基酸，較佳為3個或以下胺基酸）的取代、 刪除、加成，及/或插入。用於將一個以上胺基酸殘基取代 為其他關注之胺基酸的方法’包括例如上述方法。 本發明尚包括包含上述可變區之多胜肽。 〇 此等抗體之各重鏈及輕鏈，可作為與抗人類IL-6受體 反應的分子的一部分。使用此等重鏈及輕鏈的抗人類丨l_6 文體抗體，具有優越的結合活性、優異的藥物動力學、優 異的安全性、減低的免疫原性’及/或優越的物理化學性質。 此等重鏈及輕鍵尚可包含1個或以上胺基酸（例如，1〇 個或以下胺基酸’較佳為5個或以下胺基酸，更佳為5個 或以下胺基酸）的取代、刪除、加成，及/或插入。用於將 個以上胺基酸殘基取代為其他關注之胺基酸的方法，包 括例如上述方法。 201100100 1個或以上胺基酸的取代、孤丨丨队 J J、刪除、加成，及,/或插入， 可針對可變區、恆定區或兩者進行。 本發明更包括包含上述重鏈及輕鏈的多胜肽。 本發明之抗體較佳為人型化抗體。 人型化抗體也稱為再改造（reshaped)抗體。此一人型 化抗體係藉由將來自於非人類哺乳動物的互補決定區(cdr) 移植到人類抗體之CDR而得到。習知的用於製備此種抗體 的基因重組技術為已知（見Eur〇pean patent AppHcati〇nOligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunke 1, TA (1 985) Rapid and efficient si te-spec ific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82,488- 492). This method can be used to replace the desired amino acid in the antibody with other amino acids of interest. Furthermore, as a method of amino acid substitution, gene library technology such as framework shuffling (Mol. Immunol. 2007 Apr; 44(1 1 ): 3049-60) and CDR repair (US2006/0) can be utilized. 1 22377), amino acid substitutions are made to obtain the appropriate framework and CDRs. The invention also provides a pharmaceutical formulation comprising at least one antibody selected from the group consisting of: (a) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: comprising a sequence identifier number 1 ( CDR1 of CDR1) of VH4-M73, CDR2 comprising SEQ ID NO: 2 (CDR2 of VH4-M73), and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: comprising sequence CDR1 of SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising SEQ ID NO: 11 (CDR2 of VL1), and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1); (b) - Antibody comprising a heavy chain variable region And a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2' comprising SEQ ID NO: 5 (CDR2 of VH3-M73) and inclusion sequence recognition CDR3 of SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region comprises: CDR2 comprising SEQ ID NO: 13 (CDR1 of VL3), CDR2 comprising SEQ ID NO: SEQ ID NO: 14 (CDR2 of VL3), and a CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3); (c) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain The region comprises: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); The light chain variable region comprises: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5), CDR2' comprising SEQ ID NO: 17 (CDR2 of VL5), and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5^); An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: sequence recognition No. 22 (variable region of VL1); (e) - the antibody comprises a heavy chain variable region and a light chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73) The light chain variable region comprises: SEQ ID NO: 23 (variable region of VL3); Q (f) an antibody 'comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising : SEQ ID NO: 21 (variable region of VH5-M83); the light chain variable region comprises: SEQ ID NO: 24 (variable region of VL5); (S) - an antibody comprising a heavy chain and a light chain, The heavy chain comprises: SEQ ID NO: 25 (VH4-M73); the light chain comprises: SEQ ID NO: 28 (VL1); (h) - an antibody comprising a heavy chain and a light chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprises: SEQ ID NO: 29 (VL3); (i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); The chain contains: sequence identification number 3〇 (VL5). 35 201100100 The above-mentioned antibodies can be used as anti-humans with superior binding activity, 'Yuli' pharmacokinetics, excellent capacity, full immunogenicity, and/or superior physicochemical properties. IL-6 receptor antibody. The human antibody framework regions linked to the CDRs of the invention are selected such that the CDRs form a favorable antigen binding site. The FF of the present invention for use in such variable regions is not particularly limited, and any fr can be used; however, it is preferred to use a human-derived FR. A human-derived FR having a natural, sequence may be used or, if necessary, one or more amino acid substitutions, deletions, additions, and/or insertions may be introduced into a framework region having a native sequence such that sufficient antigen binding is formed. Part. For example, the binding activity of an antibody having an amino acid-substituted FR to an antigen can be measured and evaluated, and a mutant FR sequence having a desired property can be selected (SatQ, K. (2), Cancer ^ (1993) 53, 85 856 ). Meanwhile, the constant region used for the antibody of the present invention is not particularly limited, and any constant region can be used. Preferred for use in the constant regions of the antibodies of the invention, including, for example, human-derived constant regions (derived from IgGl, IgG2, igG3, Ig (M, c/c, c, etc. constant regions). The derivatized constant region performs substitution, deletion, addition, and/or insertion of more than one amino acid. Preferred human-derived heavy chain constant regions include 'eg, a region containing amino acid sequence number 31 ( The constant region of VH4_M73), the constant region comprising the amino acid sequence number 32 (the constant region of VH3-M73), the region containing the amino acid sequence number 33 (the constant region of VH5_M83), and preferably the human 4r The king's light-key sequence includes, for example, a constant region (VL1) comprising amino acid sequence identifier 34, a constant region (VL3) comprising amino acid sequence identifier 35, and contains 36 201100100. • Amino acid sequence recognition No. 36 constant region (VL5). Further, i or more amino acids may be substituted, deleted, added, and/or inserted in the above CDR sequence. Preferably, one or more amino acids are substituted, After deleting, adding and/or inserting a 'CDR sequence, the knot Activity, neutralizing activity, stability, immunogenicity, and/or pharmacokinetics, have equivalent activity compared to the CDR sequences prior to alteration. To replace, delete, add, and/or insert the number of amino acids, It is not particularly limited, however, it is preferably 3 or less amino acid acids, more preferably 2 amino acids or less, still more preferably CDRs per molecule, and 1 amino acid or less. a region that is part of a molecule that reacts with an anti-human IL-6 receptor. These constant regions may also contain more than one amino acid (for example, 5 or less amino acids, preferably 3 or less amino acids). Substitution, deletion, addition, and/or insertion. Methods for substituting one or more amino acid residues for other amino acids of interest' include, for example, the above methods. The present invention also encompasses the inclusion of the above variable regions. Multi-peptides. The heavy and light chains of these antibodies can be used as part of a molecule that reacts with anti-human IL-6 receptors. These anti-human 丨l_6 stuvage antibodies using these heavy and light chains are superior. Binding activity, excellent pharmacokinetics, excellent safety , reduced immunogenicity' and/or superior physicochemical properties. These heavy and light bonds may still contain one or more amino acids (for example, 1 or less amino acids are preferably 5) Or a substitution, deletion, addition, and/or insertion of an amino acid, preferably 5 or less amino acids. A method for substituting more than one amino acid residue for other amino acids of interest Including, for example, the above method. 201100100 One or more amino acid substitutions, scorpion JJ, deletion, addition, and/or insertion, can be performed for a variable region, a constant region, or both. A multi-peptide comprising the above heavy chain and light chain is included. The antibody of the present invention is preferably a humanized antibody. Humanized antibodies are also known as reshaped antibodies. This humanized anti-system is obtained by grafting a complementarity determining region (cdr) from a non-human mammal to the CDR of a human antibody. Known genetic recombination techniques for the preparation of such antibodies are known (see Eur〇pean patent AppHcati〇n)
No. EP 125023;WO 96/02576)。 尤其，例如，使關注之CDR及關注的框架區（FR)連結 的經設計的DNA序列，係使用製備的數個寡核苦酸使具有 與CDR及FR均有重疊部分的末端作為引子而以pCR合成 (見W098/13388敘述之方法）。人型化抗體係藉由以下得到： 將知·到的DNA連接到編碼為人類抗體恆定區或經修飾的人 類抗體恆定區的DNA;將上述插入到一表現載體；及將此 載體‘入到一寄主以生產此抗體（見Eur〇pean patentNo. EP 125023; WO 96/02576). In particular, for example, a designed DNA sequence in which a CDR of interest and a framework region of interest (FR) are linked is obtained by using a plurality of oligonucleotides prepared by using the oligo-nucleolytic acid having an overlap with the CDR and the FR as an primer. pCR synthesis (see method described in W098/13388). The humanized anti-system is obtained by ligating the known DNA to a DNA encoding a human antibody constant region or a modified human antibody constant region; inserting the above into a expression vector; and introducing the vector into a host to produce this antibody (see Eur〇pean patent
Application No. EP 239400,及 International patent Application Publication No. WO 96/02576)。 與CDR連結之人類抗體框架區係選取使cdr形成有利 之抗原結合部位者。視需要，可將胺基酸取代、刪除、加 成’及/或插入導入到抗體可變區的框架區。 一人類抗體恆定區’或其中1個以上胺基酸經過取 代、刪除、加成及/或插入的經改變的人類抗體恆定區，可 作為人型化抗體之恆定區。 38 201100100 例如，Η鏈可使用C T 1、C τ 2、C 7 3、C 7 4、 C mu ' C <5、Cal、Ca2、C epsi Ion，L 鏈可使用 C /c 及C λ。C /c的胺基酸序列顯示於序列識別號38，編碼 為此胺基酸序列的核苷酸序列顯示於序列識別號37。C τ 1的胺基酸序列顯示於序列識別號4 0，編碼為此胺基酸序 列的核苷酸序列顯示於序列識別號39。C τ 2的胺基酸 序列顯示於序列識別號4 2，編碼為此胺基酸序列的核普酸 序列顯示於序列識別號41。C r 4的胺基酸序列顯示於 〇 序列識別號4 4 ’編碼為此胺基酸序列的核苷酸序列顯示於 序列識別號43。 又，人類抗體C區可經修飾以改良抗體穩定性或抗體 生產穩定性。任意同型物之人類抗體，例如，IgG、IgM、 Ϊ gA、I gE或I gD，可使用在抗體人型化。然而，本發明較 佳為使用IgG。IgGl、IgG2、IgG3、IgG4等可作為igG使 用。 〇 人型化抗體的可變區（例如CDR及FR)及恆定區的胺基 酸，可在製備後刪除、加成、刪除及/或取代為胺基酸。本 發明之抗體也包括此種包含胺基酸取代等的人型化抗體。 本發明之抗體’只要具有IL_6受體結合活性及/或中 和活性，不僅包括以I gG代表的雙價抗體，也包括單價抗 體，及以IgM代表的多價抗體。本發明之多價抗體包括抗 原結合部位相同的多價抗體，以及全部或部分抗原結合部 位不同的多價抗體。本發明之抗體只要其結合於IL_6受體 蛋白質，不僅包括完整的抗體分子，也包括微小抗體 201100100 (minibody)及其修飾產物。 微小抗體係包含欠缺完整抗體的一部分(例如完整的 IgG等）之抗體片段之抗體，且只要具有IL6受體社a ,舌 性及/或中和活性且包含欠缺完整抗體之一部分的：：片 段者’即不特別限定。本發明之微小抗體不特別限定，只 要其具有完整抗體之一部分即可。然而，"微小抗體較 佳為包含VH < VL，更佳為包含VH及VL。其他本發明中之 較佳的抗體，包括例如··包含抗體CDR之微小抗體。此等微 小抗體可包含抗體之6個CDR的全部或一部分。 本發明之微小抗體較佳為具有小於完整抗體的分子 量。然而，此等微小抗體可以形成多聚物，例如二聚物、 三聚物或四聚物，且因此其分子量有時會大於完整的抗體。 尤其’抗體片段包括例如Fab、Fab’ 、F(ab，）2及 Fv。同時’微小抗體包括例如：Fab、Fab， 、F(^ab，、Application No. EP 239400, and International patent Application Publication No. WO 96/02576). The human antibody framework regions linked to the CDRs are selected such that cdr forms a favorable antigen binding site. The amino acid may be substituted, deleted, added, and/or inserted into the framework region of the variable region of the antibody, as needed. A human antibody constant region' or an altered human antibody constant region in which one or more amino acids have been substituted, deleted, added, and/or inserted can serve as a constant region for a humanized antibody. 38 201100100 For example, the chain can use C T 1 , C τ 2, C 7 3, C 7 4, C mu ' C < 5, Cal, Ca 2 , C epsi Ion, and the L chain can use C /c and C λ . The amino acid sequence of C/c is shown in SEQ ID NO: 38, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of C τ 1 is shown in SEQ ID NO: 40, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 39. The amino acid sequence of C τ 2 is shown in SEQ ID NO: 4 2, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 41. The amino acid sequence of C r 4 is shown in SEQ ID NO: 4 4 ' The nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 43. Also, the human antibody C region can be modified to improve antibody stability or antibody production stability. Human antibodies of any isoform, for example, IgG, IgM, ΪgA, IgE or IgD, can be used to humanize antibodies. However, the present invention preferably uses IgG. IgG1, IgG2, IgG3, IgG4, etc. can be used as igG. The variable regions (e.g., CDRs and FRs) of the humanized antibody and the amino acid of the constant region can be deleted, added, deleted, and/or substituted into an amino acid after preparation. The antibody of the present invention also includes such a humanized antibody comprising an amino acid substitution or the like. The antibody ' of the present invention as long as it has IL_6 receptor binding activity and/or neutralizing activity includes not only a bivalent antibody represented by I gG but also a monovalent antibody, and a multivalent antibody represented by IgM. The multivalent antibody of the present invention includes a multivalent antibody having the same antigen binding site, and a multivalent antibody having all or part of antigen binding sites. The antibody of the present invention includes not only the intact antibody molecule but also the mini antibody 201100100 (minibody) and its modified product as long as it binds to the IL-6 receptor protein. A mini-antibody system comprises an antibody lacking an antibody fragment that is devoid of a portion of an intact antibody (eg, intact IgG, etc.) and, as long as it has an IL6 receptor, a tongue and/or neutralizing activity and contains a portion lacking an intact antibody:: fragment 'It is not particularly limited. The minibody of the present invention is not particularly limited as long as it has a part of an intact antibody. However, "miniature antibodies preferably comprise VH < VL, more preferably VH and VL. Other preferred antibodies of the invention include, for example, mini-antibodies comprising the CDRs of the antibody. Such mini-antibodies may comprise all or a portion of the six CDRs of the antibody. The mini-antibody of the present invention preferably has a molecular weight smaller than that of the intact antibody. However, such mini-antibodies can form multimers, such as dimers, trimers or tetramers, and thus their molecular weight will sometimes be greater than intact antibodies. In particular, antibody fragments include, for example, Fab, Fab', F(ab,) 2 and Fv. At the same time, 'minor antibodies include, for example, Fab, Fab, F (^ab,
Fv、svFc(單鏈 Fv)、雙抗體（diab〇dy)，及 sc(Fv)2(單鏈 (Fv)2)。此等抗體的多聚物（例如：二聚物、三聚物、四聚 物’及多聚物）’也包括在本發明之微小抗體。 抗體片段可藉由例如以酵素處理抗體以生產抗體片段 而得到。已知產生抗體片段的酵素’包括例如：木瓜酵素、 胰蛋白酶，及胞漿素（plasmin)。或者，可藉由將編碼為此 抗體片段的基因導入到一表現載體，並表現在適當的寄主 細胞而構築（見例如：Co, M.S. etal.，j. Immun〇1. Π 994) 152,2968-2976； Better, Μ. SHorwitz, A. H. Methods in Enzymology (1989) 178, 476-496； Pluckthun, A. & 40 201100100Fv, svFc (single-chain Fv), diabodies (diab〇dy), and sc(Fv)2 (single-chain (Fv) 2). Multimers (e.g., dimers, trimers, tetramers, and polymers) of such antibodies are also included in the minibody of the present invention. Antibody fragments can be obtained by, for example, treating an antibody with an enzyme to produce an antibody fragment. Enzymes which produce antibody fragments are known to include, for example, papain, trypsin, and plasmin. Alternatively, it can be constructed by introducing a gene encoding the antibody fragment into a expression vector and expressing it in an appropriate host cell (see, for example, Co, MS et al., j. Immun〇1. Π 994) 152, 2968 -2976; Better, Μ. SHorwitz, AH Methods in Enzymology (1989) 178, 476-496; Pluckthun, A. & 40 201100100
Skerra， A_ Methods in Enzymol〇gy(1989) 178， 476-496;Skerra, A_ Methods in Enzymol〇gy (1989) 178, 476-496;
Lamoyi, E. , Methods in Enzynio 1 ogy ( 1 989) 1 21, 652-663；Lamoyi, E. , Methods in Enzynio 1 ogy ( 1 989) 1 21, 652-663;
Rousseaux, j. et al_，Methods in Enzymology (1989) 121， 663-669; Bird, R.E. et al. , TIBTECH(1991) 9， 132-137 。 分解酵素切割於抗體片段的特定部位，產生如下示之 特定結構的抗體片段。基因工程技術可應用於此種酵素方 法得到的抗體片段，以刪除抗體的任意的部位。 〇 使用上述分解酵素得到的抗體片段如下： 木瓜酵素消化：F(ab)2或Fab 胰蛋白酶消化：F(ab’ ）2或Fab， 胞漿素消化汴acb 本發明之微小抗體，只要有IL — 6受體結合活性及/或 中和活性，包括欠缺任一區域的抗體片段。 「雙抗體（diabody)」，意指以基因融合構築的雙價抗 〇 體片段（11〇114^?6七31.，1 993，？1*0匕1^討1.人“(1.5(^· USA 90:6444-6448; EP 404, 097; W0 93/1 1 161 等）。雙抗 體係由2條多胜肽鏈組成的二聚物。於形成二聚物的各多 胜肽鏈，VL及VH —般係以同鏈的連結子（丨inker)連結。 一般而言，雙抗體中的連結子短至使得VL及VH不能彼此 結合。詳言之，構成連結子的胺基酸殘基數，例如約5個 殘基。因此，編碼在同一多胜肽上的VL及VH，不能形成 單鏈可變區片段，且將會與另一單鏈可變區片段形成二聚 物。結果，此雙抗體具有2個抗原結合部位。 41 201100100Rousseaux, j. et al_, Methods in Enzymology (1989) 121, 663-669; Bird, R.E. et al., TIBTECH (1991) 9, 132-137. The enzyme is cleaved at a specific portion of the antibody fragment to produce an antibody fragment of a specific structure as shown below. Genetic engineering techniques can be applied to antibody fragments obtained by this enzyme method to remove any part of the antibody.抗体 The antibody fragment obtained by using the above-mentioned decomposing enzyme is as follows: Papaya enzyme digestion: F(ab) 2 or Fab Trypsin digestion: F(ab') 2 or Fab, cytosolic digestion 汴acb The mini-antibody of the present invention, as long as IL - 6 receptor binding activity and / or neutralizing activity, including antibody fragments lacking any region. "diabody" means a bivalent anti-steroidal fragment constructed by gene fusion (11〇114^?6七31.,1 993,?1*0匕1^1. person" (1.5( ^· USA 90:6444-6448; EP 404, 097; W0 93/1 1 161, etc.). The double-antibody system is a dimer composed of two multi-peptide chains. , VL and VH are generally linked by a linker (丨inker). In general, the linker in the diabody is so short that VL and VH cannot bind to each other. In detail, the amino acid constituting the linker The number of residues, for example about 5 residues. Therefore, VL and VH encoded on the same polypeptide cannot form a single-chain variable region fragment and will form a dimer with another single-chain variable region fragment. As a result, the diabody has two antigen-binding sites. 41 201100100
ScFv抗體，係藉由以連結子等連結VH及VL而產生的 單鍵多胜肽（Huston, J. S. etal.，Proc. Natl. Acad. Sci. U.S.A. (1988) 85， 5879-5883 ; P1 ueckthun>, TheThe ScFv antibody is a single-bonded multi-peptide produced by linking VH and VL with a linker or the like (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883; P1 ueckthun> , The
Pharmacology 〇f Monoclonal Antibodies” Vo 1. 113, eds. , Resenburg and Moore, Springer Verlag, New York, pp· 269-31 5，（1 994))。scFv 的 H鏈 V 區及 L 鏈 V 區，可 自任何此處所述抗體而來。用於連結V區的胜肽連結子， 不特別限定。例如，可使用包含約3至25個殘基的任何單 鏈胜肽作為連結子。詳言之，可使用以下所述胜肽連結子 〇 等。 該一鏈的V區可藉由例如上述pcR連結。首先，編碼 如下示DNA之一的完整胺基酸序列或所欲的部分胺基酸序 列的DNA作為模板，經PCR連結該v區： 編碼抗體之Η鏈或Η鏈V區的DNA序列，及 編碼抗體之L鏈或L鏈V區的DNA序列。 編碼Η鏈或L鏈之V區的DNA，使用一對引子經PCR ◎ 擴增’該對引子含有欲擴增的DNA的兩端的對應序列。然 後’可製備編碼此胜肽連結子部分的DNA。此編碼胜肽連 結子的DNA也可利用PCR合成。將可用於連結分別合成的 V區擴增產物之核苷酸序列，加到欲使用的引子5，端。然 後，使用[Η鏈V區DNA]-[胜肽連結子dna]-[L鏈V區DNA] 的各DNA及組合PCR引子進行PCR。 此組合PCR引子，包括一組引子，其中之一與[η鏈v 區DNA的5’端]黏合，另一引子與[L鏈ν區卯幻之3，端 42 201100100 黏合。換5之，此組合PCR引子為一組引子，可使用於擴 增欲合成的編碼為scFv之全長序列的dna。同時，將可用 於連結各V區DNA的核酸序列加至[胜肽連結子ΜΑ ]。然 後，將此等DNA連結，並且使用組合引子產生完整的 scFv作為最後的擴增產物。一旦編碼scFv的D產生， 可藉由習知方法得到包含此等DNA的表現載體，及經此等 表現載體形質轉換的重組細胞。又，此scFv可經由培養得 〇 到的重組細胞而表現此scFv編碼DNA而獲得。 欲連結之VH及VL的順序不特別限定，可以任意順序 安排。可能的安排如下： [VH]連結子[vl] [VL]連結子[vh] sc(Fv)2為一單鏈微小抗體，係藉由使用連結子等連 結 2 個 VH 及 2 個 VL 而形成（Hudson et al.，1 999 了 hnmnol. Methods 231:1 77-1 89)。Sc(Fv)2 可例如使用— 〇 連結子連結scFv生產。 平父1主有 饥遯之2個”及。1固VL 以從單鏈夕 肽的N端，以vh、vl、vi^vl的順序安排（[VH]連結夕 連結子[VH]連結子[VL])'然而，2個VH及2個VL的順总 不限於上述安排’且可以任意順序安排。安排的例子如下. [VL]連結子[VH]連結子[VH]連結子[vl] .Pharmacology 〇f Monoclonal Antibodies" Vo 1. 113, eds. , Resenburg and Moore, Springer Verlag, New York, pp. 269-31 5, (1 994)). The H chain V region and the L chain V region of scFv can be The peptide linker for linking the V region is not particularly limited. For example, any single-stranded peptide containing about 3 to 25 residues can be used as a linker. The following peptide peptides can be used, etc. The V region of the one strand can be linked by, for example, the above pcR. First, the complete amino acid sequence encoding one of the DNAs shown below or the desired partial amino acid sequence The DNA is used as a template to link the v region by PCR: a DNA sequence encoding the Η or Η chain V region of the antibody, and a DNA sequence encoding the L chain or L chain V region of the antibody. The V region encoding the Η chain or the L chain The DNA is amplified by PCR using a pair of primers. 'The primer contains the corresponding sequence at both ends of the DNA to be amplified. Then 'DNA can be prepared to encode the linker of this peptide. This DNA encoding the peptide linker PCR synthesis can also be used. The nucleosides that can be used to link the separately synthesized V region amplification products The sequence was added to the end of the primer 5 to be used. Then, PCR was carried out using each DNA of [Η chain V region DNA]-[peptide peptide linker dna]-[L chain V region DNA] and a combination PCR primer. PCR primers, including a set of primers, one of which binds to [the 5' end of the η chain v region DNA], and the other primer binds to the [L chain ν region 卯 之 3, end 42 201100100. For 5, this combination The PCR primer is a set of primers which can be used to amplify the DNA encoding the full length sequence of the scFv to be synthesized. Meanwhile, the nucleic acid sequence which can be used to link the DNA of each V region is added to the [peptide linker ΜΑ]. These DNAs are ligated and a complete scFv is used as the final amplification product using a combination of primers. Once the D encoding scFv is generated, a representation vector containing such DNA can be obtained by a conventional method, and the expression trait transformation is performed by such a method. Further, the scFv can be obtained by expressing the scFv-encoding DNA via the cultured recombinant cells. The order of the VH and VL to be linked is not particularly limited and can be arranged in any order. The possible arrangement is as follows: [VH ] linker [vl] [VL] linker [vh] sc(Fv)2 is a single chain Small antibodies are formed by linking two VHs and two VLs using a linker or the like (Hudson et al., 1 999 hnmnol. Methods 231:1 77-1 89). Sc(Fv)2 can be used, for example, as 〇 Linker link scFv production. Pingfu 1 has two hungers and one. 1 solid VL is arranged from the N-terminus of the single-stranded peptide, in the order of vh, vl, vi^vl ([VH] link eve link [VH] linker [VL]) 'However, the cis of 2 VH and 2 VL is not limited to the above arrangement' and can be arranged in any order. Examples of the arrangement are as follows. [VL] linker [VH] linker [VH] linker [vl ] .
[VH]連結子[VL]連結子[VL]連結子[VH] [VH]連結子[VH]連結子[VL]連結子[vl] [VL]連結子[VL]連結子[VH]連結子[VH] 43 201100100 [VL]連結子[VH]連結子[VL]連結子[VH] 微小抗體的心或VL的胺基酸序列，可包括取代、删 除、加成及/或插入…只要職VL組合時具有抗原結 口活14 ’ m可删除一部分或也可加入其他的多胜肽。又， 此等可變區可為嵌合化或人型化。 本發月中可用於連結抗體可變區的連結子，包括可 用基因工程導人的任意胜肽連結子，及合成連結子，例如， Protern Engineering, ( 1 996)9(3)，299 — 3〇5 所揭露的連 結子0 本發月中的較佳連結子，為胜肽連結子。胜肽連結子 的長度不特別限定，熟習此項技藝的人士可依用途適當選 擇長度。典型的長度為丨〜丨⑽個胺基酸，較佳為3〜5〇個胺 基酸，又較佳為5〜30個胺基酸’更佳為12〜18個胺基酸（例 如，1 5個胺基酸）。 例如’用於胜肽連結子的胺基酸序列，包括以下序列： Ser[VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [vl] [VL] linker [VL] linker [VH] link Sub-[VH] 43 201100100 [VL] linker [VH] linker [VL] linker [VH] The amino acid sequence of the core or VL of a small antibody, which may include substitution, deletion, addition and/or insertion... When the occupational VL combination has an antigen junction activity of 14 'm, a part may be deleted or other multi-peptides may be added. Moreover, the variable regions can be chimeric or humanized. Linkers that can be used to link antibody variable regions in this month, including any peptide linkers that can be used by genetic engineering, and synthetic linkers, for example, Protern Engineering, (1 996) 9(3), 299-3 Link 5 disclosed in 〇5 The preferred linker in this month is the peptide linker. The length of the peptide linker is not particularly limited, and those skilled in the art can appropriately select the length depending on the application. Typical lengths are 丨~丨(10) amino acids, preferably 3 to 5 amino acids, and more preferably 5 to 30 amino acids' more preferably 12 to 18 amino acids (for example, 1 5 amino acids). For example, the amino acid sequence for the peptide linker includes the following sequence: Ser
Gly Ser Gly Gly Ser Ser Gly GlyGly Ser Gly Gly Ser Ser Gly Gly
Gly Gly Gly Ser(序列識別號 45)Gly Gly Gly Ser (sequence identification number 45)
Ser G1 y G1 y G1 y (序列識別號 46)Ser G1 y G1 y G1 y (sequence identification number 46)
Gly Gly Gly Gly Ser(序列識別號 47)Gly Gly Gly Gly Ser (sequence identification number 47)
Ser Gly Gly Gly Gly(序列識別號 48)Ser Gly Gly Gly Gly (sequence identification number 48)
Gly Gly Gly Gly Gly Ser(序列識別號 49) 44 201100100Gly Gly Gly Gly Gly Ser (Sequence ID 49) 44 201100100
Ser Gly Gly Gly Gly Gly(序列識別號 5〇)Ser Gly Gly Gly Gly Gly (Sequence ID 5〇)
Gly Gly Gly Gly Gly Gly Ser(序列識別號 51)Gly Gly Gly Gly Gly Gly Ser (Sequence ID 51)
Ser Gly Gly Gly Gly Gly Gly(序列識別號 52) (Gly Gly Gly Gly Ser (序列識別號 a) )n (Ser Gly Gly Gly Gly(序列識別號 48))n 其中，n為1以上的整數。 胜肽連結子的胺基酸序列，可由熟習該技術領域之人 士依照用途適當選擇。例如，上述決定胜肽連結子長度 的’’ η” ，通常為1〜5，較佳為1〜3，又較佳為1〜2。 合成的連結子（化學性交聯劑），包括例行性用於交聯 胜肽的交聯劑，例如，Ν-羥基琥珀醯亞胺（NHS)、二琥珀醯 亞胺辛二酸酯（DSS)、雙（磺基琥珀醯亞胺基）辛二酸酯 (BS3)、二硫雙（琥珀醯亞胺基丙酸酯）（DSP)、二硫雙（磺基 琥轴醯亞胺基丙酸酯）（DTSSP)、乙二醇雙（琥珀醯亞胺基琥 拍酸酯）（EGS)、乙二醇雙（磺基琥珀醯亞胺基琥珀酸酯X磺 Q 基—EGS)、雙琥珀醯亞胺基酒石酸酯（DST)、雙磺基琥珀醯 亞胺基酒石酸酯（磺基—DST)、雙[2-琥珀醯亞胺基氧羰基氧] 乙基]颯（磺基-BS0C0ES)。此等交聯劑，為市售可得。 一般而言’需要3個連結子來連結4個抗體可變區。 此等多數連結子可為相同或不同的連結子。 本發明中的抗體也包括在一抗體的胺基酸序列加入1 個以上胺基酸的抗體。再者’本發明之抗體也包括上述抗 體與另一胜肽或蛋白質融合的融合蛋白質。此融合蛋白質 可藉由以下方式製備：將編碼一抗體之多核苷酸及編碼另 45 201100100 一胜狀或多胜肽的多核苷酸在框架中連接，將此dNA導入 一表現載體，並且於寄主中表現此載體。可使用熟習此項 技術領域之人士已知的技術。與本發明之抗體融合的胜肽 或多胜肽，可為一已知胜肽，例如，FLAG(H〇pp，T. p et ai.，Ser Gly Gly Gly Gly Gly (Sequence ID 52) (Gly Gly Gly Gly Ser (sequence number a)) n (Ser Gly Gly Gly Gly (sequence number 48)) n where n is an integer of 1 or more. The amino acid sequence of the peptide linker can be appropriately selected by those skilled in the art according to the use. For example, the above-mentioned ''η" which determines the length of the peptide linker is usually 1 to 5, preferably 1 to 3, and more preferably 1 to 2. Synthetic linker (chemical crosslinker), including routine Crosslinking agent for cross-linking peptides, for example, Ν-hydroxysuccinimide (NHS), disuccinimide suberate (DSS), bis(sulfosuccinimide) octane Acid ester (BS3), disulfide (amber succinimide propionate) (DSP), disulfide (sulfosuccinyl imino propionate) (DTSSP), ethylene glycol bis (amber bismuth) Iminosyl succinate) (EGS), ethylene glycol bis(sulfosuccinimide succinate X sulfo Q--EGS), adiammonium imino tartaric acid ester (DST), disulfo Amber quinone iartrate tartrate (sulfo-DST), bis[2-arene quinone oxycarbonyloxy]ethyl] sulfonium (sulfo-BS0C0ES). These crosslinkers are commercially available. In general, three linkers are required to link four antibody variable regions. These majority linkers may be the same or different linkers. The antibodies of the present invention also include the addition of one amino acid sequence to one antibody. Take An antibody to an amino acid. Further, the antibody of the present invention also includes a fusion protein in which the above antibody is fused to another peptide or protein. The fusion protein can be produced by: encoding a polynucleotide encoding an antibody and encoding another 45 201100100 A polynucleotide of a single or multiple peptide is ligated in a framework, the dNA is introduced into a performance vector, and the vector is expressed in a host. Techniques known to those skilled in the art can be used. The peptide or multi-peptide of the antibody fusion of the invention may be a known peptide, for example, FLAG (H〇pp, T. p et ai.,
BioTechnology 6，1 204-1 21 0( 1 988)) ; 6xHis，由 Θ 個組胺 酸（histidine)殘基組成；i〇x His;流感病毒凝集素（HA)、 人類 c-myc 片段、VSV-GP 片段、pi8HIV 片段、T7-tag、 HSV-tag、E-tag、SV40T 抗原片段、lcktag、a_tubuHn 片^、B-tag，及蛋白c片段。與本發明之抗體融合的多 Ο 胜肽，例如，GST(谷胱甘肽-S-轉移酶）、HA(流感病毒凝集 素）、免疫球蛋白恆定區、石-半乳糖苷酶，及MBp(麥芽糖 結合蛋白質）。可將市售可得的編碼為此等胜肽或多胜肽的 多核苷酸與編碼為本發明所述抗體之多核苷酸融合。融合 多胜肽可藉由表現如此得到的融合多核苷酸而製備。 又，本發明所述抗體也可為連結到各種分子的接合抗 體，各種分子例如，聚合物，包括聚乙二醇（pEG)及透明質 酸；放射性物質；螢光物質；冷光物質；酵素，及毒素。此等1 接合抗體可藉由將得到的抗體以化學性修飾獲得。抗體修 飾的方法在此技術領域中已建立（見例如US5〇573i3， US51 56840)。本發明之，，抗體，，也包括此等接合抗體。 又，本發明之抗體包括具有經改變之糖鏈的抗體。 又，本發明中使用的抗體可為雙特異性抗體 (bispecific antibody)。雙特異性抗體係指在同一抗體分 子上具有可辨識不同抗原決定位的可變區。本發明中之雙 46 201100100 特異性抗體可為辨識IL-6受體分子上之不同抗原決定位 的雙特異性抗體’或者其中一抗原結合部位辨識〗l_6受體 且另一抗原結合部位辨識另一物質的雙特異性抗體。結合 於包括本發明之IL-6受體辨識抗體的雙特異性抗體的^ 一抗原結合部位的抗原，包括例如，iL_6、TNF α、tnfri、 TNFR2、CD80、CD86、CD28、CD20、CD19、IL-l α、IL_beta IL-1R 、 RANKL 、 RANK 、 IL-17 、 IL-17R 、 IL—23 、 IL—23R 、BioTechnology 6, 1 204-1 21 0 (1 988)); 6xHis, consisting of a few histidine residues; i〇x His; influenza virus lectin (HA), human c-myc fragment, VSV - GP fragment, pi8 HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lcktag, a_tubuHn fragment, B-tag, and protein c fragment. Polypeptides fused to the antibody of the present invention, for example, GST (glutathione-S-transferase), HA (influenza lectin), immunoglobulin constant region, stone-galactosidase, and MBp (maltose binding protein). Commercially available polynucleotides encoding such peptides or peptides can be fused to polynucleotides encoding the antibodies of the invention. The fusion multi-peptide can be prepared by expressing the fusion polynucleotide thus obtained. Further, the antibody of the present invention may also be a conjugated antibody linked to various molecules, such as polymers, including polyethylene glycol (pEG) and hyaluronic acid; radioactive substances; fluorescent substances; luminescent substances; enzymes, And toxins. These 1 conjugated antibodies can be obtained by chemically modifying the obtained antibodies. Methods of antibody modification have been established in the art (see, for example, US 5 〇 573i3, US 51 56840). In the present invention, antibodies, also including such conjugated antibodies. Further, the antibody of the present invention includes an antibody having an altered sugar chain. Further, the antibody used in the present invention may be a bispecific antibody. A bispecific anti-system refers to a variable region on the same antibody molecule that recognizes different epitopes. In the present invention, the double 46 201100100 specific antibody may be a bispecific antibody that recognizes different epitopes on the IL-6 receptor molecule or one of the antigen binding sites recognizes the l_6 receptor and another antigen binding site recognizes another A bispecific antibody of a substance. An antigen that binds to an antigen binding site of a bispecific antibody comprising an IL-6 receptor recognition antibody of the present invention, including, for example, iL_6, TNFα, tnfri, TNFR2, CD80, CD86, CD28, CD20, CD19, IL -l α, IL_beta IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R,
il—15、IL—15R、Biys、淋巴毒素《、淋巴毒素石、UGHT 配體、LIGHT、VLA-4、CD25、IL-12、IL-12R、CD40、CD40L、 BAFF、CD52、CD22、IL-32、IL-21、IL-21R、GM-CSF、GM-CSFR、 M-CSF、IFN- a、VEGF、VEGFR、VEEGF、EGFR、CCR5、APRIL， 及 APRILR 。 用於產生雙特異性抗體之方法為已知。雙特異性抗體 可藉由例如將兩種類型的辨識不同抗原的抗體連結而製 備。欲連結的抗體可為半分子，各包含一Η鏈及一l鏈， 〇或四分之一分子，僅包括一Η鏈。或者，可利用融合生產 不同單株抗體之融合瘤，而製備生產雙特異性抗體的融合 、田胞又，可利用基因工程技術生產雙特異性抗體。 如下述，本發明使用的抗體視精製方法或用於生產此 抗體的細胞或寄主，在胺基酸序列、分子量、等電點、存 在/不存在糖鏈，及型態，可能有所不同。然而，只要所得 J的抗體在功忐上等同於本發明所述抗體，則應視為包含 於本毛明中。例如，當於原核細胞例如大腸桿菌中表現一 抗體則甲硫胺酸會加到原始的抗體胺基酸序列的Ν末 47 201100100 端。此等抗體也包括在本發明所述抗體。 本發明之抗IL-6受體抗體之多胜肽等，可利用熟習該 技術領域之人士已知方法生產。 抗IL-6受體抗體，可利用熟習該技術領域之人士已知 的基因重組技術，依據所得到的抗IL — 6受體抗體的序列製 備。詳言之，抗IL-6受體抗體可藉由依據IL_6受體辨識 抗體的序列’構築編碼該抗體之多核苷酸，將此多核苷酸 插入一表現載體，然後將其於適當的寄主細胞中表現而製 備（參見例如 ’ Co, Μ· S. et al .，J. Immunol. (1994) 152, 2968-2976；Better, M. and Horwitz, A. H. , Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515；Il-15, IL-15R, Biys, lymphotoxin, lymphotoxin, UGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL- 32. IL-21, IL-21R, GM-CSF, GM-CSFR, M-CSF, IFN-a, VEGF, VEGFR, VEEGF, EGFR, CCR5, APRIL, and APRILR. Methods for producing bispecific antibodies are known. Bispecific antibodies can be prepared, for example, by linking two types of antibodies that recognize different antigens. The antibodies to be linked may be half molecules, each comprising an Η chain and an l chain, 〇 or a quarter molecule, including only one Η chain. Alternatively, a fusion-producing fusion of different monoclonal antibodies can be used to prepare a fusion-producing bispecific antibody, and the cell can be produced by genetic engineering techniques. As described below, the antibody used in the present invention may differ depending on the purification method or the cell or host used to produce the antibody in the amino acid sequence, molecular weight, isoelectric point, presence/absence of a sugar chain, and form. However, as long as the antibody of the obtained J is functionally equivalent to the antibody of the present invention, it should be considered to be included in the present invention. For example, when an antibody is expressed in a prokaryotic cell such as E. coli, methionine is added to the end of the original antibody amino acid sequence at the end of the cell 47 201100100. Such antibodies are also included in the antibodies of the invention. The multipeptide or the like of the anti-IL-6 receptor antibody of the present invention can be produced by a method known to those skilled in the art. The anti-IL-6 receptor antibody can be prepared based on the sequence of the obtained anti-IL-6 receptor antibody by genetic recombination techniques known to those skilled in the art. In particular, an anti-IL-6 receptor antibody can be constructed by constructing a polynucleotide encoding the antibody based on the sequence of the IL_6 receptor recognition antibody, inserting the polynucleotide into a expression vector, and then placing it in an appropriate host cell. Prepared for performance (see, for example, 'Co, Μ·S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476- 496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515;
Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;
Rousseaux, J. et al., Methods Enzyraol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W. , TrendsRousseaux, J. et al., Methods Enzyraol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W. , Trends
Biotechnol. (1991) 9，132-137)。 因此，本發明提供（i)生產本發明之多胜肽之方法，或 (i i)生產一多胜肽之方法，該多胜肽係由編碼本發明之多 胜肽之基因編碼，其中’該等方法包含以下步驟：培養包含 一載體之寄主細胞’其中，該載體中導入有編碼本發明之 多胜肽的多核苷酸。 詳言之’本發明提供生產本發明之多胜肽之方法，包 含以下步驟： (a)培養一寄主細胞，該寄主細胞包含一載體，該載體 48 201100100 中導入有編碼為本發明之多胜肽的基因； (b)得到由此基因編碼的多胜肽。 载體之例子，包括：M13型載體、pUC型載體、pBR322、 pBluescript及pCR_Script。或者，當目標經由次選殖及 切下cDNA，上述以外的載體例子尚包括：pgem-T、pDIRECT 及pT7。表現載體尤其有用於生產本發明所述抗體。例如， 當表現載體用於表現在大腸桿菌，則此載體應具備能在大 〇 腸桿菌中擴增的特徵。此外，當寄主為大腸桿菌例如 JM1 09、DH5 a、HB101或XU-Blue，具有能使其有效率地 在大腸桿菌中表現的啟動子為必要，例如，1 acZ啟動子 (Ward et al., Nature(1989) 341, 544-546; FASEB J. ( 1 992) 6，2422-2427)、araB 啟動子（Better et al·， Science( 1 988)240，1 041-1 043)、T7 啟動子等。除 了上述 以外，此種載體包括 pGEX-5X-l(PharmaCia)、，，QlAexpress system”（Quiagen)、pEGFP，及 pET(於此情形，寄主較佳 Q 為BL21 ’其表現T7 RAN聚合酶）。 又’表現質體載體可包含針對抗體分泌的訊息序列。 就用於抗體分泌的訊息序列而言，可使用pe丨B訊息序列 (Lei, S.P. et al., J. Bacteriol. (1987) 169, 4379) 以提供在大腸桿菌胞外質内生產。載體可利用例如氣化躬 法或電穿孔法導入到寄主細胞内。 除了針對大腸桿菌的載體，用於生產本發明所述抗體 的載體’包括例如：哺乳動物衍生的表現載體（例如Biotechnol. (1991) 9, 132-137). Accordingly, the present invention provides (i) a method of producing a multi-peptide of the present invention, or (ii) a method of producing a multi-peptide, which is encoded by a gene encoding a multi-peptide of the present invention, wherein The method comprises the steps of: cultivating a host cell comprising a vector into which a polynucleotide encoding the multi-peptide of the present invention is introduced. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing a multi-peptide of the present invention comprising the steps of: (a) cultivating a host cell comprising a vector, the vector 48 201100100 incorporating a coding for the invention a gene for the peptide; (b) a polypeptide encoded by the gene. Examples of vectors include: M13 type vector, pUC type vector, pBR322, pBluescript, and pCR_Script. Alternatively, when the target is sub-selected and the cDNA is excised, examples of vectors other than the above include: pgem-T, pDIRECT, and pT7. Expression vectors are especially useful for the production of the antibodies of the invention. For example, when the expression vector is used for expression in E. coli, the vector should be characterized by amplification in E. coli. Further, when the host is Escherichia coli such as JM1 09, DH5 a, HB101 or XU-Blue, it is necessary to have a promoter capable of efficiently expressing it in Escherichia coli, for example, an acZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1 992) 6, 2422-2427), araB promoter (Better et al, Science (1 988) 240, 1041 043), T7 promoter Wait. In addition to the above, such vectors include pGEX-5X-1 (PharmaCia),, QlAexpress system" (Quiagen), pEGFP, and pET (in this case, the host preferably Q is BL21 'which expresses T7 RAN polymerase). Further, the 'plasty vector can contain a message sequence for antibody secretion. For a message sequence for antibody secretion, a pe丨B message sequence can be used (Lei, SP et al., J. Bacteriol. (1987) 169, 4379) for production in the extracellular cytoplasm of E. coli. The vector can be introduced into the host cell by, for example, gasification hydrazine or electroporation. In addition to the vector for E. coli, the vector used to produce the antibody of the present invention includes For example: mammalian derived expression vectors (eg
PcDNA3(Invitrogen) 、 pEF-BOS(Nucleic Acids. 49 201100100PcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. 49 201100100)
Res. ( 1 990) 1 8( 1 7)，p5322) ' pEF 及 pCDM8)、昆蟲細胞衍生 之表現載體（比如 Bac-to-BAC baculovirus expression system”（Gibco-BRL)及pBacPAK8)、植物衍生之表現載體 (例如’ pMHl及pMH2)、動物病毒衍生之表現載體（比如， pHSV、pMV及pAdexLcw)、反轉錄病毒衍生之表現載體（比 如pZIPneo)、酵母菌衍生之表現載體（比如” PichiaRes. ( 1 990) 1 8( 1 7), p5322) 'pEF and pCDM8), insect cell-derived expression vectors (eg Bac-to-BAC baculovirus expression system) (Gibco-BRL) and pBacPAK8), plant-derived Expression vectors (eg 'pMH1 and pMH2), animal virus-derived expression vectors (eg pHSV, pMV and pAdexLcw), retroviral-derived expression vectors (eg pZIPneo), yeast-derived expression vectors (eg "Pichia"
Expression Kit”（Invitrogen)、pNVll 及 SP-Q01)，及枯 草桿菌（Bacillus subtil is)衍生之表現載體（例如ppl608 及 PKTH50)。 當使用表現質體載體於動物細胞例如CH0、C0S及 NIH3T3中表現時，必需具有用於在此等細胞中表現的啟動 子，例如：SV40 啟動子（Mul 1 igan et al.，Nature( 1 979)277, 108)、MMLV-LTR 啟動子、EF1 <2 啟動子（Miziishimaetal.， Nucleic Acids Res_ ( 1 990) 18，5322，或 CMV 啟動子。更 佳為’此載體具有用於選擇經形質轉換之細胞的基因（例 如’抗藥性基因’其容許以藥劑（新徽素（ne〇myc i n)、G418 等）區分。具有此等特徵的載體包括例如：ρΜΑΜ、pI)R2、 pBK-RSV 、 pBK-CMV 、 p〇PRSV ，及 p0P13 。 此外，當目標穩定地表現基因及在細胞中擴增基因複 製數’可使用一方法，對於欠缺核酸合成路徑之CH〇細胞 $入具有DHFR基因之載體以彌補此欠缺（例如， pSV2-dfhr( “Molecular Cloning 2nd edition” Cold Spring Harbor Laboratory Press，（1989))，並將此載體 使用胺甲葉酸（methotrexate，MTX)擴增。又，當目標為過 50 201100100 渡性的基因表現，可使用一方法，將帶有表現sv 4〇 τ抗Expression Kit" (Invitrogen), pNVll and SP-Q01), and Bacillus subtil is derived expression vectors (eg, ppl608 and PKTH50). When expressed using plastid vectors in animal cells such as CH0, COS and NIH3T3 At the time, it is necessary to have a promoter for expression in such cells, for example: SV40 promoter (Mul 1 igan et al., Nature (1 979) 277, 108), MMLV-LTR promoter, EF1 < 2 promoter (Miziishima et al., Nucleic Acids Res_ (1 990) 18, 5322, or CMV promoter. More preferably 'this vector has a gene for selecting cells that have undergone morphogenetic transformation (eg 'drug resistance gene' which allows for the administration of a drug ( New 素素 (ne〇myc in), G418, etc.). Carriers with such characteristics include, for example, ρΜΑΜ, pI)R2, pBK-RSV, pBK-CMV, p〇PRSV, and p0P13. A gene expression gene and a gene copy number in a cell can be used to compensate for this deficiency by using a vector having a DHFR gene for a CH cell lacking a nucleic acid synthesis pathway (for example, pSV2-dfhr ("Molecular Cloning" 2nd edition" Cold Spring Harbor Laboratory Press, (1989)), and this vector is amplified using methotrexate (MTX). Also, when the target is over 50 201100100, the gene expression can be used, With performance sv 4〇τ
原於其染色體的基因的cos細胞以帶有SV4〇複製起點（pcD 等）的載體形質轉換。可使用衍生自多瘤病毒、腺病毒、牛 乳突瘤病毒（BPV)等的複製起點。又，為了擴增寄主細胞株 中的基因複製數，此表現載體可包含胺基配糖體轉移酶 (ΑΡΗ)基因、胸腺嘧啶激酶（τκ)基因、大腸桿菌黃嘌呤—鳥 糞嘌呤磷酸核糖基轉移酶（Ec〇gpt)基因、二氫葉酸還原酶 (dhfr)基因等，作為選擇標記。 〇 _ 得到之本發明所述抗體，可從寄主細胞或從細胞外 (培養基等）單離’並且純化成實質上為純且同質的抗體。 此等抗體可使用習知之分離及用於抗體精製的方法精製， 而不限定。例如’抗體可藉由適當選擇及組合管柱層析、 過濾、超過濾、鹽析、溶劑沉澱、溶劑萃取、蒸餾、免疫 沉澱、SDS聚丙烯醯胺凝膠電泳、等電聚膠、透析、再結 晶等，而分離及精製。 〇 層析包括例如：親和性層析、離子交換層析、疏水性 層析、凝膠過濾、反向層析，及吸附層析（Strategies for Protein Purification and Characterization : A Laboratory Course Manual . Ed Daniel R. Marshak et al. Cold Spring Harbor Laboratory Press, 1 996)。此等層 析可使用液相層析例如，HPLC、FPLC實施。用於親和性層 析的管柱，包括proteinA管柱，及proteinG管柱。使用 proteinA 管柱之例，包括 HyperD、P0R0S 及 Sepharose FF(GE Amersham Biosciences)。本發明尚包括使用此等精 51 201100100 製方法向度精製的抗體。 得到的抗體的IL-6受體結合活性，可利用熟習該技術 領域之人士已知的方法測定。用於測定抗體之抗原結合活 性的方法，包括例如：酵素連結免疫吸附試驗（EUsa)、 酵素免疫分析（EIA)、放射性免疫分析（RIA)及螢光抗 體方法。例如’當使用酵素免疫分析，係將含有抗體之: 本例如精製過的抗體及抗體生產細胞的培養上清，加至塗 佈抗原的平盤。加入標記有酵素例如鹼性磷解酶的二次抗 體。並將此等平盤溫育。清洗後，加入酵素基質例如對： 基苯基磷酸酯，並且測定吸光值以評估抗原結合活性。 醫藥組合物 本發明提供醫藥組合物，其包含上述多胜肽作為有效 成分。本發明之醫藥組合物，可使用在IL_6相關疾病，例 如類風濃性關節炎。因此，本發明也提供一種藥劑，用於 治療疾病例如，類風濕性關節炎，其包含—種上述抗體作 為有效成分。較佳的目標疾病之例子’包括但不限於:類風 濕性關m年特發性關節炎、全身性幼年特發性關節 炎、卡斯托曼病（Castleman disease)氏病、全身性紅斑性 狼瘡（SLE)、狼瘡腎炎、Crc)hn氏病、淋巴瘤、潰癌性大腸 炎、貧血、血管炎、川崎病、stiu氏病、類澱粉沉積症、 夕發性硬化症、移殖、年齡相關之視網膜斑退化、僵直性 脊椎炎、牛皮癖、牛皮癬關節炎、慢性阻塞性肺病（c〇pD)、 IgA腎病 '骨性關節炎、氣喘病、糖尿病性腎病、㈣D、 内臈異位、肝炎咖）、心肌梗塞 '動脈硬化、敗血症、 52 201100100 骨質疏鬆症、糖尿病、多重骨髓瘤、前列腺癌、腎癌、Β 細胞非Hodgkin氏淋巴瘤、胰臟癌、肺癌、食道癌、直腸 癌、癌惡病質、癌神經侵入、心肌梗塞、近視脈絡膜新生 血管、特發性脈絡膜血管新生、葡萄膜炎、慢性甲狀腺炎、 延遲的過敏、接觸性皮膚炎、過敏性皮膚炎、間皮瘤、多 發性肌炎、皮肌炎、全葡萄膜炎、前葡萄膜炎、中葡萄膜 炎、鞏膜炎、角膜炎、眼窩發炎、視神經炎、糖尿病性視 Ο 〇 網膜病變、增殖性玻璃體視網膜病變、乾眼症，及手術後 發炎。 用語「包含一抗IL-6受體抗體作為有效成分」，係指 包含一抗IL — 6受體抗體作為至少一種有效成分，但不特別 限制其内容。X，本發明之醫藥組合物，可以組合上述多 胜肽及其他有效成分。 本發明之醫藥組合物，可使用於治療用途，也可用於 預防用途。 本發明之胺基酸序列包括的胺基酸，可經轉譯後修 飾。例如，利用焦谷胺醯胺酸化將N末端的谷酿胺（Gin) 殘基修飾為焦谷胺酸（pGIu)殘基，為熟習該技術領域之人 士周知的方法。自然地，此種經轉譯後修飾之胺基酸也包 括在本發明之胺基酸序列。 結合到本發明之抗體的糖鏈，可為任意結構。297位 ⑽編號法）的糖鏈，可為任意糖鏈結構（較 糖鍵），或在該位置不具糖鍵(例如，可藉由在大腸桿菌中 ^體’或藉由導入改變使得在297位⑽編號法）無糖 53 201100100 鏈結合）。 所有在此引用的先前技術，引入於此說明書作為參考。 實施例 以下，將由實施例協助說明本發明，但應了解不限定 於此等實施例。 實施例1 鑑別可變區之突變部位以供增強T0CILIZUMAB對於 IL-6受體之親和性 構築一已導入有突變之CDR序列之基因庫，並分析以 改良TOCILIZUMABCH鏈WT-IgGl/序列識別號53;L鏈 WT-/C /序列識別號54)對於IL-6受體之親和性。CDR突變 庫之篩選，找出改良針對IL-6受體之親和性的突變。此突 變顯示於圖1。此等突變的組合得到高親和性 T0CILIZUMAB，例如，RDC-23(H 鏈 RDC23H-IgGl/序列識別 號55;L鏈RDC-23L-/c /序列識別號56)。在RDC-23及 T0CILIZUMAB之間，比較對可溶性IL-6受體之親和性，以 及使用BaF/gp 1 30確認的生物活性（見此方法之參考實施 例）。 親和性測定的結果顯示於表1。使用BaF/gpl30確認 之生物學活性結果（IL-6之最終濃度為3〇ng/ml)，顯示於 圖2。結果顯示’與T0CILIZUMAB相比，RDC-23之親和性 約南6 0倍’且以B a F / g p 1 3 0之1 〇 〇 %抑制濃度表示的活性， 約高100倍。 '201100100 ' [表 1] ·~ ~—— ______ _K^Cl/MS) _J^U/S)_KD(M) TOCILIZUMAB 4. 9E + 05 4. QE-09 _gDC-23 6.4E + 05 4. 6.7E-11 實施例2 鑑別突變以供經由減低等電點而改良T〇CilIZUMAB之 藥物動力學 〇 為了改良TOCILIZUMAB之藥物動力學，檢查以鑑別會 降低可變區之等電點但不會顯著降低對於I L_6受體之結 合的突變部位。篩選可變區中的突變部位，其係依照 TOC ILIZUMAB之三維結構模型預測者，找出會降低可變區 之等電點但不會顯著降低對於IL-6受體之結合的突變部 位。此等顯示於圖3。組合此等突變，得到具有降低之等 電點的 TOCILIZUMAB，包括例如：H53/L28CH 鏈 H53-IgGl/ 〇 序列識別號57;L鏈L28-/C /序列識別號58)。在H53/L28 及T0CILIZ丽AB之間，比較針對il-6受體之親和性、等電 點、於小鼠中之藥物動力學，及使用BaF/gpl 30確認之生 物學活性（見本方法之參考實施例）。 親和性測定之結果，如表2所示。使用BaF/gpl 30得 到之生物學活性之測定結果（IL-6之最終濃度為30ng/ml) 如圖4所示。結果顯示，比起TOCILIZUMAB，H53/L28之親 和性高約6倍’且以BaF/gpl3〇之100%抑制濃度表示之活 性高約數倍。 55 201100100 [表2 ]The cos cells of the gene originally derived from its chromosome are transformed with a vector having a SV4〇 replication origin (pcD, etc.). An origin of replication derived from polyomavirus, adenovirus, bovine papilloma virus (BPV), or the like can be used. Furthermore, in order to amplify the number of gene copies in the host cell strain, the expression vector may comprise an aminoglycoside transferase (ΑΡΗ) gene, a thymidine kinase (τκ) gene, Escherichia coli, guanosine-guanine phosphoribosyl The transferase (Ec〇gpt) gene, the dihydrofolate reductase (dhfr) gene, and the like are used as selection markers. The antibody of the present invention obtained can be isolated from the host cell or from the outside of the cell (medium, etc.) and purified into a substantially pure and homogeneous antibody. Such antibodies can be purified using conventional isolation and methods for antibody purification, without limitation. For example, 'antibody can be selected and combined by column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric polymerization, dialysis, Recrystallization, etc., separation and purification. Helium chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R Marshak et al. Cold Spring Harbor Laboratory Press, 1 996). Such stratification can be carried out using liquid chromatography, for example, HPLC, FPLC. Columns for affinity chromatography, including proteinA columns, and proteinG columns. Examples of proteinA columns are used, including HyperD, P0R0S, and Sepharose FF (GE Amersham Biosciences). The present invention also encompasses antibodies that have been refined to a degree using the method of the method of Precision 51 201100100. The IL-6 receptor binding activity of the obtained antibody can be measured by a method known to those skilled in the art. Methods for determining the antigen binding activity of an antibody include, for example, an enzyme-linked immunosorbent assay (EUsa), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), and a fluorescent antibody method. For example, when an enzyme immunoassay is used, an antibody containing the antibody, such as a purified antibody and an antibody-producing cell, is added to a flat plate on which an antigen is applied. A secondary antibody labeled with an enzyme such as alkaline phospholyase is added. And these flats are incubated. After washing, an enzyme substrate such as p-phenyl phosphate was added, and absorbance was measured to evaluate antigen binding activity. Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising the above polypeptide as an active ingredient. The pharmaceutical composition of the present invention can be used in diseases related to IL-6, such as rheumatoid arthritis. Accordingly, the present invention also provides an agent for treating a disease such as rheumatoid arthritis, which comprises the above-mentioned antibody as an active ingredient. Examples of preferred target diseases include, but are not limited to, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, systemic erythema Lupus (SLE), lupus nephritis, Crc)hn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, stiu's disease, amyloidosis, sclerosing sclerosis, colonization, age Related retinal plaque degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (c〇pD), IgA nephropathy 'osteoarthritis, asthma, diabetic nephropathy, (d) D, internal hemorrhoids, Hepatitis), myocardial infarction, arteriosclerosis, sepsis, 52 201100100 Osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, sputum cells, non-Hodgkin's lymphoma, pancreatic cancer, lung cancer, esophageal cancer, rectal cancer, Cancer cachexia, cancerous nerve invasion, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed allergy, contact dermatitis, allergies Dermatitis, mesothelioma, polymyositis, dermatomyositis, uveitis, anterior uveitis, uvitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic paralytic sputum omental lesions Proliferative vitreoretinopathy, dry eye, and inflammation after surgery. The phrase "containing a primary antibody against IL-6 receptor as an active ingredient" means containing a primary antibody against an IL-6 receptor as at least one active ingredient, but the content thereof is not particularly limited. X. The pharmaceutical composition of the present invention may be combined with the above-mentioned multi-peptide and other active ingredients. The pharmaceutical composition of the present invention can be used for therapeutic purposes as well as for prophylactic use. The amino acid included in the amino acid sequence of the present invention can be modified after translation. For example, the modification of the N-terminal glutamine (Gin) residue to a pyroglutamic acid (pGIu) residue by pyroglutamine guanamine is a method well known to those skilled in the art. Naturally, such post-translationally modified amino acids are also included in the amino acid sequences of the present invention. The sugar chain which binds to the antibody of the present invention may be of any structure. The 297 (10) numbered sugar chain may be any sugar chain structure (more sugar linkage), or may have no sugar linkage at this position (for example, by electrophoresis in E. coli) or by introduction of a change in 297 Bit (10) numbering method) No sugar 53 201100100 Chain combination). All prior art cited herein is incorporated herein by reference. EXAMPLES Hereinafter, the invention will be described by the examples, but it should be understood that the invention is not limited thereto. Example 1 Identification of Mutant Sites of Variable Regions for Enhancing the Affinity of TOCILIZUMAB for IL-6 Receptor Construction of a Genebank into Which a Mutant CDR Sequence Has Been Introduced, and Analysis to Improve TOCILIZUMABCH Chain WT-IgGl/SEQ ID NO: 53 ; L chain WT-/C / SEQ ID NO: 54) Affinity for IL-6 receptor. Screening of the CDR Mutant Library to identify mutations that improve the affinity for the IL-6 receptor. This mutation is shown in Figure 1. The combination of these mutations results in a high affinity T0CILIZUMAB, for example, RDC-23 (H chain RDC23H-IgGl/SEQ ID NO: 55; L chain RDC-23L-/c/SEQ ID NO: 56). The affinity for the soluble IL-6 receptor was compared between RDC-23 and T0CILIZUMAB, and the biological activity confirmed using BaF/gp 1 30 (see the reference example of this method). The results of the affinity measurement are shown in Table 1. The biological activity results confirmed by BaF/gpl30 (the final concentration of IL-6 was 3 ng/ml) are shown in Fig. 2. The results showed that the affinity of RDC-23 was about 60 times higher than that of TOCILIZUMAB and the activity expressed by the inhibition concentration of B a F / g p 1 3 0 〇 〇 % was about 100 times higher. '201100100 ' [Table 1] ·~ ~—— ___ _K^Cl/MS) _J^U/S)_KD(M) TOCILIZUMAB 4. 9E + 05 4. QE-09 _gDC-23 6.4E + 05 4. 6.7 E-11 Example 2 Identification of mutations for improved pharmacokinetics of T〇CilIZUMAB via reduced isoelectric point 〇 In order to improve the pharmacokinetics of TOCILIZUMAB, examination to identify decreases the isoelectric point of the variable region but does not significantly decrease Mutation site for binding of the I L_6 receptor. Mutation sites in the variable region were screened according to the predictors of the three-dimensional structural model of TOC ILIZUMAB to identify mutation sites that would reduce the isoelectric point of the variable region but did not significantly reduce binding to the IL-6 receptor. These are shown in Figure 3. Combining these mutations yields TOCILIZUMAB with reduced isoelectric point, including, for example, H53/L28CH chain H53-IgGl/[SEQ ID NO: 57; L chain L28-/C/SEQ ID NO: 58). The affinity for the il-6 receptor, the isoelectric point, the pharmacokinetics in mice, and the biological activity confirmed using BaF/gpl 30 were compared between H53/L28 and T0CILIZ AB (see method Reference example). The results of the affinity measurement are shown in Table 2. The results of the biological activity obtained using BaF/gpl 30 (the final concentration of IL-6 was 30 ng/ml) are shown in Fig. 4. The results showed that the affinity for H53/L28 was about 6 times higher than that of TOCILIZUMAB and the activity expressed by the 100% inhibitory concentration of BaF/gpl3 was about several times higher. 55 201100100 [Table 2]
Ka(l/MS) Kd(l/S) KD(M) T0CILIZUMAB 4.9E+05 2.0E-03 4.0E-09 H53/L28 7.6E+05 5.2E-04 6.8E-10 以熟習該技術領域之人士已知的等電點電泳確認的等 電點結果，顯示T0CILIZUMAB及H53/L28的等電點各為約 9. 3，及 6. 5〜6. 7。因此，比起 T0CILIZUMAB，H53/L28 的 等電點減低約 2. 7。又’使用 GENETYX(GENETYX CORPORATION) 計算 VH/VL 的可變區的理論等電點。結果顯 示：T0CILIZUMAB及H53/L28的理論等電點各為9.20及 4. 52。因此，與T0CILIZUMAB相比，H53/L28的等電點減 低約4. 7。 為了評估具有減低的等電點的經改變的抗體Η 5 3 / L 2 8 的藥物動力學’比較正常小鼠的T0CILIZUMAB與H53/L28 的藥物動力學。對於小鼠（C57BL/6J; Charles River japan， Inc.)以靜脈内（IV)或皮下（SC)投予lmg/kg的單一劑量的 T0CILIZUMAB或H53/L28，以評估血漿濃度的時間變化。針 對T0CILIZUMAB及H53/L28靜脈内投予或皮下投予後之血 蒙濃度的時間變化，各顯示於圖5及圖6。使用 WinNonlinCPharsight)獲得之藥物動力學參數（清除率 (clearance，CL)及半衰期（T1/2)顯示於表 3。h53/l28 靜 脈内投予後之血漿半衰期⑺/2)延長為T〇cluz_之約 1.3倍，而清除率減少、約1>7倍。咖心皮下投予後之 56 .201100100 .T1/2增加為T〇CILIZUMAB之約2倍，而清除率減少約2>1 ^因此，發現到：可經由胺基酸取代，使T0CILIZUMAB之 等電點降低， [表3] 藉此顯著改良藥物動力學。 I V SC CL mL/h/kg T1/2 曰 CL/F T1/2 mL/h/kg 曰 TOCILIZUMAB H53/L28 0. 177 0. 102 18. 5 23. 5 0.18 14. 7 0.086 29.7 實施例3 鑑別減低T0CILIZUMAB之免疫原性的突變部位 鐶別減少存在於可變區之τ細胞抗原決定位之免疫原 性風險的突變 使用 TEPITOPE(Methods. 2004 Dec;34(4):468-75)分 析TOC ILIZUMAB序列的可變區存在的τ細胞抗原決定位。 Q 結果’預測L鏈CI)R2具有許多結合於HLA的T細胞抗原決 定位（即，具有高免疫原性風險的序列）。因此，實施 ΤΕΡΙΤ0ΡΕ分析以檢查會降低l鏈CDR2之免疫原性風險但 不會降低穩定性、結合活性或中和活性的胺基酸取代。 如下所述’篩選結果證明可藉由取代T0CILIZUMAB之 L鏈CDR2(序列識別號59)L51的蘇胺酸為甘胺酸，及L53 之精胺酸為谷胺酸（序列識別號60)，以減低免疫原性但不 降低穩定性、結合活性或中和活性（Kabat，s numbering; Kabat et al. , (1991) Sequence of Proteins 〇f 57 201100100Ka(l/MS) Kd(l/S) KD(M) T0CILIZUMAB 4.9E+05 2.0E-03 4.0E-09 H53/L28 7.6E+05 5.2E-04 6.8E-10 is familiar with the technical field The singularity of the T0CILIZUMAB and H53/L28 is about 9.3, and 6. 5~6. Therefore, the isoelectric point of H53/L28 is reduced by about 2. 7 compared to T0CILIZUMAB. Also, use GENETYX (GENETYX CORPORATION) to calculate the theoretical isoelectric point of the variable region of VH/VL. The results show that the theoretical isoelectric points of T0CILIZUMAB and H53/L28 are 9.20 and 4.52, respectively. 0。 Thus, compared with T0CILIZUMAB, the isoelectric point of H53/L28 is reduced by about 4.7. To assess the pharmacokinetics of the altered antibody Η 5 3 / L 2 8 with reduced isoelectric point, the pharmacokinetics of TOCCILIZUMAB and H53/L28 in normal mice were compared. A single dose of T0CILIZUMAB or H53/L28 of 1 mg/kg was administered intravenously (IV) or subcutaneously (SC) to mice (C57BL/6J; Charles River japan, Inc.) to assess temporal changes in plasma concentrations. The temporal changes in blood concentration after intravenous administration or subcutaneous administration of T0CILIZUMAB and H53/L28 are shown in Fig. 5 and Fig. 6 , respectively. The pharmacokinetic parameters (clearance, CL) and half-life (T1/2) obtained using WinNonlinCPharsight are shown in Table 3. The plasma half-life (7)/2) after intravenous administration of h53/l28 was extended to T〇cluz_ About 1.3 times, and the clearance rate is reduced by about 1 > 7 times. After the subcutaneous administration of the coffee, 56.201100100.T1/2 increased to about 2 times that of T〇CILIZUMAB, and the clearance rate decreased by about 2>1 ^ Therefore, it was found that the isoelectric point of T0CILIZUMAB can be replaced by amino acid. Decrease, [Table 3] thereby significantly improving pharmacokinetics. IV SC CL mL/h/kg T1/2 曰CL/F T1/2 mL/h/kg 曰TOCILIZUMAB H53/L28 0. 177 0. 102 18. 5 23. 5 0.18 14. 7 0.086 29.7 Example 3 Identification Mutations that reduce the immunogenicity of T0CILIZUMAB Screening for mutations that reduce the immunogenicity risk of the tau cell epitopes present in the variable region. Analysis of TOC ILIZUMAB using TEPITOPE (Methods. 2004 Dec; 34(4): 468-75) The tau cell epitope is present in the variable region of the sequence. The Q results 'predicted L chain CI) R2 have a number of T cell antigen binding sites that bind to HLA (i.e., sequences with a high risk of immunogenicity). Therefore, ΤΕΡΙΤ0ΡΕ analysis was performed to examine amino acid substitutions which would reduce the immunogenicity risk of l-chain CDR2 without reducing stability, binding activity or neutralizing activity. As shown below, 'screening results demonstrate that the sulphonic acid of the L chain CDR2 (SEQ ID NO: 59) L51 of T0CILIZUMAB can be glycine acid, and the arginine of L53 is glutamic acid (SEQ ID NO: 60). Reduces immunogenicity without reducing stability, binding activity or neutralizing activity (Kabat, s numbering; Kabat et al., (1991) Sequence of Proteins 〇f 57 201100100
Immunological Interest, NIH))° TOCILIZUMAB L 鏈 CDR2(序列識別號 59) TOCILIZUMAB L鏈CDR2，移除T細胞抗原決定位（序 列識別號60)。 實施例4 藉由將TOCILIZUMAB之可變區框架序列完全人型化， 而減低免疫原性風險 於TOCILIZUMAB人型化的處理，保留一些小鼠序列於 框架序列中以維持結合活性（Cancer Res. 1993 Feb 15; 53(4) :85卜6)。此等序列為TOCILIZUMAB之可變區序列 之Η鏈FR1中的H27、H28、H29、H30及Η鏈FR3中的 H7l(Kaba1:，s numbering; Kabai EA et al.， （1991) Sequences of Proteins of Immunological Interest, NIH))。保留的小鼠序列’為潛在的增加免疫原性風險的原 因。因此’吾人評估是否可將框架序列完全人型化以進一 步減低TOCILIZUMAB之免疫原性風險。 結果顯示’ TOCILIZUMAB之完整框架可以藉由取代 TOCILIZUMAB之Η鏈FR1 (序列識別號61)為以下之人型化η 鏈FR1-Α(序列識別號62)，及取代Η鏈FR3(序列識別號63) 為以下之人型化Η鏈FR3(序列識別號64)，而完全人型化 但不減低穩定性、結合活性或中和活性。 TOCILIZUMAB Η 鏈 FR1 (序列識別號 61) 人型化Η鏈F R1 - Α (序列識別號6 2 )(衍生自生殖細胞系 IMGT hVH—4) 58 、201100100 TOCILIZUMBA Η 鏈 FR3(序列識別號 63) 人型化Η鏈FR3(序列識別號64)(衍生自Μο1· Immunol. 2007. 44(4):412-422) 實施例5 依據TOC ILIZUMAB之pH依存性連接於il-6受體，辦 別改善藥物動力學之突變部位 改善T0CILIZUMAB之藥物動力學之方法之一，為改良 〇 該分子，使得T0CILIZUMAB之單一分子重複地連接並中和 數分子的IL-6受體。假設當TOCILIZUMAB連接於膜型IL_6 受體後，T0CILIZUMAB藉由與膜型IL-6受體連接，經由内 化而進入胞内内囊胞，然後以連接於膜型IL—6受體之狀態 傳遞到溶菌體，由溶菌體所分解。詳言之，一分子的 TOCILIZUMAB通常與一或二個膜型IL-6受體分子連接（以 單價或雙價方式），且在内化後於溶菌體中分解。因此，一 分子的TOCILIZUMAB僅能連接並中和一個或二個分子的膜 Q 型IL-6受體。 因此’本案發明人想到是否可創造使TOCILIZUMAB以 pH依存的方式結合，其中，ILIZUMAB的結合可維持於 中性條件下，而於酸性條件下則該連接顯著減低，以pH依 存方式結合的TOCILIZUMAB能在内囊胞中從膜型IL — 6受體 (抗原）分離’並藉由連接存在於内囊胞的FcRn而返回血漿 中，如圖7所示。一旦返回血漿中，以邱依存方式結合的 TOCILIZUMAB可再次與膜型IL_6受體連接。利用此重複於 血毅中之結合及於内囊胞中之分離，推想一分子的 59 201100100 T0CILIZUMAB可重複地連接/ 禪/中和數個分子的IL-6受體。 因此以pH依存方式結合 的T0CILIZUMAB推測比扭 T0CILIZUMAB具有較佳的藥物動力學。 j比起 於内囊胞中酸性條株丁 ττ 怿件下從IL-6受體分離的 T0CILIZUMAB，其結合必相較於在φ 離的 钗於在中性條件下顯著地減弱。 於細胞表面上，需要強力的Τϊ ， 洩刀的IL —6党體結合以中和化；因 此’於細胞表面pH，p{j7 4，姑§*谢ττ r» - P 4抗體與几-6受體之結合必需Immunological Interest, NIH)) ° TOCILIZUMAB L-chain CDR2 (SEQ ID NO: 59) TOCILIZUMAB L-chain CDR2, removes the T cell epitope (SEQ ID NO: 60). Example 4 Reducing the risk of immunogenicity to the humanization of TOCILIZUMAB by completely humanizing the variable region framework sequences of TOCILIZUMAB, retaining some mouse sequences in the framework sequence to maintain binding activity (Cancer Res. 1993) Feb 15; 53(4): 85 Bu 6). These sequences are H27, H28, H29, H30 in the Η chain FR1 of the variable region sequence of TOCILIZUMAB and H71 in the Η chain FR3 (Kaba1:, s numbering; Kabai EA et al., (1991) Sequences of Proteins of Immunological Interest, NIH)). The retained mouse sequence' is a potential cause of increased risk of immunogenicity. Therefore, we have assessed whether the framework sequence can be fully humanized to further reduce the immunogenic risk of TOCILIZUMAB. The results show that the complete framework of 'TOCILIZUMAB can be replaced by the Η chain FR1 (SEQ ID NO: 61) of TOCILIZUMAB as the following humanized η chain FR1-Α (SEQ ID NO: 62), and the substituted Η chain FR3 (SEQ ID NO: 63) It is a humanized Η chain FR3 (SEQ ID NO: 64) which is completely humanized but does not reduce stability, binding activity or neutralizing activity. TOCILIZUMAB Η chain FR1 (SEQ ID NO: 61) Humanized Η chain F R1 - Α (SEQ ID NO: 6 2 ) (derived from germ cell line IMGT hVH-4) 58 , 201100100 TOCILIZUMBA Η Chain FR3 (SEQ ID NO: 63) Humanized Η chain FR3 (SEQ ID NO: 64) (derived from Μο1· Immunol. 2007. 44(4): 412-422) Example 5 Depending on the pH dependence of TOC ILIZUMAB, it is linked to the il-6 receptor. One of the methods for improving the pharmacokinetics of the pharmacokinetic mutation site to improve the pharmacokinetics of TOCILIZUMAB is to modify the molecule to allow a single molecule of TOCILIZUMAB to repeatedly link and neutralize several molecules of IL-6 receptor. It is hypothesized that when TOCILIZUMAB is linked to the membrane-type IL-6 receptor, T0CILIZUMAB is linked to the membrane-type IL-6 receptor, enters the intracellular inner capsule via internalization, and is then transmitted in a state linked to the membrane-type IL-6 receptor. To the lysate, it is decomposed by the lysate. In particular, one molecule of TOCILIZUMAB is usually linked to one or two membrane-type IL-6 receptor molecules (in a monovalent or bivalent manner) and decomposed in lysosomes after internalization. Therefore, one molecule of TOCILIZUMAB can only bind and neutralize one or two molecules of the membrane Q-type IL-6 receptor. Therefore, the inventors of the present invention thought whether it is possible to create a combination of TOCILIZUMAB in a pH-dependent manner, wherein the binding of ILIZUMAB can be maintained under neutral conditions, and under acidic conditions, the connection is significantly reduced, and TOCILIZUMAB can be combined in a pH-dependent manner. It is separated from the membrane type IL-6 receptor (antigen) in the inner capsule and returned to the plasma by ligating the FcRn present in the inner capsule, as shown in FIG. Once returned to the plasma, the TOCILIZUMAB bound in a Qiu-dependent manner can be ligated to the membrane type IL-6 receptor again. Using this repetitive combination in blood stasis and isolation in the inner vesicles, one molecule of 59 201100100 T0CILIZUMAB can be repeatedly linked/zen/neutralized to several molecules of IL-6 receptor. Therefore, T0CILIZUMAB, which is combined in a pH-dependent manner, is presumed to have better pharmacokinetics than T0CILIZUMAB. j is significantly weaker than T0CILIZUMAB isolated from the IL-6 receptor under the acid squirrel in the inner capsule, which is significantly weaker than the φ-dissociated enthalpy under neutral conditions. On the cell surface, a strong sputum is needed, and the IL-6 body of the ventilator is combined to neutralize; therefore, 'the pH on the cell surface, p{j7 4, §*谢ττr» - P 4 antibody with a few - 6 receptor binding required
同等或強於T0CILIZUMAB。已右郝主瓶-士 A 巳有報告顯不内囊胞pH 一般為 5.5~6.0(Nat Rev Mol Cell R·,Equivalent or stronger than T0CILIZUMAB. The right Hao bottle - Shi A has reported that the pH of the inner capsule is generally 5.5~6.0 (Nat Rev Mol Cell R·,
Leu Biol. 2004Leu Biol. 2004
Feb;5(2):121-32)。因此，甚 u ntr /+· 士 D右以Ρίί依存方式結合的 T0CILIZUMAB修飾成於ΡΗ5.5〜6.0微弱地連接於IL_6受 體，則可預測於内囊胞中會在酸性條件下從IL — 6受體= 離。詳言之，若以pH依存方式結合的T〇CIUZUMAB修飾1 於ρΗ7· 4，其為細胞表面PH，強力地連接於IL 6受體，且 於内囊胞pH，pH5.5〜6.0，微弱地連接於IL —6受體，則一 分子的TOC ILIZUMAB可結合及中和數分子的η —6受體，且 藥物動力學可因此而改善。 對於結合T0CILIZUMAB至IL-6受體提供PH依存性之 可能方法之一，為在T0CILIZUMAB之可變區引入組胺酸殘 基’因為組胺酸殘基的pKa為約6.0〜6.5，且其質子分離 狀態在中性（ρΗ7·4)與酸性（pH5.5〜6.0)之間改變。故，依 據TOC ILIZUMAB之三維結構模型，針對鑑別在可變區引入 組胺酸的部位進行篩選。又，將T0CILIZUMAB之選擇的可 變區序列隨機以組胺酸取代以設計一篩選之庫。篩選係使 60 .201100100 用於PH7. 4結合於IL-6受體且於PH5. 5〜5. 8從IL-6受體 分離或親和力降低，作為指標。 結果’本案發明人等發現：提供pH依存性（於PH7. 4結 合、於PH5.8分離的性質）結合tocILIZUMAB至IL-6受體 之突變部位，顯示於圖8。於圖8中，H27的酪胺酸取代為 組胺酸，為Η鏈FR1的突變，並非在CDR。然而，如Eur.夂 Immun〇i( 1 992) 22:1 71 9_1 728 所述，在 H27 具有組胺酸 〇 之序列為—人類序列（序列識別號65)。因此，藉由使用以 下框架組合實施例4，可將該抗體完全人型化。 人型化Η鏈FR1-B(序列識別號65)Feb; 5(2): 121-32). Therefore, even u ntr /+· 士 D right Ρίί dependent combination of T0CILIZUMAB modified to ΡΗ5.5~6.0 weakly linked to IL_6 receptor, it can be predicted that the inner sac will be under acidic conditions from IL — 6 receptor = away. In particular, if pH-dependently bound T〇CIUZUMAB is modified to ρΗ7·4, it is a cell surface PH, strongly linked to the IL 6 receptor, and is at a pH of the inner capsule, pH 5.5 to 6.0, weak. Connected to the IL-6 receptor, one molecule of TOC ILIZUMAB can bind and neutralize several molecules of the η-6 receptor, and the pharmacokinetics can be improved accordingly. One of the possible ways to provide pH dependence in combination with TOCILIZUMAB to IL-6 receptor is to introduce a histidine residue in the variable region of TOCILIZUMAB because the histidine residue has a pKa of about 6.0 to 6.5 and its protons The separation state changes between neutral (ρΗ7·4) and acidity (pH 5.5 to 6.0). Therefore, according to the three-dimensional structural model of TOC ILIZUMAB, screening was performed for identifying the site where histidine was introduced in the variable region. Further, the selected variable region sequence of TOCILIZUMAB was randomly substituted with histidine to design a library of screening. The screening system used 60.201100100 for PH7. 4 binding to IL-6 receptor and at pH 5. 5~5. 8 from IL-6 receptor separation or affinity reduction as an indicator. As a result, the inventors of the present invention found that a pH-dependent (the property of binding at pH 7.4 and separating at pH 5.8) binds to a mutant site of tocILIZUMAB to IL-6 receptor, and is shown in Fig. 8 . In Figure 8, the tyrosine acid of H27 is substituted with histidine, which is a mutation of the Η chain FR1, not in the CDR. However, as described in Eur. 夂 Immun〇i (1 992) 22:1 71 9_1 728, the sequence having histidine in H27 is the human sequence (SEQ ID NO: 65). Therefore, the antibody can be completely humanized by combining Example 4 using the following framework. Humanized Η chain FR1-B (sequence identification number 65)
突變之組合’包括例如：H3pI/L73(H鏈H3pI-IgGl/序 列識別號66;L鏈L73-A： /序列識別號67)可產生具有pH依 存性結合性質的 T〇CILIZUMAB。H3pl/L73 及 TOCILIZUMAB 對於可溶性IL-6受體於pfj7· 4的親和性、於pH7. 4及pH5_ 8 從膜型IL-6受體分離之速率、使用BaF/gpl3〇之生物學活 〇性，及於食蟹猴及人類IL-6受體基因轉殖小鼠中之藥物動 力學被進行比較（見此方法之參考實施例）。 於ρΗ7· 4針對於可溶性il_6受體之親和性試驗結果， 如表4所不。使用BaF/gp丨3〇得到之生物學活性之試驗結 果（最終IL-6濃度為3〇ng/ml )如圖9所示。此等結果顯示， H3PI/L73在針對可溶性IL_6受體於ρΗ7·4之親和性及於 BaF/gpl30之活性上，可與tocILIZUMAB相比。 61 201100100 [表4]The combination of mutations' includes, for example, H3pI/L73 (H chain H3pI-IgGl/SEQ ID NO: 66; L chain L73-A: / SEQ ID NO: 67) to produce T〇CILIZUMAB having pH-dependent binding properties. The affinity of H3pl/L73 and TOCILIZUMAB for the soluble IL-6 receptor in pfj7·4, the rate of isolation from the membrane type IL-6 receptor at pH 7.4 and pH5_8, and the biological activity of BaF/gpl3〇 , and pharmacokinetics in cynomolgus and human IL-6 receptor gene-transferred mice were compared (see Reference Example of this method). The results of the affinity test for ρΗ7·4 against the soluble il_6 receptor are as shown in Table 4. The results of the biological activity obtained using BaF/gp丨3〇 (final IL-6 concentration was 3〇ng/ml) are shown in Fig. 9. These results show that H3PI/L73 can be compared to tocILIZUMAB in the affinity for soluble IL-6 receptor for ρΗ7·4 and for BaF/gpl30. 61 201100100 [Table 4]
Ka(l/MS) Kd( 1 /s ) KD(M) TOCILIZUMAB 5.1E+05 1. 0 E - 0 3 2.IE-09 H3pl/L73 5.4E+05 7.4E-04 1.4E-09 T0CILIZUMAB 或 H3pl/L73 於 pH7.4 及 pH5. 8 從膜型 il-6 受體分離之速率，測定結果如表5所示。與TOCILIZUMAB 相比’於pH5.8的H3pl/L73的分離速率較快，且從膜型 IL-6受體分離之速率的pH依存性增加約2. 6倍。 [表5 ] pH7. 4 pH5. 8 Kd(pH5. 8)/kd(pH7. 4) Kd(l/S) Kd(l/S) p H依存性 TOCILIZUMAB 2.5E-04 2.5E-04 1. 00 H3pl/L73 2.6E-04 6. 7E-04 2.59 單一劑量的 TOCILIZUMAB 或 H3pl/L73 以 lmg/kg 靜脈 内投予到食蟹猴以評估血漿濃度的時間變化。T〇c ILIZUMAB 或H3pl/L73以靜脈投予後之血漿濃度時間變化，顯示於圖 10°結果顯示’於食蟹猴，H3pl/L73之藥物動力學，比起 TOCILIZUMAB顯著改良。 單一劑量的 TOCILIZUMAB 或 H3pl/L73 以 25mg/kg 靜 脈内投予到人類IL-6受體基因轉殖小鼠（hIL-6R tg小 鼠；Proc Natl Acad Sci USA· 1 995 May 23; 92( 1 1 ):4862-6) 以S平估血聚濃度的時間變化。TOCILIZUMAB或H3p I/L73以 靜脈投予後之血漿濃度時間變化，顯示於圖11。結果顯示， 62 201100100 於人類IL-6受體基因轉殆，丨、白„ 锝殖小乳，H3PI/L73之藥物動力學， 比起T0CILIZUMAB顯著改良。 H3pl/L73 ，一且右 „ ^ ^ ，、有PH依存性結合性質之 T0CILIZUMAB，於食蟹猴及人相ττ ρ - 箪猴及人頰IL-6受體基因轉殖小鼠， 比起TOC ILIZUMAB顯示顯英并* & # 只丁顯者改良的藥物動力學。此代表藉 由提供於ρΗ7.4結合於括盾；^ nc。 柷原及PH5. 8從抗原分離之性質， 可利用單一分子將數分子IL— 典 — L b又體t合及中和。亦可認為 Ο 可藉由使IL-6受體結合比起與H3pI/L73更具邱依存性， 而更進一步改良藥物動力學。 實施例6 T0CILIZUMAB恆定區之最適化 減低TOCILIZUMAB Η鏈C端之異質性 已有人報告，針對IgG抗體之Η鏈c端序列之異質性， 由於刪除兩C端胺基酸，甘胺酸及離胺酸，刪除c端胺基 酸離胺酸殘基及醢胺化C端敌基。（Anai Bi〇 chem. 2007 Jan 〇 1; 360(1) :75-83)。又，於 TOCILIZUMAB，其主要成分為 一序列’其核苷酸序列中的C端胺基酸離胺酸藉由轉譯後 修飾而刪除；然而，次要成分中的離胺酸保留，且次要成分 中的C端羧基由於刪除甘胺酸及離胺酸兩者而醯胺化也貢 獻於異質性。欲以大規模製備其為醫藥品而維持在生產間 之目標物質/相關物質相關之異質性，並不容易且成本高。 若有可能，希望開發抗體為醫藥品時，為單—物質且異質 性減小。因此，當開發抗體為醫藥品時，較佳為Η鏈c端 異質性不存在。 63 201100100 c端胺基酸經改變以減少c端胺基酸異質性。結果顯 示’ C端衍生之異質性，可藉由預先從核苷酸序列刪除 T0CILIZUMAB之Η鏈恆定區之C端之離胺酸及甘胺酸殘基 而避免。以陽離子交換層析評估以下之異質 性：T0CILIZUMAB 、缺少 C 端離胺酸殘基之 TOCILIZUMABCTOCILIZUMAB 5 Κ:Η 鏈 WT-IgGl 5 Κ/序列 識別號6 8 ; L鏈W T - /c /序列識別號5 4)、缺少C端離胺酸及 甘胺酸殘基之 TOCILIZUMAB(TOCILIZUMAB δ GK:H 鍵 WT-IgGl (5 GK/序列識別號69;L鏈WT-/c /序列識別號 〇Ka(l/MS) Kd( 1 /s ) KD(M) TOCILIZUMAB 5.1E+05 1. 0 E - 0 3 2.IE-09 H3pl/L73 5.4E+05 7.4E-04 1.4E-09 T0CILIZUMAB or The rate of separation of H3pl/L73 from the membrane type il-6 receptor at pH 7.4 and pH 5.8 is shown in Table 5.倍倍。 The pH dependence of the rate of isolation of the membrane type IL-6 receptor increased by about 2.6 times compared with the TOCILIZUMAB. [Table 5] pH 7. 4 pH 5. 8 Kd (pH 5. 8) / kd (pH 7. 4) Kd (l / S) Kd (l / S) p H dependency TOCILIZUMAB 2.5E-04 2.5E-04 1. 00 H3pl/L73 2.6E-04 6. 7E-04 2.59 A single dose of TOCILIZUMAB or H3pl/L73 was administered intravenously to cynomolgus monkeys at 1 mg/kg to assess temporal changes in plasma concentrations. The plasma concentration of T〇c ILIZUMAB or H3pl/L73 after intravenous administration was shown in Fig. 10°. The results showed that the pharmacokinetics of H3pl/L73 was significantly improved compared to TOCILIZUMAB. A single dose of TOCILIZUMAB or H3pl/L73 was administered intravenously to human IL-6 receptor gene-transferred mice at 25 mg/kg (hIL-6R tg mice; Proc Natl Acad Sci USA· 1 995 May 23; 92 ( 1 1): 4862-6) The temporal change of blood concentration was evaluated by S. The temporal change in plasma concentration of TOCILIZUMAB or H3p I/L73 after intravenous administration is shown in Figure 11. The results showed that 62 201100100 was converted to human IL-6 receptor gene, 丨, 白 锝 小 small milk, H3PI/L73 pharmacokinetics, significantly improved compared to T0CILIZUMAB. H3pl/L73, one and right „ ^ ^ , T0CILIZUMAB with PH-dependent binding properties, in cynomolgus monkeys and human phase ττ ρ - simian and human buccal IL-6 receptor gene-transferred mice, compared with TOC ILIZUMAB shows sin and * &# 丁Significantly improved pharmacokinetics. This representative is provided by Η Η 7.4 in combination with the shield; ^ nc. The nature of the separation of prion and PH5.8 from the antigen, a single molecule can be used to combine and neutralize several molecules of IL-Lb. It is also believed that Ο can further improve pharmacokinetics by making IL-6 receptor binding more hygienic than H3pI/L73. EXAMPLE 6 Optimization of the T0CILIZUMAB constant region reduces the heterogeneity of the C-terminus of the TOCILIZUMAB Η chain. It has been reported that the heterogeneity of the c-terminal sequence of the IgG antibody is deleted by the removal of the two C-terminal amino acids, glycine and the amine. The acid removes the c-terminal amino acid amide acid residue and the guanidine C-terminal group. (Anai Bi〇 chem. 2007 Jan 〇 1; 360(1): 75-83). Further, in TOCILIZUMAB, the main component is a sequence whose C-terminal amino acid lysine in the nucleotide sequence is deleted by post-translational modification; however, the amide acid retention in the minor component is secondary. The C-terminal carboxyl group in the component also contributes to heterogeneity due to the deletion of both glycine and lysine. It is not easy and costly to maintain the heterogeneity of the target substance/related substance in the production room for large-scale preparation of the drug. If it is possible to develop an antibody as a pharmaceutical, it is a single substance and the heterogeneity is reduced. Therefore, when the antibody is developed into a pharmaceutical product, it is preferred that the c-terminal heterogeneity of the oxime chain does not exist. 63 201100100 The c-terminal amino acid was altered to reduce the c-terminal amino acid heterogeneity. The results show that the heterogeneity of the 'C-terminal derivation can be avoided by deleting the amino acid and glycine residues at the C-terminus of the constant chain region of T0CILIZUMAB from the nucleotide sequence in advance. The following heterogeneity was evaluated by cation exchange chromatography: TOCILIZUMAB, TOCILIZUMABCTOCILIZUMAB 5 lacking the C-terminal lysine residue Η: Η chain WT-IgGl 5 Κ/SEQ ID NO: 6 8 ; L chain WT - /c / sequence identification number 5 4), TOCILIZUMAB lacking C-terminal lysine and glycine residues (TOCILIZUMAB δ GK: H-bond WT-IgGl (5 GK/SEQ ID NO: 69; L-chain WT-/c/SEQ ID NO:
54)。使用 ProPac WCX-10 4x250mm(Dionex)管柱；移動相 a 使用 25mmol/LMES/Na0H(pH6.1)及移動相 B 使用 25mmol/L MES/NaOH’ 250mmol/L NaCl (ρΗ6· 1)。使用適當的流速及梯 度。陽離子交換層析獲得之評估結果顯示於圖12。結果顯 示’ C端胺基酸異質性可藉由從核苷酸序列，預先刪除η 鏈怪定區之C端之離胺酸及甘胺酸殘基而減低，而非僅預 先删除Η鏈恆定區之C端之離胺酸殘基。所有的人類抗體 IgG 1、IgG2、IgG4 t亙定區之C端序列依照EU編號法（見54). A ProPac WCX-10 4x250mm (Dionex) column was used; mobile phase a using 25 mmol/L MES/NaOH (pH 6.1) and mobile phase B using 25 mmol/L MES/NaOH' 250 mmol/L NaCl (ρΗ6.1). Use the appropriate flow rate and gradient. The evaluation results obtained by cation exchange chromatography are shown in Fig. 12. The results show that 'C-terminal amino acid heterogeneity can be reduced by pre-delete the amino acid and glycine residues at the C-terminus of the η chain from the nucleotide sequence, rather than pre-delete only the Η chain constant. The amino acid residue at the C-terminus of the region. C-terminal sequences of all human antibodies IgG 1, IgG2, IgG4 t-definite regions according to EU numbering (see
Sequences of proteins of immunological interest NIHSequences of proteins of immunological interest NIH
Publi cat ion No· 91-3242)在447位及446位各具有離胺酸 及甘胺酸。因此，本研究中發現的用於減低C端胺基酸異 質性的方法，期待使用在IgG2及IgG4恆定區及其變異體。 減少IgG2同型物TOCILIZUMAB中之雙硫鍵衍生的異 質性 64 201100100Publi cat ion No. 91-3242) has lysine and glycine at 447 and 446, respectively. Therefore, the method for reducing the heterogeneity of the C-terminal amino acid found in this study is expected to be used in the IgG2 and IgG4 constant regions and variants thereof. Reduced heterogeneity derived from disulfide bonds in the IgG2 isoform TOCILIZUMAB 64 201100100
T0CILIZUMAB之同屯】私迭T Ν坦物為IgGl。因為T0CILIZUMAB為 一中和性抗體，連接於F - ' Fc 7受體，在免疫原性及不利效 果的觀點上’可能不利。用於減⑯Fc r受體結合之可能 方法，為冑IgG抗體之同型㈣IgGl變換為IgG2或 IgGVAnnHematol. 1 998 Jun; 76(6):231_48)。從 Fc 了 又體I結合及藥物動力學之觀點，比起IgG4更希望為The same as T0CILIZUMAB] private T Ν 物 is IgGl. Since T0CILIZUMAB is a neutralizing antibody linked to the F - 'Fc 7 receptor, it may be disadvantageous in terms of immunogenicity and adverse effects. A possible method for reducing 16Fc r receptor binding is the homotypic 胄 IgG antibody (IV) IgGl conversion to IgG2 or IgGVAnnHematol. 1 998 Jun; 76(6): 231_48). From the point of view of Fc and I binding and pharmacokinetics, it is more desirable than IgG4.
IgG2(Nat Biotechnol. 2〇〇7 Dec;25(12):1369_72)。同IgG2 (Nat Biotechnol. 2〇〇7 Dec; 25(12): 1369_72). with
時’蛋白質之物理化學性質，尤其是同質性及穩定性於開 發抗體為醫藥品時非常重要。IgG2同型物據報告由於在欽 鍵區的雙硫鍵而具有非常高的異質性（J Bi〇1 Chem. 2008The physical and chemical properties of proteins, especially homogeneity and stability, are very important when developing antibodies are pharmaceuticals. The IgG2 isoform is reported to have very high heterogeneity due to the disulfide bond in the primordial region (J Bi〇1 Chem. 2008).
Jun 6; 283(23): 1 6206-1 5 )。欲大規模生產其為醫藥品而同 時在生產間維持目標物質/相關物質相關的衍生自雙硫鍵 的異質性，並不容易且成本高。因此，儘可能希望為單一 物質。因此，當開發IgG2同型物抗體為醫藥品時，較佳為 減少衍生自雙硫鍵之異質性而不降低穩定性。 對於降低IgG2同型物之異質性的用途，對各種變異體 進行評估。結果發現，使用WT-SKSC恆定區（序列識別號 70)能降低異質性而不降低穩定性之方法，其中，IgG2恆 定區序列Η鏈CH1區之131位之半胱胺酸殘基及133位之 精胺酸殘基（EU編號法），各取代為絲胺酸及離胺酸，且Η 鏈上部鉸鏈中219位之半胱胺酸殘基，取代為絲胺酸。製 備 TOCILIZUMAB-IgGl(H 鏈 WT-IgGl/序列識別號 53;L 鏈 WT- /c /序列識別號 54)、T0CILIZUMAB-IgG2(H 鏈 WT-IgG2/ 序列識別號 71 ;L鏈 WT- /c /序列識別號 54)及 65 201100100 TOCILIZUMAB-SKSCCH 鏈 WT-SKSC/序列識別號 70 ;L 鏈 WT- /c /序列識別號54)並用於評估異質性及穩定性。異質性係 以陽離子交換層析評估。使用propac WCX-lO(Dionex)管 柱：移動相A使用20mM乙酸鈉（PH5. 0)及移動相B使用20mM 乙酸鈉、1M NaCl(pH5. 〇)。使用適當的流速及梯度。由陽 離子交換層析得到的評估結果，如圖13所示。穩定性依據 以差示掃描熱量計（DSC)(VP-DSC:M i croca 1)決定之熱變性 (Tm值）中的中間溫度§平估。於2〇mM乙酸納、150mMNaCl、 pH6. 0及Fab區之Tm值的DSC測定結果，如圖14所示。 結果顯示，比起 TOCILIZUMAB-IgGl，T0CILIZUMAB-IgG2 的異質性明顯增加；然而，異質性可藉由轉變為 TOCILIZUMAB-SKSC 而顯著減低。又，與 TOCILIZUMAB-IgGl 相較，T0CILIZUMAB-IgG2之DSC在Fab區之熱變性峰部， 有一低穩定性的肩峰（Fab*)成分，即低Tm，其推測係由於 異質性成分而生。然而’當轉變為TOCILIZUMAB-SKSC，認 為由於異質成分而產生的肩峰（低Tm)消失，且Tm值約 94°C，等同於 TOCILIZUMAB-IgGl 及 T0CILIZUMAB-IgG2 之 Fab區。故，T0CILIZUMAB-SKSC顯示高穩定性。 鑑別T0CIL1ZUMAB之恆定區之藥物動力學改良突變部 位 如上述，起自IgGl，其為TOCILIZUMAB之同型物，減 低C端異質性及減低IgG2同型物之抗體恆定區的異質性而 同時減少結合至Fc τ受體並維持高穩定性，可以達成。 66 201100100 又，較佳為該恆定區也較IgGl，T0CILIZUMAB之同型物， 具有更優越的藥物動力學性質。 為了尋找比具有IgGl同型物恆定區之抗體具有更優 越之血漿半衰期的恆定區，實施篩選以鑑別用於改良 TOCILIZUMAB-SKSC之藥物動力學之突變部位，該 TOCILIZUMAB-SKSC具有與上述具有lgG2同型物恆定區之 抗體相關的高穩定性及減低的異質性。結果，發現到 WT-M58(序列識別號72(胺基酸序列），其中，相較於 WT-SKSC ’ EU編號的137位的谷胺酸取代為甘胺酸，138位 之絲胺酸取代為甘胺酸，268位之組胺酸取代為谷醯胺， 355位之精胺酸取代為谷醯胺，419位之谷醯胺取代為谷胺 酸’其中’ 446位之甘胺酸及447位之離胺酸被刪除以減 少Η鏈C端的異質性。此外，製備WT-M44C序列識別號 73)(胺基酸序列），相對於IgG1，具有434位的天冬醯胺 取代為丙胺酸。又，從M44刪除446位之甘胺酸及447位 〇 之離胺酸以產生WT-M83C序列識別號74(胺基酸序列）），以 減低Η鏈C端之異質性。此外，藉由將wt-M58的434位的 天冬醯胺以丙胺酸取代，而生產WT-M73C序列識別號75(胺 基酸序列））。 製備 TOCILIZUMAB Μ-44(Η 鏈 WT-M44/序列識別號 73; L 鏈 WT- /c /序列識別號 54)、T0CILIZUMAB M-58CH 鏈 WT-M58/ 序列識別號58/序列識別號72;L鏈WT-/C /序列識別號54) 及 T0CILIZUMAB-M73CH 鏈 WT-M73/序列識別號 75;L 鏈 WT-/c /序列識別號54) ’並使用人類FcRn基因轉殖小鼠評估 67 201100100 其對於人類FcRn及藥物動力學（見本方法之參考實施例）。 使用 Biacore 評估 T0CILIZUMAB-IgGl 、 T0CILIZUMAB-M44 、 TOCILIZUMAB-M58 及 TOCILIZUMAB-M73 於人類FcRn的結合。如表6所示，TOCILIZUMAB-M44、 TOCILIZUMAB-M58 及 T0CILIZUMAB-M73 分別優於 TOCILIZUMAB-IgGl 之結合的約 2. 7 倍、1. 4 倍、3. 8 倍。 [表6 ] KD( β Μ) TOCILIZUMAB-IgGl 1. 62 T0CILIZUMAB-M44 0. 59 TOCILIZUMAB-M58 1. 17 TOCILIZUMAB-M58 0. 42 就 TOCILIZUMAB-IgGl .、 TOC ILIZUMAB-M44 、 T0CILIZUMAB-M58，及 T0CILIZUMAB-M73 於人類 FcRn 基因 轉殖小鼠中的藥物動力學進行評估。結果如圖1 5所示。如 圖 15 所示，比起 TOCILIZUMAB-IgGl，TOCILIZUMAB-M44、 T0CILIZUMAB-M58 ’ 及 TOCILIZUMAB-M73，均顯示較佳的藥 物動力學。改良藥物動力學之效果與結合至人類FcRn之能 力係具相關性。尤其，28天後在血漿中的TOCILIZUMAB-M73 的濃度，比起TOCILIZUMAB-IgG卜改良約16倍。因此， 推測具有M73之恆定區之抗體在人類，也比起具有IgG1恆 定區之抗體’具有顯著改良的藥物動力學性質。 實施例7 製備具有改良的PK/PD的全人型化IL-6受體抗體 68 201100100Jun 6; 283(23): 1 6206-1 5 ). It is not easy and costly to mass-produce the heterogeneity derived from the disulfide bond associated with maintaining the target substance/related substance in the production while maintaining the drug. Therefore, as much as possible, it is desirable to be a single substance. Therefore, when an antibody of IgG2 isoform is developed as a pharmaceutical, it is preferred to reduce the heterogeneity derived from the disulfide bond without lowering the stability. Various variants were evaluated for the use of reducing the heterogeneity of IgG2 isoforms. As a result, it was found that the use of the WT-SKSC constant region (SEQ ID NO: 70) can reduce the heterogeneity without lowering the stability, wherein the IgG2 constant region sequence 131 chain CH1 region at position 131 of the cysteine residue and 133 The arginine residues (EU numbering method) are each substituted with a serine and an lysine, and the cysteine residue at position 219 in the upper hinge of the oxime chain is substituted with serine. Preparation of TOCILIZUMAB-IgG1 (H chain WT-IgG1/SEQ ID NO: 53; L chain WT- /c / SEQ ID NO: 54), TOCILIZUMAB-IgG2 (H chain WT-IgG2/SEQ ID NO: 71; L chain WT- /c /SEQ ID NO: 54) and 65 201100100 TOCILIZUMAB-SKSCCH chain WT-SKSC/SEQ ID NO: 70; L chain WT- /c / sequence identification number 54) and used to assess heterogeneity and stability. Heterogeneity was assessed by cation exchange chromatography. A propac WCX-lO (Dionex) column was used: mobile phase A using 20 mM sodium acetate (pH 5.0) and mobile phase B using 20 mM sodium acetate, 1 M NaCl (pH 5. 〇). Use the appropriate flow rate and gradient. The evaluation results obtained by cation exchange chromatography are shown in Fig. 13. Stability basis The intermediate temperature in the thermal denaturation (Tm value) determined by differential scanning calorimetry (DSC) (VP-DSC: M croca 1) is estimated. The results of DSC measurement of Tm values in 2 mM sodium acetate, 150 mM NaCl, pH 6.0 and Fab regions are shown in FIG. The results showed that the heterogeneity of TOCILIZUMAB-IgG2 was significantly increased compared to TOCILIZUMAB-IgGl; however, the heterogeneity was significantly reduced by conversion to TOCILIZUMAB-SKSC. Further, compared with TOCILIZUMAB-IgG1, the DSC of TOCILIZUMAB-IgG2 has a low-stability shoulder (Fab*) component, that is, a low Tm, in the heat-denatured peak of the Fab region, which is presumed to be due to a heterogeneous component. However, when converted to TOCILIZUMAB-SKSC, it is considered that the shoulder (low Tm) due to the heterogeneous component disappears, and the Tm value is about 94 ° C, which is equivalent to the Fab region of TOCILIZUMAB-IgG1 and T0CILIZUMAB-IgG2. Therefore, T0CILIZUMAB-SKSC shows high stability. Identification of the pharmacokinetically modified mutation site of the constant region of TOCIL1ZUMAB as described above, starting from IgG1, which is an isoform of TOCILIZUMAB, which reduces C-terminal heterogeneity and reduces the heterogeneity of the antibody constant region of the IgG2 isoform while reducing binding to Fc τ Receptors and maintaining high stability can be achieved. 66 201100100 Further, it is preferred that the constant region has superior pharmacokinetic properties as compared with the IgGl, T0CILIZUMAB isoform. In order to find a constant region having a superior plasma half-life than an antibody having an IgGl isoform constant region, screening was performed to identify a mutation site for improving the pharmacokinetics of TOCILIZUMAB-SKSC having the same lgG2 isoform as described above. High stability associated with antibody in the constant region and reduced heterogeneity. As a result, WT-M58 (SEQ ID NO: 72 (amino acid sequence)) was found in which glutamic acid was replaced with glycine at position 137 of WT-SKSC 'EU number, and serine acid at position 138 was substituted. For glycine, the histidine acid at position 268 is substituted with glutamine, the arginine at position 355 is substituted with glutamine, and the glutamine at position 419 is substituted with glutamic acid, which is the '446 glycine and The 447 acid lysine was deleted to reduce the heterogeneity of the C-terminus of the Η chain. In addition, WT-M44C SEQ ID NO: 73) (amino acid sequence) was prepared, and the 434-position of aspartame was substituted with propylamine relative to IgG1. acid. Further, the 446-position glycine and the 447-position lysine were removed from M44 to produce WT-M83C SEQ ID NO: 74 (amino acid sequence) to reduce the heterogeneity of the C-terminus of the Η chain. Further, WT-M73C SEQ ID NO: 75 (amino acid sequence) was produced by substituting the 434-position of aspartame of wt-M58 with alanine. Preparation of TOCILIZUMAB Μ-44 (Η chain WT-M44/SEQ ID NO: 73; L chain WT- /c / SEQ ID NO: 54), T0CILIZUMAB M-58CH chain WT-M58/ SEQ ID NO: 58/SEQ ID NO: 72; Chain WT-/C/SEQ ID NO: 54) and T0CILIZUMAB-M73CH chain WT-M73/SEQ ID NO: 75; L-chain WT-/c/SEQ ID NO: 54) 'And evaluated using human FcRn gene transgenic mice 67 201100100 It is for human FcRn and pharmacokinetics (see reference examples of the method). The binding of T0CILIZUMAB-IgGl, T0CILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 to human FcRn was assessed using Biacore. As shown in Table 6, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and T0CILIZUMAB-M73 were respectively superior to TOCILIZUMAB-IgGl by about 2. 7 times, 1.4 times, and 3.8 times. [Table 6] KD(β Μ) TOCILIZUMAB-IgGl 1. 62 T0CILIZUMAB-M44 0. 59 TOCILIZUMAB-M58 1. 17 TOCILIZUMAB-M58 0. 42 on TOCILIZUMAB-IgGl., TOC ILIZUMAB-M44, T0CILIZUMAB-M58, and T0CILIZUMAB -M73 was evaluated for pharmacokinetics in human FcRn gene-transferred mice. The result is shown in Figure 15. As shown in Figure 15, TOICLIZUMAB-Ml, TOCILIZUMAB-M44, T0CILIZUMAB-M58' and TOCILIZUMAB-M73 showed better pharmacokinetics compared to TOCILIZUMAB-IgGl. The effect of improved pharmacokinetics is related to the ability to bind to human FcRn. In particular, the concentration of TOCILIZUMAB-M73 in plasma after 28 days was about 16 times better than that of TOCILIZUMAB-IgG. Therefore, it is speculated that an antibody having a constant region of M73 has significantly improved pharmacokinetic properties in humans as compared with an antibody having a constant region of IgG1. Example 7 Preparation of a fully humanized IL-6 receptor antibody with improved PK/PD 68 201100100
利用組合上述實施例中發現到的TOC ILIZUMAB的可變 區及恆定區的多個突變，製備T〇CIUZUMAB變異體。從許 多筛選中找到的完全人型化IL-6受體抗體，為：Fv3-M73(H 鏈VH4-M73/序列識別號25 ; L鏈VU- /c /序列識別號28)、 Fv4-M73(H鏈VH3-M73/序列識別號26;L鏈VL3-/C /序列識 別號29)及Fv5-M83(H鏈VH5-M83/序列識別號27 ;L鏈VL5- κ /序列識別號30)。 比較製備的 Fv-M73、Fv4-M73、Fv5-M83 對 IL-6 受體 〇 之親和性與T0CILIZUMAB對IL-6受體之親和性（見本方法 之參考實施例）。此等抗體對可溶性IL-6受體於pH7.4的 親和性，如表7所示。又’比較其與TOC ILIZUM AB及對照 組（已知的參考實施例中所述高親和性抗IL_6受體，及 US2007/0280945 所述 VQ8F11-21 hlgGl)的 BaF/gpl30 中和 活性（見本發明之參考實施例）。使用BaF/gpl30決定此等 抗體之生物學活性的結果如圖16所示（T0CILIZUMAB、對照 Q 組、Fv5-M83最終IL-6濃度為300ng/ml)及圖17所示 (TOCILIZUMAB、Fv3-M73、Fv4-M73 最終 IL-6 濃度為 30ng/ml)。如表 7 所示，Fv3-M73、Fv4-M73 比起 T0CILIZUMAB 的親和性高約2〜3倍，而Fv5-M83比起TOCILIZUMAB親和 性高約100倍（因難以測定Fv5-M83之親和性，使用 Fv5-IgGl測定親和性（H鏈VH5-IgGl/序列識別號76;L鏈 VL5-/C /序列識別號30)，Fv5-M83具有一 IgGl形式的恒定 區；該恆定區通常認為對親和性無影響）。如圖1 7所示， Fv3-M73、Fv4-M73 比起 TOCILIZUMAB 的活性稍高。如圖 16 69 201100100 所示’ FV5-M83的活性非常強，以5G%抑制濃度計，高於 T0CILIZUMAB 100倍。Fv5_M83也以5〇%抑制濃度計，比起 對照組（已知的高親和性抗IL_6受體抗體）顯示約1〇倍高 的中和活性。 [表7 ]The T〇CIUZUMAB variant was prepared by combining multiple mutations of the variable and constant regions of TOC ILIZUMAB found in the above examples. The fully humanized IL-6 receptor antibody found in many screens is: Fv3-M73 (H chain VH4-M73/SEQ ID NO: 25; L chain VU- /c / SEQ ID NO: 28), Fv4- M73 (H chain VH3-M73/SEQ ID NO: 26; L chain VL3-/C/SEQ ID NO: 29) and Fv5-M83 (H chain VH5-M83/SEQ ID NO: 27; L chain VL5-κ/SEQ ID NO: 30). The affinity of the prepared Fv-M73, Fv4-M73, Fv5-M83 for IL-6 receptor 〇 and the affinity of TOCILIZUMAB for IL-6 receptor were compared (see Reference Example of the method). The affinity of these antibodies for the soluble IL-6 receptor at pH 7.4 is shown in Table 7. 'Compare the BaF/gpl30 neutralizing activity with TOC ILIZUM AB and the control group (the high-affinity anti-IL_6 receptor described in the known Reference Examples, and the VQ8F11-21 hlgGl described in US2007/0280945) (see the present invention) Reference example). The results of determining the biological activity of these antibodies using BaF/gpl30 are shown in Figure 16 (T0CILIZUMAB, control Q group, Fv5-M83 final IL-6 concentration 300 ng/ml) and Figure 17 (TOCILIZUMAB, Fv3-M73). , Fv4-M73 final IL-6 concentration of 30 ng / ml). As shown in Table 7, Fv3-M73 and Fv4-M73 have an affinity of about 2 to 3 times higher than that of TOCILIZUMAB, and Fv5-M83 is about 100 times more affinity than TOCILIZUMAB (due to difficulty in determining the affinity of Fv5-M83, Affinity was determined using Fv5-IgG1 (H chain VH5-IgGl/SEQ ID NO: 76; L chain VL5-/C/SEQ ID NO: 30), Fv5-M83 has a constant region in the form of IgGl; this constant region is generally considered to be affinity Sex has no effect). As shown in Figure 17, Fv3-M73 and Fv4-M73 are slightly more active than TOCILIZUMAB. As shown in Figure 16 69 201100100 'FV5-M83 is very active, with a concentration of 5G% inhibition, 100 times higher than T0CILIZUMAB. Fv5_M83 also showed about 1 〇 higher neutralizing activity than the control group (known high-affinity anti-IL_6 receptor antibody) at a concentration of 5〇%. [Table 7]
Ka(l/MS) Ka(l/s) KD(M) TOCILIZUMAB 4.OE+5 1. IE-03 2.7E-09 Fv3-M73 8.5E+5 8.7E-04 · 一 1.0E-09 Fv4-M73 7.5E+5 1.0E-03 . — --- 1.4E-09 Fv5-M83 1.1E+06 2.8E-05 2·5E-11 決定 T0CILI2UMAB、Fv3-M73 及 Fv4-M73 於 pH7. 4 及 PH5. 8從膜型IL-6受體分離之速率。如表8(見本方法參考 實施例）所示結果證明，Fv3-M73及Fv4-M73從膜型il-6 受體之分離速率之pH依存性，比起T0CILIZUMAB，各改善 約11倍及10倍。相對於如實施例5所述H3pl/L73之分離 速率的pH依存性有相當大的改善，顯示當相較於 H3pI/L73，Fv3-M73及Fv4-M73之藥物動力學可顯著改善。 [表8 ] TOCILIZUMAB pH7. 4 kd( 1 /s) pH5. 8 kd(l/s) Kd(pH5. 8)/kd(pH7. 4) pH依存性 2.5E-04 2.5E-04 1. 00 Fv3-M73 4.9E-04 5.3E-03 10. 88 Fv4-M73 5.IE-04 5.IE-03 10. 06 '201100100 使用熟習該技術領域之人士已知的方法，以等電聚焦Ka(l/MS) Ka(l/s) KD(M) TOCILIZUMAB 4.OE+5 1. IE-03 2.7E-09 Fv3-M73 8.5E+5 8.7E-04 · One 1.0E-09 Fv4- M73 7.5E+5 1.0E-03 . — --- 1.4E-09 Fv5-M83 1.1E+06 2.8E-05 2·5E-11 Determine T0CILI2UMAB, Fv3-M73 and Fv4-M73 at pH 7.4 and PH5 . 8 The rate of separation from membrane type IL-6 receptor. As shown in Table 8 (see Reference Example of the Method), the pH dependence of the separation rate of Fv3-M73 and Fv4-M73 from the membrane type il-6 receptor was improved by about 11 times and 10 times compared with TOCILIZUMAB. . The pH dependence of the separation rate of H3pl/L73 as described in Example 5 was considerably improved, indicating that the pharmacokinetics of Fv3-M73 and Fv4-M73 were significantly improved compared to H3pI/L73. [Table 8] TOCILIZUMAB pH 7. 4 kd ( 1 /s) pH 5. 8 kd (l / s) Kd (pH 5. 8) / kd (pH 7. 4) pH dependence 2.5E-04 2.5E-04 1. 00 Fv3-M73 4.9E-04 5.3E-03 10. 88 Fv4-M73 5.IE-04 5.IE-03 10. 06 '201100100 Using methods known to those skilled in the art to isoelectrically focus
電泳決定 T0CILIZUMAB、對照組、Fv3-M73、Fv4-M73、Fv5-M83 的等電點。結果顯示，T0CILIZUMAB之等電點約9. 3;對照 組之等電點約 8. 4〜8. 5;Fv3-M73 之等電點約 5.7〜5.8;Fv4-M73之等電點約5·6〜5.7;Fv5-M83之等電點 約5.4〜5_5。因此，比起T0CILIZUMAB及對照組，各抗體 具有顯著較低的等電點。又，以GENETYXCGENETYX CORPORATION)計算可變區VH/VL之理論等電點。結果顯 示，T0CILIZUMAB之等電點約9. 20;對照組之等電點為 7.79;Fv3-M73之等電點為5.49;Fv4-M73之等電點為 5· 01 ;Fv5-M83 之等電點為 4.27。因此，比起 T0CILIZUMAB 及對照組’各抗體具有顯著較低的等電點。由實施例2中 顯示藉由降低等電點，藥物動力學改良，故Fv3-M73、Electrophoresis determines the isoelectric point of T0CILIZUMAB, control group, Fv3-M73, Fv4-M73, Fv5-M83. The results show that the isoelectric point of T0CILIZUMAB is about 9.3; the isoelectric point of the control group is about 8. 4~8. 5; the isoelectric point of Fv3-M73 is about 5.7~5.8; the isoelectric point of Fv4-M73 is about 5· 6~5.7; the isoelectric point of Fv5-M83 is about 5.4~5_5. Therefore, each antibody has a significantly lower isoelectric point than the T0CILIZUMAB and the control group. Further, the theoretical isoelectric point of the variable region VH/VL is calculated by GENETYXCGENETYX CORPORATION). The results show that the isoelectric point of T0CILIZUMAB is about 9.20; the isoelectric point of the control group is 7.79; the isoelectric point of Fv3-M73 is 5.49; the isoelectric point of Fv4-M73 is 5.01; the isoelectricity of Fv5-M83 The point is 4.27. Therefore, each antibody has a significantly lower isoelectric point than the T0CILIZUMAB and the control group. It is shown in Example 2 that by reducing the isoelectric point, the pharmacokinetics are improved, so Fv3-M73,
Fv4-M73及Fv5-M83被認為比起T0CILIZUMAB及對照組， 藥物動力學改善。 〇 使用 TEPITOPE (Methods. 2004 Dec;34(4):468-75) 分析 TOCILIZUMAB、Fv3-M73、Fv4-M73 及 Fv5-M83 的可變 區序列的T細胞抗原決定位。結果，預測T〇c〗L〗ZUMAB具 有T細胞抗原決定位’其中有許多結合於hla，如實施例3 所示。反之’預測結合於T細胞抗原決定位之序列數目， 在 Fv3-M73、Fv4-M73 及 Fv5-M83 顯著減少。此外，Fv3-M73、Fv4-M73 and Fv5-M83 were considered to have improved pharmacokinetics compared to T0CILIZUMAB and the control group. T T cell epitopes of 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, it was predicted that T〇c〗L] ZUMAB has a T cell epitope ‘and many of them bind to hla, as shown in Example 3. Conversely, the number of sequences predicted to bind to the T cell epitope was significantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83. In addition, Fv3-M73,
Fv4-M73或Fv5-M83的框架不具有小鼠序列，且因此完全 人型化。此等表示比起 TOCILIZUMAB，Fv3-M73、FV4-M73 及Fv5-M83的免疫原性風險可能顯著減低。 71 201100100 實施例8 於猴中之完全人型化IL —6受體抗體之PK/PD測試 T0CILIZUMAB、對照組、Fv3-M73、Fv4-M73 及 Fv5-M83， 以靜脈内注射一次劑量1 mg/kg到食蟹猴以評估血漿濃度 之時間變化（見本方法之參考實施例）。靜脈内注射後之 T0CILIZUMAB、Fv3-M73、FV4-M73 及 Fv5-M83 的血衆濃度 時間變化，如圖18所示。結果顯示，Fv3_M73、Fv4_M73 及Fv5-M83，比起T0CILIZUMAB及對照組，在食蟹猴中顯 不顯著改良的藥物動力學。其中，比起T〇CIUZUMAB， Fv3-M73及Fv4-M73顯示高度改良的藥物動力學。 評估各抗體中和膜型食蟹猴IL-6受體之效力。於抗體 才又予後（T0CILIZUMAB為第3日至第1〇日），將食蟹猴il-6 從第6至第18曰，以5 // g/kg每天以皮下投予在下背， 並於24小時後決定各動物的CRP濃度（見本方法之參考實 施例）。各抗體投予後之CRP濃度之時間變化，顯示於圖 19。為了評估各抗體中和可溶性食蟹猴IL_6受體之效力， 決定食蟹猴中之游離可溶性食蟹猴IL_6受體的血漿濃 度，並且，計算游離可溶性IL — 6受體之百分比（見本方法 之參考實施例）。各抗體投予後之游離可溶性IL_6受體之 百分比時間變化，如圖20所示。 比起 IL-6The framework of Fv4-M73 or Fv5-M83 does not have a mouse sequence and is therefore fully humanized. These indicate that the risk of immunogenicity may be significantly reduced compared to TOCILIZUMAB, Fv3-M73, FV4-M73 and Fv5-M83. 71 201100100 Example 8 PK/PD test of fully humanized IL-6 receptor antibody in monkeys T0CILIZUMAB, control group, Fv3-M73, Fv4-M73 and Fv5-M83, administered intravenously at a dose of 1 mg/ Kg to cynomolgus monkeys to assess temporal changes in plasma concentration (see reference examples of the method). The blood concentration of T0CILIZUMAB, Fv3-M73, FV4-M73 and Fv5-M83 after intravenous injection was changed as shown in Fig. 18. The results showed that Fv3_M73, Fv4_M73 and Fv5-M83 showed no significant improved pharmacokinetics in cynomolgus monkeys compared to T0CILIZUMAB and the control group. Among them, Fv3-M73 and Fv4-M73 showed highly improved pharmacokinetics compared to T〇CIUZUMAB. The potency of each antibody to neutralize the membrane type cynomolgus IL-6 receptor was assessed. After the antibody was added (T0CILIZUMAB for the 3rd to the 1st day), the cynomolgus monkey il-6 was administered subcutaneously from the 6th to the 18th day at 5 // g/kg in the lower back. The CRP concentration of each animal was determined after 24 hours (see reference example of the method). The time change of the CRP concentration after administration of each antibody is shown in Fig. 19. To assess the potency of each antibody to neutralize the soluble cynomolgus IL-6 receptor, the plasma concentration of the free soluble cynomolgus IL-6 receptor in cynomolgus monkeys was determined and the percentage of free soluble IL-6 receptor was calculated (see method) Reference example). The percentage change in the percentage of free soluble IL-6 receptor after administration of each antibody is shown in Figure 20. Compared to IL-6
T0CILIZUMAB 及對照組（已知 的高親和性抗 受體抗體），FV3-M73、Fv4-M73及Fv5 —M83以較持續方式 中和膜型食蟹猴IL-6受體，且在較長期間内抑制cRp增 加。 又，比起T0CILIZUMAB及對照組T0CILIZUMAB and the control group (known high-affinity anti-receptor antibodies), FV3-M73, Fv4-M73 and Fv5-M83 neutralize the membrane-type cynomolgus IL-6 receptor in a more sustained manner, and for a longer period of time Internal inhibition of cRp increases. Also, compared to T0CILIZUMAB and the control group
Fv3~M73 ' Fv4-M73 72 .201100100 及Fv5-M83以較持續方式中和游離可溶性食蟹猴受 體’且在較長期間内抑制游離可溶性食蟹猴IL-6受體增 加。此等發現證明，Fv3-M73、Fv4-M73及Fv5-M83比起 TOC ILIZUMAB及對照組，在持續中和膜型及可溶性I 受 體方面為佳。其中，Fv3-M73、Fv4-M73在持續中和方面顯 著優越。同時，Fv5-M83比起Fv3-M73、Fv4-M73，更強力 抑制CRP及游離可溶性食蟹猴IL_6受體。因此，Fv5M83 據認為在中和膜型及可溶性IL-6受體方面，強於 FV3-M73、FV4-M73及對照組（已知的高親和性抗IL 6受體 抗體）。據認為此等食蟹猴體内的結果，反映相對於對照 ’卫Fv5 M83對IL-6受體的較強親和力及Fv5_M83在 BaF/gpl 39試驗系中的較強生物學活性。 此等發現代表，比起T〇CIUZUMAB及對照組，Fv3~M73 'Fv4-M73 72 .201100100 and Fv5-M83 neutralized free soluble cynomolgus monkey recipients in a more sustained manner and inhibited the increase of free soluble cynomolgus IL-6 receptor over a longer period of time. These findings demonstrate that Fv3-M73, Fv4-M73 and Fv5-M83 are superior to TOC ILIZUMAB and the control group in terms of sustained neutralization of membrane type and soluble I receptor. Among them, Fv3-M73 and Fv4-M73 are superior in continuous neutralization. At the same time, Fv5-M83 inhibited CRP and free soluble cynomolgus IL_6 receptor more strongly than Fv3-M73 and Fv4-M73. Therefore, Fv5M83 is considered to be stronger than FV3-M73, FV4-M73 and control (known high-affinity anti-IL 6 receptor antibody) in neutralizing membrane type and soluble IL-6 receptor. It is believed that the results in these cynomolgus monkeys reflect the stronger affinity for the IL-6 receptor relative to the control defensive Fv5 M83 and the stronger biological activity of Fv5_M83 in the BaF/gpl 39 test line. These findings represent that compared to T〇CIUZUMAB and the control group,
Fv3_M73、Fv4~M73 於作么 ττ β > a* i 、作為抗IL-6抗體中和抗體的活性具 有高度優越的持續性’且因此能顯著減低劑量及投予頻 〇率。又’陶3證明在作為抗R_6受體中和抗體之活性 強度及強度持續性方面，顯著優越。因此， 及FV5-M83期待作為醫藥品IL 6枯抗劑為有用。 實施例9 ^擇Fv4 M73用之穩定的緩衝液種類 、上所述方法製備FV4-M73。由於Fv4-M73 ’如實施 例6 7由非天然恆定區M73組成以改良異質性、穩定性、 安全性及藥物會j六風 動力予，Fv4-M73之穩定性概況（比如，最穩 定缓衝液種類），可能淪山工& J此與由天然恆定區組成之抗體例如Jg(n 73 201100100 不同。據報告，由天缺i + 6 ’、、g 1匣疋區組成的抗體，通常在組 胺酸~乙㈣緩衝液中為最穩定⑽讓編9G8)。因此， 測定緩衝液種類對於Fv4_M73之穩定性的效果。 將Fv4-M73配方於4錄τ m 乃4種不同的ρΗ6·〇的緩衝液配方 中’使最終濃度為37mg/mU^〇 I η 、 λ 。 g mu如表9所述）。將此等樣本於Fv3_M73, Fv4~M73 are used for ττ β > a* i , and the activity as an anti-IL-6 antibody neutralizing antibody has a highly superior persistence' and thus can significantly reduce the dose and the frequency of administration. Further, 'Tao 3 proved to be superior in terms of the activity intensity and strength persistence of the neutralizing antibody against the R_6 receptor. Therefore, FV5-M83 is expected to be useful as a pharmaceutical IL 6 antagonist. Example 9 Selection of a stable buffer for Fv4 M73 FV4-M73 was prepared as described above. Since Fv4-M73' consists of the non-native constant region M73 as in Example 67 to improve heterogeneity, stability, safety, and drug efficiencies, Fv4-M73 stability profile (eg, most stable buffer) Type), may be 沦山工& J This is different from an antibody consisting of a natural constant region such as Jg (n 73 201100100. It is reported that an antibody consisting of genus i + 6 ', g 1 匣疋 region, usually The most stable in histidine-B (tetra) buffer (10) let 9G8). Therefore, the effect of the buffer type on the stability of Fv4_M73 was measured. The Fv4-M73 formulation was prepared in 4 buffers of 4 different ρΗ6·〇 buffer solutions to give a final concentration of 37 mg/mU^〇 I η , λ . g mu is as described in Table 9). Sample these samples
4 0 C的保存條件保存超過2個B AA 丨丁廿& m z個月的期間，以uv檢測，以尺 寸排除層析及陰離子交換層虹八 又棵瓚析分析，此等配方中形成的凝 集物百分比，以時間顯示於固 门，、肩不於圖2丨。凝集物之百分比增加， 代表抗體之穩定性下降的如辨 _ Μ伞的才曰軚。因此，使用百分比增加， 作為比較不同緩衝液的穩定效果的指標。 如圖21所示，具址脸酿_Hri '版is· HC1緩衝液（配方a)及檸檬酸 鹽緩衝液)之配方，最為穩定，而具乙酸鹽緩衝液 之配方（配；ίτ C)最為不穩定。、組胺酸_乙酸鹽（配方b)比起 組胺酸-HC1緩衝液稍為妨；., 明馮1乂不穩定，據推測係由於乙酸鹽缓 衝液之去穩定化作用所致。 因此，發現到：具有非天然恆定區之Fv4 —Μ73在組胺酸 -HC1緩衝液及檸檬酸鹽緩衝液中為最穩定，而非如報導的 組胺酸-乙酸鹽緩衝液為天然⑽抗體最為敎之緩衝液 種類。 [表9 ]實施例H用之配方 mAb Cone. mg/mL 配方 緩衝液 NaCl pH Fv4-M73 37 A 20mM組胺酸-ηπ 150mM 6 0 B 20mM組胺酸-乙酸鹽 C 20mM乙酸鹽 D _?0mM檸檬酸鹽 201100100 方法 排除層析（sec) ,^ ，Α，以師選在 在抗體凝集物及片段的抗體配方。將樣本注人尺寸排卜 G3000 SWu管柱（T0S0H)。移動相A u Μ汾 示 杪動相為5〇mM磷酸鈉、3〇〇 氯化納(PH7.0)、以流速〇.5inL/min等梯度流動。提 Ο Ο 白質於2〇〇nm…吸光度。檢測到的任何蛋白質種類的 相對量’係以產物峰部相對於所有其他檢測到之峰部的納 面積百分比表現。早於抗體單體峰部提取出的峰部，記錄 ,凝集物百分位，而晚於抗體單體峰部但早於緩衝液峰部 提取出的峰部，記錄為片段百分位。 樣本製備··將樣本以移動相稀釋為〇 3〜2mg/mL，並注 射15#L至管柱中。 ' ^陰離子交換層析：實施陰離子交換層析以分析存在異 質性之抗體配方’尤其是存在去醯胺化(係在主峰部後以酸 性種類提出的經去酿胺化者）。將樣本於4〇。。注入 DEAE-NPR管柱（T〇s〇H)。移動相⑴為The storage conditions of 4 0 C were saved in more than 2 B AA 丨 廿 amp & mz months, detected by uv, by size exclusion chromatography and anion exchange layer rainbow analytic analysis, formed in these formulations The percentage of agglutination is shown in time at the solid door, and the shoulder is not as shown in Figure 2. The percentage of agglomerates increases, which means that the stability of the antibody is reduced. Therefore, the percentage increase is used as an indicator for comparing the stabilizing effects of different buffers. As shown in Figure 21, the formulation of the address _Hri 'is · HC1 buffer (formulation a) and citrate buffer) is the most stable, and the formulation with acetate buffer (with ίτ C) Most unstable. , histidine-acetate (formulation b) is slightly better than the histidine-HC1 buffer;., Ming Feng 1乂 is unstable, presumably due to destabilization of acetate buffer. Thus, it was found that Fv4-Μ73 with a non-native constant region is most stable in histidine-HC1 buffer and citrate buffer, whereas the non-naminant-acetate buffer as reported is a native (10) antibody. The most ambiguous type of buffer. [Table 9] Formulation for Example H mAb Cone. mg/mL Formulation buffer NaCl pH Fv4-M73 37 A 20 mM histidine-ηπ 150 mM 6 0 B 20 mM histidine-acetate C 20 mM acetate D _? 0 mM Citrate 201100100 Methods Exclude chromatography (sec), ^, Α, to select antibody formulations in antibody agglutinates and fragments. Place the sample in the size of the G3000 SWu column (T0S0H). The mobile phase A u 示 shows that the turbulent phase is 5 mM sodium phosphate, 3 〇〇 sodium chloride (pH 7.0), and the flow rate is 〇5 inL/min.提 Ο White matter at 2 〇〇 nm... absorbance. The relative amount of any protein species detected is expressed as a percentage of the product peak relative to all other detected peaks. The peak extracted from the peak of the antibody monomer was recorded, and the percentile of the aggregate was recorded, and the peak extracted from the peak of the antibody monomer but earlier than the peak of the buffer was recorded as the percentile of the fragment. Sample preparation · Dilute the sample to 〇 3~2 mg/mL with mobile phase and inject 15#L into the column. 'Anion exchange chromatography: Anion exchange chromatography was carried out to analyze the heterogeneous antibody formulation', especially the presence of deamidation (de-aminated by the acid species after the main peak). Take the sample at 4〇. . Inject the DEAE-NPR column (T〇s〇H). Mobile phase (1) is
TriS HC1(PH7.5)，B) 、 10mM Tris-HC1 、 50〇mM 氣化鈉TriS HC1 (pH 7.5), B), 10 mM Tris-HC1, 50 mM sodium sulphate
(PH7.5) ’梯度為100%移動相A，之後30分鐘70%移動相A 及3〇%移動相B，然後1分鐘100%移動相B，然後維持4分 鐘將Β柱以流速L 0mL/min清洗。提取的蛋白質於28〇nm 檢測UV吸光度。 樣本製備：將樣本以移動相稀釋為 05〜〇.33mg/mL’並注射100#L至管柱中。 實施例1 〇 75 201100100 pH、NaCl 及精胺酸-HC1 對於 Fv4_M73 於 1〇〇mg/mL 之 穩定性的作用 將Fv4-M73配方在不同的緩衝液配方（如表1〇所示， 使最終濃度為10〇mg/mL’並以尺寸排除層析法及陰離子交 換層析法’以UV檢測保存在25及4〇t超過2個月的樣本， 並且也檢測冷凍-解凍的樣本（於_2〇t冷凍〇. 5天再於室 溫解凍30分鐘。配方中存在的凝集物百分比及於及 40 C保存2/4/8週後比起起初增加的凝集物百分比，如圖 22-25所示。凝集物增加量作為穩定性降低的指標。 [表1 0 ]於實施例1 〇使用的配方 緩衝液 20mM組胺酸 - HC1 -----1 20mM MM m m NaCl 50mM 150mM 5〇mM 5 OmM 1 R/lmM 精胺S LHC1 - l〇〇mM — 50mM 目標 4. 5 4. 3 4. 3 -— — 4. 0 4. 3 4 S •i uuinM ——___ A r pH 5. 0 4. 9 4. 9 5. 〇 " _ 4 Q 4. 5 A 〇 5. 5 5.4 5.4 5.4 5.4 5 4 4. 〇 C 4 广6. 0 6. 0 5. 8 5. 8 6. 0 5 Q 〇. 4 —-- C 7 6. 5 6.4 6. 3 6. 3 6. 2 6 2 ^，7 — R 9 7. 0 6. 6 6. 7 6. 6 6. 5 0 · ^ -Ai__ 實際值（pf 0 圖26中的低分子量種類侧，推測係弱點例如鉸鏈 部在酸性或鹼性條件下直接水解胜肽鍵的結果。此對於液 體配方中的單株抗㈣高溫為尤其常見。酸性種類於約< 22. 5〜26分鐘提取出，其於鹼性條件增加，且見於圖27， 推測係由於天冬醯胺殘基之非酵素性去醯胺化（常見的抗 體修飾），其據報告，藉由將抗體在高溫於鹼性pH溫育後^ 201100100 觀察更為酸性之種類，係參與單株抗體之電荷異質性。考(pH 7.5) 'The gradient is 100% mobile phase A, then 70% mobile phase A and 3〇% mobile phase B after 30 minutes, then 100% phase B in 1 minute, then 4 minutes to maintain the column at flow rate L 0mL /min cleaning. The extracted protein was tested for UV absorbance at 28 〇 nm. Sample preparation: The sample was diluted to a mobile phase of 05~〇.33 mg/mL' and 100#L was injected into the column. Example 1 〇75 201100100 pH, NaCl and arginine-HC1 For the stability of Fv4_M73 at 1〇〇mg/mL, Fv4-M73 was formulated in different buffer formulations (as shown in Table 1〇, Samples were stored at 25 and 4 〇t for more than 2 months by UV exclusion at a concentration of 10 〇mg/mL ' and by size exclusion chromatography and anion exchange chromatography, and also for frozen-thawed samples (in _ 2〇t frozen 〇. After thawing for 30 minutes at room temperature for 5 minutes, the percentage of agglutinate present in the formulation and the percentage of agglutinated substance increased after 2/4/8 weeks of storage at 40 C, as shown in Figure 22-25 The increase in agglutination was used as an indicator of the decrease in stability. [Table 1 0] Formulation buffer used in Example 1 20 20 mM histidine-HC1 -----1 20 mM MM mm NaCl 50 mM 150 mM 5 mM 5 OmM 1 R/lmM Spermine S LHC1 - l〇〇mM — 50mM Target 4. 5 4. 3 4. 3 -- — 4. 0 4. 3 4 S •i uuinM ——___ A r pH 5. 0 4. 9 4. 9 5. 〇" _ 4 Q 4. 5 A 〇5. 5 5.4 5.4 5.4 5.4 5 4 4. 〇C 4 广六. 0 6. 0 5. 8 5. 8 6. 0 5 Q 〇. 4 —-- C 7 6. 5 6.4 6. 3 6. 3 6 2 6 2 ^,7 — R 9 7. 0 6. 6 6. 7 6. 6 6. 5 0 · ^ -Ai__ Actual value (pf 0 The low molecular weight type side in Fig. 26 is estimated to be a weak point such as a hinge The result of direct hydrolysis of the peptide bond under acidic or basic conditions. This is especially common for single plant anti-(four) high temperatures in liquid formulations. Acidic species are extracted at about < 22. 5~26 minutes, under alkaline conditions. Increased, and seen in Figure 27, is presumed to be due to non-enzymatic deamidation of the indoleamine residue (common antibody modification), which has been reported to be incubated after incubation at elevated temperatures at alkaline pH ^ 201100100 Observing the more acidic species is involved in the charge heterogeneity of individual antibodies.
慮圖22-26之結果’發現Fv4-M73關於凝集及分解，在pH 值約5. 5〜6. 3於溶液中為穩定的。觀察到，關於相對於總 峰部面積之主峰部比值，pH約5. 5~6. 3為最適穩定度（圖 27-28)。 在本研究中，將lOOmM NaCl添加到含有20mM緩衝劑 及50mM NaCl(20mM緩衝液，i5〇mM NaCl)的緩衝液中，能 帶來與含有20mM緩衝劑及50mM NaCl(20mM緩衝液，5〇mM ® NaC1)同等穩定的抗體溶液，表示NaCl沒有穩定作用。而 添加100mM精胺酸至含有2〇mM緩衝劑及50mM NaCl(20mM 緩衝液、50mM NaCl及l〇〇mM精胺酸-HC1)之緩衝液中，顯 示相對於凝集，該抗體溶液之顯著穩定作用，代表精胺酸 HC1具有顯著的穩疋效果。就緩衝劑而言，使用組胺酸—Hu 緩衝液比起檸檬酸鹽緩衝液的溶液穩定性稍佳。 關於冷凍-解凍研究（“FT”代表冷凍/解凍循環之 〇 數），如圖29所示，較低PH或較低鹽濃度之配方中觀察到 顯著量的凝集物。添加l00mM精胺酸到含有2〇祕緩衝劑及 50mM NaCl(20inM 緩衝液、5〇mM NaCl、lOOmM 精胺酸—HC1) 之緩衝液中，顯示在形成凝集物方面，比起添加i 〇〇mM NaCK20mM緩衝液、150mM NaC1)更為有效，顯示精胺酸_hci 對冷凍-解凍具有顯著的穩定作用。最適穩定性在含有 1〇〇1111^精胺酸-11(31之約5.〇〜6.6之011觀察到。 實施例11 糖及精胺酸-HC1對於Fv4-M73在l〇〇mg/mL的作用 77 201100100 *將FV4-M73配方於4種不同的緩衝液配方中 浪度為l〇〇mg/mL(如表U所述）。將此等樣本 最終 °C的保存條件保存超過2個月的_，以uv_及4〇 排除層析法及陰離子交換層析法分析，並使進行二寸 竭循環(於—2。度冷;東〇.5曰，並於室溫 :里。配方中含的凝集物的百分比’從起初之增加 、會圖並顯示於圖30及31。凝集物量之增加百分比二 定性降低之指標。 穩 如圖30及31所示，配方FAG比起配μ提供較少 的穩定性，顯示嚴糖及海藻糖比起精胺酸—HC1於保存在= 及40°C的液體條件下，具有較小的穩定作用，而於冷凍一 解凍，配方F及G比起配方η提供可匹敵的或更高的穩定 性，顯示蔗糖或海藻糖在冷凍_解凍具有顯著的穩定作用。 [表11 ]實施例11之配方 mAb Cone. mg/mL 配方 賦形劑 緩衝液 pH Fv4-M73 100 E M. 20mM組胺 酸-HC1 、 50mM NaCl 6. 5 F l〇〇mM蔗糖 G lOOmM海藻糖 H lOOmM精胺酸-HC1 實施例125〜6. 3。 Stable in the solution. The solution was found to be stable in the solution. It is observed that, with respect to the ratio of the main peaks of the total peak area, the pH is about 5. 5 to 6. 3 is the optimum stability (Fig. 27-28). In the present study, 100 mM NaCl was added to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, i5 mM mM NaCl) to bring with 20 mM buffer and 50 mM NaCl (20 mM buffer, 5 〇). mM ® NaC1) An equally stable antibody solution, indicating that NaCl has no stabilizing effect. Adding 100 mM arginine to a buffer containing 2 mM mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl, and 1 mM arginine-HC1) showed significant stability of the antibody solution relative to agglutination. The role of arginine HC1 has a significant stabilizing effect. In the case of buffers, the stability of the solution using histidine-Hu buffer was slightly better than that of the citrate buffer. Regarding the freeze-thaw study ("FT" represents the number of freeze/thaw cycles), as shown in Figure 29, a significant amount of agglutinate was observed in formulations with lower or lower salt concentrations. Adding l00 mM arginine to a buffer containing 2 〇 secret buffer and 50 mM NaCl (20 inM buffer, 5 mM mM NaCl, 100 mM arginine-HC1) showed that in the formation of agglutination, compared with the addition of i 〇〇 mM NaCK 20 mM buffer, 150 mM NaCl) was more effective, indicating that arginine _hci has a significant stabilizing effect on freeze-thaw. The optimum stability was observed in 011 containing 9.111 arginine-11 (31 of about 5. 〇~6.6. Example 11 Sugar and arginine-HC1 for Fv4-M73 at l〇〇mg/mL Role 77 201100100 *FV4-M73 formulation in 4 different buffer formulations with a wave duration of l〇〇mg/mL (as described in Table U). Save the final °C storage conditions for these samples for more than 2 The _ of the month is analyzed by uv_ and 4〇 exclusion chromatography and anion exchange chromatography, and the cycle of two inches is performed (in the case of -2. degree cold; Dongpu. 5曰, and at room temperature:). The percentage of agglomerates contained in the formula 'increased from the beginning, will be shown and shown in Figures 30 and 31. The percentage increase in the amount of agglomerate is two indicators of qualitative reduction. As shown in Figures 30 and 31, the formulation FAG is better than the match. Provides less stability, showing that the sugar and trehalose have less stabilizing effect than the arginine-HC1 stored in liquid conditions at = and 40 ° C, while freezing-freezing, formula F and G Provides comparable or higher stability than formulation η, showing that sucrose or trehalose has a significant stabilizing effect on freeze-thaw. [Table 11] Example 1 Formula 1 mAb Cone. mg/mL Formulation Excipient Buffer pH Fv4-M73 100 E M. 20 mM histidine-HC1, 50 mM NaCl 6. 5 F l mM sucrose G lOOmM trehalose H lOO mM arginine -HC1 Example 12
Fv4-M73於20 0mg/mL之穩定性：pH、精胺酸-HC1及海 藻糖的作用 將Fv4-M73配方在如表12所述6種不同的配方，最 終濃度200mg/mL。 .201100100 施例12使用的配方 mAb Con. mg/mL 配方 Γ — 緩衝液 Arg-HCl(mM) 賦形劑 pH Fv4-M73 200 1 2 20mM 組胺酸 100 5.5 6.0 _ 3 6.5 4 50 6.0 5 6 150 100 50mM海藻糖 〇 將樣本各於5°C及-2(TC溫育3個月及6個月，將3個 月樣本及6個月樣本以如實施例9所述使用尺寸排除層析 法分析，以UV檢測。3個月及6個月後，於代在此配方 中凝集物從起始增加的百分比，如圖32所示。3個月及6 個月後於2 0 C在此配方中凝集物從起始增加的百分比， 如圖3 3所示。 如圖32所示，於保存在5°c之液體條件下，Fv4_M73 顯然在較低pH較穩定（於PH5.5最穩定，於pH6.5最不穩 定），且於較高精胺酸濃度較穩定（於150mM精胺酸-HC1最 穩定，於50mM精胺酸-HC1最不穩定）。添加5〇ωΜ海藻糖 顯示對於保存於5t:之液體條件中的Fv4—Μ73有一些穩定 作用。因為抗體之醫藥液體配方常保存在5t，故較佳的 FV4-M73的液體配方應至少包含1〇〇mM精胺酸_Ηπ及組胺 酸缓衝液PH5.5〜ΡΗ6·0，及視需要的額外的穩定劑。 如圖33所示，於-20t之冷凍保存條件，Fv4_M73在 較南pH較為穩定，且明顯在較高精胺酸濃度較為穩定（在 Ί9 201100100 15〇mM精胺酸—HC1最穩定，且於精胺酸_ΗΠ最不穩 定）。添加50mM海藻糖於—2吖保存之冷凌保存條件下，顯、 示對於Fv4.有顯著的穩定作用。抗體的大批物質時常 二冷滚狀態保存在-2卜㈣。考量在冷綠態的保存及運 送成本希望為-20 C保存。較佳的用於在_2〇它保存 FV4-M73之大批物質配方，應至少包含1〇〇mM精胺酸邗q 及組胺酸緩衝液PH5. 5〜pH6. 5及糖（例如海藻糖）。 參考實施例 製備可溶性重組人類1L-6受體 依如下方式生產係為抗原之人類IL —6受體之可溶性 重組人類IL-6受體。產生結構性地表現含有來自Stability of Fv4-M73 at 20 mg/mL: pH, arginine-HC1 and trehalose The Fv4-M73 formulation was formulated in six different formulations as described in Table 12, with a final concentration of 200 mg/mL. .201100100 Formulation mAb used in Example 12 Con. mg/mL Formulation Γ - Buffer Arg-HCl (mM) Excipient pH Fv4-M73 200 1 2 20 mM Histamine 100 5.5 6.0 _ 3 6.5 4 50 6.0 5 6 150 100 50 mM trehalose 〇 Each sample was incubated at 5 ° C and -2 (TC was incubated for 3 months and 6 months, and the 3 month sample and the 6 month sample were used as described in Example 9 using size exclusion chromatography. Method analysis, UV detection. After 3 months and 6 months, the percentage of agglutination increased from the initial in this formula, as shown in Figure 32. After 3 months and 6 months at 20 C The percentage of agglomerate increase in the formulation from the beginning, as shown in Figure 33. As shown in Figure 32, Fv4_M73 is clearly stable at lower pH (see pH 5.5 at the pH of 5 °c). Stable, most unstable at pH 6.5, and stable at higher arginine concentration (most stable at 150 mM arginine-HC1, most unstable at 50 mM arginine-HC1). Add 5 〇 ω Μ trehalose It shows some stabilizing effect on Fv4-Μ73 stored in liquid conditions of 5t: Since the pharmaceutical liquid formulation of antibody is often stored at 5t, the preferred liquid formula of FV4-M73 Contains at least 1 mM arginine _ Η π and histidine buffer pH 5.5 ~ ΡΗ 6 · 0, and additional stabilizer as needed. As shown in Figure 33, at -20t cryopreservation conditions, Fv4_M73 It is more stable than the south pH, and is obviously stable at higher arginine concentration (in Ί9 201100100 15 mM arginine-HC1 is the most stable, and arginine _ ΗΠ is the most unstable). Add 50mM trehalose to - 2吖Under the storage condition of cold storage, the display shows a significant stabilizing effect on Fv4. The bulk of the antibody is often stored in the cold-rolled state at -2b (4). The consideration of storage and transportation cost in the cold green state is expected. 5〜pH6. 5和。 The -20 C. It is preferably used in the _2 〇 保存 保存 保存 保存 保存 保存 保存 保存 大 大 大 大 大 大 大 大 大 及 及 及 及 及 及 及 及 及 及 及 及 及 及Sugar (e.g., trehalose). Reference Example Preparation of Soluble Recombinant Human 1L-6 Receptor A soluble recombinant human IL-6 receptor producing human IL-6 receptor which is an antigen is produced in the following manner.
Biochem.(1990) 108, 673-676(Yamasaki et al.,Biochem. (1990) 108, 673-676 (Yamasaki et al.,
Science(1 988) 241，825-828(GenBank *X1 2830)之 N 末端 第1〜344位胺基酸序列之可溶性人類IL_6受體的CH〇細胞 株。將可溶性人類IL-6受體從表現SR344之CH0細胞培養 上m ’以3管柱層析精製：b 1 ue Sepharose 6 FF管柱層析、 使用固定化有專一於SR344之抗體的管柱進行親和性層 析’及凝膠過濾管柱層析。以主峰部提取的分部，使用作 為最終精製樣本。 製備可溶性重組食蟹猴IL-6受體（CIL-6R) 依據已揭露的恒河猴（Rhesus monkey)IL-6受體的基 因序列（Birney et al.，Ensembl 2006, Nucleic Acids Res. 2006 Jan l;34(Database issue:D556-61 )，製備募 DNA 引 80 .201100100 子。使用此引子’以從食蟹猴之胰臟製備之cDNA為模板， 以PCR製備編碼為完整食蟹猴IL 6受體基因之片段。 將得到的DNA片段插入哺乳動物表現載體，並使用此載體 製作穩定的表現CH0細胞株（cyn〇 sIL_6R生產CH0細胞 株）。將cyno. SIL-6R生產CH0細胞株使用HisTrap管柱（GE Healthcare Bioscience)精製，並以 Amicon Ultra-15 U1 tracel-l〇k(Mi11ipore)濃縮。進一步在 〇 Superdex20〇Pgl 6/60 凝膠管柱（ge Healthcare Bioscience) 精製後’得到最終精製的可溶性食蟹猴IL_6受體的樣本 (以下稱為cIL-6R)。 製備重組食蟹猴a-6(clL—6) 以如下程序製備食蟹猴IL-6。製備寄存於SWISSPR0T 存取編號P79341之編碼為21 2個胺基酸的核苷酸序列，並 選殖到一哺乳動物表現載體。將得到的載體導入CH0細 Q 胞’以製備一穩定的表現細胞株（cynoIL-6生產CH0細胞 株）。將 cynoIL-6生產 CH0細胞株之培養基使用 SP-Sepharose/FF 管柱（GE Healthcare Bioscience)精製， 並以 Amicon Ultra-15 Ultracel-5k(Millip〇re)濃縮。進 一步在 Superdex200pgl6/60 凝膠管柱（GE HealthcareScience (1 988) 241, 825-828 (GenBank * X1 2830) N-terminal CH 〇 cell line of soluble human IL_6 receptor at amino acid sequence 1 to 344. The soluble human IL-6 receptor was cultured from CH0 cells expressing SR344 on m' by 3-column chromatography: b 1 ue Sepharose 6 FF column chromatography, using a column packed with an antibody specific for SR344 Affinity chromatography' and gel filtration column chromatography. The fraction extracted by the main peak was used as the final refined sample. Preparation of Soluble Recombinant Cynomolgus IL-6 Receptor (CIL-6R) Based on the Revealed Gene Sequence of the Rhesus Monkey IL-6 Receptor (Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan l; 34 (Database issue: D556-61), preparation of DNA primer 80.201100100. Using this primer 'to prepare cDNA from the pancreas of cynomolgus monkey as a template, PCR was prepared to encode the complete cynomolgus IL 6 Fragment of the receptor gene. The obtained DNA fragment was inserted into a mammalian expression vector, and the vector was used to produce a stable CHO cell line (cyn〇sIL_6R producing CH0 cell line). The cyno. SIL-6R production CH0 cell line was used with HisTrap. The column (GE Healthcare Bioscience) was refined and concentrated with Amicon Ultra-15 U1 tracel-l〇k (Mi11ipore). Further refined by 〇Superdex20〇Pgl 6/60 gel column (ge Healthcare Bioscience) A sample of the soluble cynomolgus monkey IL_6 receptor (hereinafter referred to as cIL-6R). Preparation of recombinant cynomolgus monkey a-6 (clL-6) The cynomolgus monkey IL-6 was prepared by the following procedure. Preparation was deposited in the SWISSPR0T accession number. The code of P79341 is 21 2 amino acids. The nucleotide sequence was cloned into a mammalian expression vector. The resulting vector was introduced into CH0 fine Q cells to prepare a stable expression cell line (cynoIL-6 producing CH0 cell line). The cynoIL-6 production CH0 cell line was prepared. The medium was refined using SP-Sepharose/FF column (GE Healthcare Bioscience) and concentrated with Amicon Ultra-15 Ultracel-5k (Millip〇re). Further on Superdex 200pgl6/60 gel column (GE Healthcare)
Bioscience)精製後，再以 Amicon Ultra-15 U1 tracel-5k(Mi 11 ipore)濃縮後，得到最終精製的食蟹猴 IL-6樣本（以下稱為cIL-6)。 81 201100100 製備已知的南親和性抗IL ~6受體抗體After purification, Bioscience was concentrated in Amicon Ultra-15 U1 tracel-5k (Mi 11 ipore) to obtain a finally purified cynomolgus IL-6 sample (hereinafter referred to as cIL-6). 81 201100100 Preparation of known Southern affinity anti-IL-6 receptor antibodies
hlgGl ， VQ8F11-21hlgGl, VQ8F11-21
尚親和性抗IL-6受體Affinity anti-IL-6 receptor
現載體。以熟習該技術領域之人士已知的方法決定得到的 表現載體的核苷酸序列。使用如實施例丨所述方法，構築 表現載體，表現及純化該高親和性抗〗L_6受體抗體（以下 簡稱為”對照組”）。 製備、表現及精製TOCILIZUMAB變異體 依照 QuikChange Site-Directed Mutagenesis ❹ Kit(Stratagene)所附指導手冊，製備TOCILIZUMAB變異 體。將得到的質體片段插入哺乳動物細胞表現載體，以構 築供關注之Η鏈及L鏈的表現載體。以熟習該技術領域之 人士已知的方法確認得到之表現載體的核苷酸序列。抗體 以如下方法表現。將人類胚胎腎癌衍生之ΗΕΚ293Η細胞株 (Invitrogen)懸浮於補充有10%胎牛血清（Invitrogen)之 DMEMUnvitrogen)。將細胞以細胞密度 5〜6xl05 cells/ml 各注10mL於供黏著細胞之培養皿（直徑l〇cm;Corning)， 82 201100100 m 並在C〇2培養箱（37°c，5%C〇2)培養一整天及一整夜。然後， 以抽吸除去培養基，添加6.9mL CH0-S-SFM-II培養基 (Invitrogen)。將製備的質體以脂轉染法導入細胞。收集 的到的培養上清’離心（約20 0Og ’ 5mi η，室溫）以移除細 胞’並經 0.22/zm 渡膜 MILLEX®-GV(Millipore)過濾、以除 菌’以得到上清。將抗體從得到之培養上清，使用rPr〇tein A Sepharose TM Fast Flow(Amersham Biosciences)以熟習 〇 該技術領域之人士所知方法精製。為了決定精製抗體的濃 度，使用光譜分光計測定280nm的吸光度。以PACE方法 (Protein Science 1 995;4:241 1-2423)計算吸光度係數， 由決定的值計算抗體濃度。 建立人類gp130-表現BaF3細胞株 使用以下所述程序建立表現人類gp 之BaF3細胞 株，以得到以IL-6依存方式增殖的細胞株。 〇 以 PCR 擴增全長人類 gpl30 cDNA(Hibi et al.，Current carrier. The nucleotide sequence of the resulting expression vector is determined by methods known to those skilled in the art. The expression vector was constructed by the method described in Example ,, and the high-affinity anti-L_6 receptor antibody (hereinafter referred to as "control group") was expressed and purified. Preparation, performance, and purification of the TOCILIZUMAB variant The TOCILIZUMAB variant was prepared according to the instruction manual attached to the QuikChange Site-Directed Mutagenesis ❹ Kit (Stratagene). The obtained plasmid fragment is inserted into a mammalian cell expression vector to construct a expression vector for the Η chain and the L chain of interest. The nucleotide sequence of the resulting expression vector is confirmed by methods known to those skilled in the art. The antibody was expressed as follows. Human embryonic kidney cancer-derived 293 Η cell line (Invitrogen) was suspended in DMEM Unvitrogen supplemented with 10% fetal bovine serum (Invitrogen). Inject the cells at a cell density of 5~6xl05 cells/ml into 10mL of culture cells for the adherent cells (diameter l〇cm; Corning), 82 201100100 m and in a C〇2 incubator (37 ° C, 5% C 〇 2 ) Train all day and all night. Then, the medium was removed by suction, and 6.9 mL of CH0-S-SFM-II medium (Invitrogen) was added. The prepared plastids were introduced into the cells by lipofection. The collected culture supernatant was centrifuged (about 20,000 g 5 η, room temperature) to remove the cells and filtered through a 0.22/zm membrane MILLEX®-GV (Millipore) to sterilize 'to obtain a supernatant. The antibody was cultured from the obtained supernatant and purified using rPr〇tein A SepharoseTM Fast Flow (Amersham Biosciences) by a method known to those skilled in the art. In order to determine the concentration of the purified antibody, the absorbance at 280 nm was measured using a spectrometer. The absorbance coefficient was calculated by the PACE method (Protein Science 1 995; 4:241 1-2423), and the antibody concentration was calculated from the determined values. Establishment of human gp130-expressing BaF3 cell line A BaF3 cell line expressing human gp was established using the procedure described below to obtain a cell line which proliferated in an IL-6-dependent manner. 〇 Amplification of full-length human gpl30 cDNA by PCR (Hibi et al.,
Cell( 1 990) 63:1 1 49-1 1 57(GenBank #NM_002184)，並選殖Cell( 1 990) 63:1 1 49-1 1 57 (GenBank #NM_002184), and colonization
到表現載體 pC0S2Zeo 以構築 pCOS2Ze〇/gpl3〇cpC〇S2Ze〇 為一表現載體，係藉由從pCH〇i(Hirate et al.，FEBSTo the expression vector pC0S2Zeo to construct pCOS2Ze〇/gpl3〇cpC〇S2Ze〇 as a performance vector, from pCH〇i (Hirate et al., FEBS)
Letter( 1 994) 456:244-248)移除DHFi?基因表現區並插入Letter (1 994) 456:244-248) Remove the DHFi? gene expression region and insert
Zeocin抗藥基因之表現區而構築。全長人類cdNA 以PCR擴增，並選殖到pcDNA31( + ) (Invitr〇gen)以構築 hIL-6R/pcDNA3.1(+)。 將 pC0S2Zeo/gp!30 l〇# g 與懸浮在 pBS 之 BaF3 細胞 83 201100100 (0·8χ107 cells)混合，然後，於 0 33kV 及 95〇micr〇 Fd 使用Gene Pu 1 ser (Bi〇-Rad)脈衝。將具有以電穿孔法導入 基因之BaF細胞於補充有〇.2ng/ml小鼠IL_3(Pepr〇tech) 及10%胎牛血清（以下稱為FBS，HyClone)之RPMI 1 640培 養基（Invitrogen)培養一整天及一晚，並藉由添加補充有 100ng/ml 人類 IL-6(R&D systems)、100ng/ml 人類 IL-6 可溶性受體（R&D systems)及10% FBS之RPMI 1 640培養 基’以建立一表現人類gpl3〇之BaF3細胞株（以下稱Constructed by the expression region of the Zeocin drug resistance gene. The full-length human cdNA was amplified by PCR and cloned into pcDNA31(+) (Invitr〇gen) to construct hIL-6R/pcDNA3.1(+). Mix pC0S2Zeo/gp!30 l〇# g with BaF3 cells 83 201100100 (0·8χ107 cells) suspended in pBS, then use Gene Pu 1 ser (Bi〇-Rad) pulse at 0 33kV and 95〇micr〇Fd . BaF cells having a gene introduced by electroporation were cultured in RPMI 1 640 medium (Invitrogen) supplemented with ng2 ng/ml mouse IL_3 (Pepr〇tech) and 10% fetal bovine serum (hereinafter referred to as FBS, HyClone). All day and night, by adding RPMI 1 supplemented with 100 ng/ml human IL-6 (R&D systems), 100 ng/ml human IL-6 soluble receptor (R&D systems) and 10% FBS 640 medium' to establish a BaF3 cell line expressing human gpl3〇 (hereinafter referred to as
為 BaF3/gpl30”）。此 BaF/gpi3〇 於存在人類 IL-6(R&D sy sterns)及可溶性人類IL-6受體時增殖，且可用於評估抗 IL - 6受體抗體之生長抑制活性（或I l — 6受體中和活性）。 以表現人類gpl30之BaF細胞株（BaF/gpl30)評估生物 學活性 使用以IL-6/IL-6受體依存方式增殖之BaF3/gpl30， 評估IL-6受體中和活性。以補充有i〇%FBS之rpmi 1640 清洗3次後，將BaF/gpl30於補充有600ng/ml或60ng/ml 人類IL-6CT0RAY)、適量可溶性人類IL-6受體及i〇%FBS 之 RPMI 1640 懸浮成 5xl04cells/ml(最終濃度為 300ng/ml 或30ng/m 1)。將細胞懸浮液分注（5〇 mi cro 1 i ter/we 11 )於 96井平盤（CORNING)。然後’將經精製的抗體以含有i〇%FBS 之 RPMI 1640 稀釋’並添加至各井（5〇microliter/well)。 將細胞於5%C〇2下於37。(：溫育3天。將WST-8試劑（Cell Counting Kit-8; Doji ndo Laboratories)以 PBS 稀釋 2 倍。 84 1 201100100 δ 2〇" L藥劑添加至各井後，立即使用SUNRISE CLASSIC(TECAN)測量45〇nm(參考波長：62〇nm)之吸光度。 培養2小時後，再於45 0nm(參考波長：62〇nm)測量吸光度。 在2小時期間，使用吸光度之改變作為指標評估丨L_6受體 中和活性。 依據Biacoare分析對可溶性人類IL_6受體之結合 使用Biac〇re T100(GE Healthcare)分析抗原-抗體反 0 應動力學。藉由以胺偶合方法固定化適當量的蛋白A或蛋 白A/G或抗IgG(r鏈專一性）F(ab，）2在感應晶片上，將 該關注的抗體於PH7.4結合在晶片上，並在該晶片上於 PH7.4跑動調整為各種濃度的可溶性R —6受體作為分析 物，測疋可洛性人類IL - 6受體抗體交互作用。所有的測定 係於37 C進行。由測定值得到之感應譜，計算動力學參數， 結合速率常數ka(l/Ms)及解離速率常數kd(1/sh然後，依 據此速率常數，決定Kd(M)。使用Biacore T1〇〇 Evaluati〇n 〇 Software(GE Healthcare)決定各自的參數。 使用Biacore評估從膜型受體之pH依存性解離 使用 Biacore T100(GE Healthcare)觀察於 ρΗ5· 8 及 ΡΗ7.4 ’與膜型IL-6受體的抗原抗體交互作用。藉由評估 與固定化在感應晶片上的可溶性人類〗L — 6受體的結合，來 評估與膜型IL-6受體之結合。SR344係以熟習該技術領域 之人士所知的方法生物素化（bi〇tinyiate)。基於生物素與 鏈親和素（Streptavidin)間的親和性，經由鏈親和素將經 85 201100100It is BaF3/gpl30"). This BaF/gpi3 is proliferated in the presence of human IL-6 (R&D sy sterns) and soluble human IL-6 receptor, and can be used to evaluate growth inhibition of anti-IL-6 receptor antibodies. Activity (or I 6-6 receptor neutralizing activity). Evaluation of biological activity using BaF cell line (BaF/gpl30) expressing human gpl30 using BaF3/gpl30 proliferating in IL-6/IL-6 receptor-dependent manner, The IL-6 receptor neutralizing activity was evaluated. After washing 3 times with rpmi 1640 supplemented with i〇% FBS, BaF/gpl30 was supplemented with 600 ng/ml or 60 ng/ml human IL-6 CT0RAY), and the appropriate amount of soluble human IL- 6 receptor and i〇%FBS RPMI 1640 was suspended in 5xl04cells/ml (final concentration was 300ng/ml or 30ng/m 1 ). The cell suspension was dispensed (5〇mi cro 1 i ter/we 11 ) at 96 CORNING. Then 'Dilute the purified antibody with RPMI 1640 containing i〇% FBS' and add to each well (5〇microliter/well). Place the cells at 5% C〇2 at 37. : Incubate for 3 days. WST-8 reagent (Cell Counting Kit-8; Doji ndo Laboratories) was diluted 2 times with PBS. 84 1 201100100 δ 2〇" L agent was added to each well, The absorbance at 45 〇 nm (reference wavelength: 62 〇 nm) was measured immediately using SUNRISE CLASSIC (TECAN). After 2 hours of incubation, the absorbance was measured at 45 0 nm (reference wavelength: 62 〇 nm). During 2 hours, absorbance was used. The change was used as an indicator to evaluate the neutralizing activity of the 丨L_6 receptor. The binding of the soluble human IL_6 receptor to the soluble human IL-6 receptor was analyzed by Biac〇re T100 (GE Healthcare). The antigen-antibody anti-zero kinetics was analyzed by immobilization by amine coupling. Appropriate amount of protein A or protein A/G or anti-IgG (r chain specificity) F(ab,) 2 on the sensing wafer, the antibody of interest is bound to the wafer at pH 7.4, and on the wafer PH7.4 was adjusted to various concentrations of soluble R-6 receptor as an analyte to measure the interaction of the human IL-6 receptor antibody. All assays were performed at 37 C. The induction was obtained from the measured values. Spectral, calculated kinetic parameters, combined with rate constant ka (l/Ms) and dissociation rate constant kd (1/sh then, based on this rate constant, determines Kd(M). Use Biacore T1〇〇Evaluati〇n 〇Software (GE Healthcare) determines the respective parameters. Evaluation of pH-dependent dissociation from membrane-type receptors using Biacore The antigen-antibody interactions of ρΗ5·8 and ΡΗ7.4′ with membrane-type IL-6 receptors were observed using Biacore T100 (GE Healthcare). Binding to the membrane-type IL-6 receptor was assessed by assessing binding to the soluble human L-6 receptor immobilized on the sensing wafer. SR344 is biotinylated by methods known to those skilled in the art. Based on the affinity between biotin and streptavidin, via streptavidin will be 85 201100100
生物素化的可溶性人類IL-6受體固定化在感應晶片上。所 有的測定係於37 t進行。移動相緩衝液為1〇mM MES(pH5. 8)、150mM NaCl 及 0. 05% Tween20。將顯示 PH 依The biotinylated soluble human IL-6 receptor is immobilized on an inductive wafer. All assays were performed at 37 t. The mobile phase buffer was 1 mM MES (pH 5.8), 150 mM NaCl, and 0.05% Tween20. Will show PH
存性結合的選殖體（clone)，於pH7. 4的條件注入，以連接 到可溶性人類IL-6受體（注射樣本緩衝液為1〇mM MES(pH7.4)、150mM NaCl 及 0.05% Tween20)。然後，於 ρΗ5· 8觀察各選殖體的pH依存性解離，此pH為移動相之 pH 。使用 Biacore T100 Evaluation Software(GE Healthcare)’藉由僅符合在pH5.8的解離相，計算於pH5.8 之解離速率常數（kd(l/s))。樣本濃度為〇· 25 # g/ml。結 合實施於 10mM MES(pH7. 4)、150mM NaCl、及 〇. 05% Tween 20 ’ 解離實施於 i〇mM MES(pH5. 8)、150mM NaCl 及 〇· 05% Tween20。同樣地，藉由僅符合在pH7. 4的解離相，計算於 PH7.4之解離速率常數（kd(1/s))。樣本濃度為〇.5 microgramme/ml。結合實施於 lOmM MES(pH7.4)、150mM NaCl、及 0. 05% Tween 20，解離實施於 10mM MES(pH7. 4)、 150niM NaCl 及 〇. 05% Tween20。 評估對於人類FcRn之結合The adherent clone was injected at pH 7.4 to connect to the soluble human IL-6 receptor (injection sample buffer was 1 mM MES (pH 7.4), 150 mM NaCl, and 0.05%). Tween20). Then, the pH-dependent dissociation of each of the colonies was observed at ρΗ5·8, which is the pH of the mobile phase. The dissociation rate constant (kd(l/s)) at pH 5.8 was calculated using Biacore T100 Evaluation Software (GE Healthcare) by only meeting the dissociated phase at pH 5.8. The sample concentration is 〇· 25 # g/ml. The combination was carried out at 10 mM MES (pH 7.4), 150 mM NaCl, and 0. 05% Tween 20 ′ dissociation was carried out at i〇mM MES (pH 5.8), 150 mM NaCl, and 〇·05% Tween20. Similarly, the dissociation rate constant (kd (1/s)) at pH 7.4 was calculated by only meeting the dissociated phase at pH 7.4. The sample concentration was 〇.5 microgramme/ml. The binding was carried out in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, and dissociation was carried out at 10 mM MES (pH 7.4), 150 niM NaCl, and 5%. 05% Tween20. Assessing the binding of human FcRn
FcRn為FcRn及/32-微球蛋白之複合體。依已揭露的 人類 FcRn 基因序列（J· Exp. Med. (1994) 1 80(6):2377-2381 )製備寡 DNA 引子。使用人類 cDNA(Human Placenta Marathon-Ready cDNA，Clontech)作為模板及已 製備的引子，以PCR製備編碼為完整基因的DNA片段。使 86 201100100FcRn is a complex of FcRn and /32-microglobulin. An oligo DNA primer was prepared according to the disclosed human FcRn gene sequence (J. Exp. Med. (1994) 1 80(6): 2377-2381). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and a prepared primer. Make 86 201100100
寡DNA引子。使用人類cDNA(Hu —piacentaOligo DNA primer. Use human cDNA (Hu — piacenta
Placenta Marathon-ReadyPlacenta Marathon-Ready
擴增含有信息區（Metl-Metll9)之編碼為完整点2_ 微球蛋白之DNA片段，並插入— 哺乳動物細胞表現載體（列 於序列識別號80之人類2-微球蛋白之胺基酸序列）。 以如下程序表現可溶性人類FCRn。將針對人類FcRn 及/52-微球蛋白構築之質體’使用fbs以脂質轉染法 導入人類胚胎腎癌衍生之細胞株HEK293H(Invitrogen)之 細胞中。收集得到之培養上清’依照j. Immun〇12〇〇2 〇 Novl;169(9h517卜80所述方法，精製FcRn係使用igG Sepharose6 Fast Flow(Ainershain Biosciences)，巧 γ吏用 HiTrap Q HP(GE Healthcare)進一步精製。 決定小鼠血漿中之抗體濃度 依照熟習該技術領域之人士所知的方法，以EL ISA決 定小鼠血漿中之抗體濃度。 以PK/PD測試確認猴血漿中之抗體濃度、CRP濃度， 87 201100100 及游離可溶性il—6受體 使用熟習該技術領域之人士已知方法以EL ISA確認食 蟹猴中的血漿濃度。 以自動化分析儀（TBA-120FR; Toshiba Medical Systems Co.)使用 Cias R CRPCKANTO CHEMICAL CO.，Inc.) 決定CRP之濃度。 以如下程序確認食蟹猴中游離的可溶性食蟹猴il_6 受體之血漿濃度。藉由載入30 食蟹猴血漿於0.22Amplification of a DNA fragment encoding the information region (Metl-Metll9) encoded as a complete point 2_ microglobulin, and insertion into a mammalian cell expression vector (amino acid sequence of human 2-microglobulin listed in SEQ ID NO: 80) ). The soluble human FCRn was expressed in the following procedure. The plastids constructed for human FcRn and /52-microglobulin were introduced into cells of human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) by lipofection using fbs. The collected culture supernatant was collected according to the method of j. Immun〇12〇〇2 〇Novl; 169 (9h517, 80, refined FcRn using igG Sepharose6 Fast Flow (Ainershain Biosciences), and γ吏 using HiTrap Q HP (GE) Further purification. Determine the antibody concentration in mouse plasma. Determine the antibody concentration in mouse plasma by EL ISA according to a method known to those skilled in the art. The antibody concentration in monkey plasma is confirmed by PK/PD test. CRP concentration, 87 201100100 and free soluble il-6 receptors Plasma concentrations in cynomolgus monkeys were confirmed by EL ISA using methods known to those skilled in the art. Automated analyzers (TBA-120FR; Toshiba Medical Systems Co.) The concentration of CRP is determined using Cias R CRPCKANTO CHEMICAL CO., Inc.). The plasma concentration of the free soluble cynomolgus il_6 receptor in cynomolgus monkeys was confirmed by the following procedure. By loading 30 cynomolgus monkey plasma at 0.22
濾-杯乾燥（Mi llipore)之一適當量的 rProtein A Sepharose Fast Flow樹脂（GE Healthcare)，使所有在血漿中之I gG 型之抗體（食蟹猴IgG、抗人類il-6受體抗體，及抗人類 IL-6受體抗體-可溶性食蟹猴IL_6受體複合體）吸附到蛋 白A °然後’將杯中之溶液使用高速離心轉下以收集通過 的溶液。通過的溶液不含蛋白A連接的抗人類IL_6受體抗 體-可溶性食蟹猴IL-6受體複合體。因此，可藉由測定通 過ProteinA之溶液中的可溶性食蟹猴IL_6受體濃度，決 定游離之可溶性IL — 6受體濃度。使用熟習此項技術領域之 人士已知用於測定可溶性人類IL_6受體之濃度的方法，確 ⑽可各性食蟹猴IL-6受體之濃度。使用如上述製備之可溶 !生食蟹猴IL-6受體（cil-6R)作為標準。以下式計算游離之 可溶性IL-6受體之百分比。Filter-cup dry (Mi llipore) an appropriate amount of rProtein A Sepharose Fast Flow resin (GE Healthcare) to make all I gG-type antibodies (cynomolgus monkey IgG, anti-human il-6 receptor antibody, in plasma, And the anti-human IL-6 receptor antibody-soluble cynomolgus IL_6 receptor complex) was adsorbed to protein A ° and then the solution in the cup was transferred using high speed centrifugation to collect the passed solution. The resulting solution contained no protein A-linked anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex. Therefore, the free soluble IL-6 receptor concentration can be determined by measuring the concentration of the soluble cynomolgus IL-6 receptor in the solution through Protein A. The method for determining the concentration of the soluble human IL-6 receptor is known to those skilled in the art to determine (10) the concentration of the individual cynomolgus IL-6 receptor. The soluble cynomolgus monkey IL-6 receptor (cil-6R) prepared as described above was used as a standard. The percentage of free soluble IL-6 receptor is calculated by the following formula.
xlOO 88 201100100 【圖式之簡單說明】 圖1顯示改良T0CILIZUMAB對IL-6受體之親和性的突 變部位表。T0CILIZUMAB之HCDR2序列顯示於序列識別號 81 ;突變後的HCDR2序列（上列）顯示於序列識別號82;突變 後的HCDR2序列（下列）顯示於序列識別號83;T0CILIZUMAB 之HCDR3序列顯示於序列識別號84;突變後之HCDR3序列 (上列）顯示於序列識別號85 ;突變後之HCDR3序列（下列） 顯示於序列識別號86;TOCILIZUMAB之LCDR1序列顯示於序 〇 列識別號87 ;突變後之LCDR1序列（上列）顯示於序列識別 號88;突變後之LCDR1序列（下列）顯示於序列識別號 89;T0CILIZUMAB之LCDR3序列顯示於序列識別號90 ;突變 後之LCDR3序列（上列）顯示於序列識別號91 ;突變後之 LCDR3序列（下列）顯示於序列識別號92。 圖 2 顯示於 BaF/gP130 中之 T0CILIZUMAB 及 RDC-23 之 中和活性之圖。 〇 圖3顯示能夠減低可變區之等電點而不顯著減低 T0CILIZUMAB對IL-6受體結合的突變部位表。圖中的星號 代表對於等電點無影響但是為了轉變為人類序列而突變的 突變部位。T0CILIZUMAB之HFR1序列顯示於序列識別號93 ; 犬變後之HFR1序列顯示於序列識別號94; tocilizumAB之 HCDR1序列顯示於序列識別號95 ;突變後之hcdri序列顯示 於序列識別號96;T〇CILIZUMAB之HFR2序列顯示於序列識 別號97’大變後之HFR2序列顯示於序列識別號 ITOCILIZUMAB之隱2序列顯示於序列識別號81;突變 89 201100100 後之HCDR2序列顯示於序列識別號99 ; T0CILIZUMAB之HFR4 序列顯示於序列識別號1 00 ;突變後之HFR4序列顯示於序 列識別號101 ;T0CILIZUMAB之LFR1序列顯示於序列識別號 102;突變後之 LFR1序列顯示於序列識別號 103 ; T0CILIZUMAB之LCDR1序列顯示於序列識別號87;突變 後之LCDR1序列顯示於序列識別號1〇4;TOCILIZUMAB之 LFR2序列顯示於序列識別號105 ;突變後之LFR2序列顯示 於序列識別號106;TOCILIZUMAB之LCDR2序列顯示於序列 識別號1 07 ;突變後之LCDR2序列顯示於序列識別號1 08及 109;TOCILIZIMAB之LFR3序列顯示於序列識別號110;突變 後之LFR3序列顯示於序列識別號111 ;TOCILIZUMAB之LFR4 序列顯示於序列識別號11 2 ;突變後之LFR4序列顯示於序 列識別號11 3。 圖 4 顯示於 BaF/gpl30 中之 TOCILIZUMAB 及 H53/L28 的中和活性。 圖5顯示靜脈内投予之後小鼠體内的TOCILIZUMAB及 H53/L28的血漿濃度的時間變化。 圖6顯示皮下投予之後小鼠體内的TOCILIZUMAB及 H5 3/L28的血漿濃度的時間變化。 圖7圖示IgG分子能藉由在内囊胞（endosome)中從膜 型抗原分離而再結合於另一抗原。 圖8顯示能對TOCILIZUMAB結合於IL-6受體提供pH 依存性的突變部位（結合於ρΗ7· 4，分離於ρΗ5· 8)。 TOCILIZUMAB之HFR1序列顯示於序列識別號93;突變後之 90 201100100 ' HFR1序列顯示於序列識別號114;T0CILIZUMAB之HCDR1序 列顯示於序列識別號95 ;突變後之HCDR1序列顯示於序列 識別號115;T0CILIZUMAB之LCDR1序列顯示於序列識別號 87;突變後之 LCDR1 序列顯示於序列識別號 116;T0CILIZUMBA之LCDR2序列顯示於序列識別號1〇7; 且，突變後之LCDR2序列顯示於序列識別號11 7。 圖 9 顯示於 BaF/gpl30 中之 T0CILIZUMAB 及 H3pl/L73 之中和活性。 〇 / 圖10顯示靜脈内投予後，於食蟹猴體内的 T0CILIZUMAB及H3pl/L73的血漿濃度的時間變化。 圖11顯示靜脈内投予後’於人類IL-6受體基因轉殖 小鼠體内的T0CILIZUMAB及H3pl/L73的血漿濃度的時間變 圖12顯示利用陽離子交換層析評估T0CILIZUMAB、 T0CILIZUMAB (5 K、T0CILIZUMAB (5 GK 的 C 端衍生的異 Q 質性的結果。 圖 13 顯示利用陽離子交換層析評估XlOO 88 201100100 [Simplified description of the schema] Figure 1 shows a table of mutation sites that improve the affinity of TOCILIZUMAB for IL-6 receptor. The HCDR2 sequence of T0CILIZUMAB is shown in SEQ ID NO: 81; the mutated HCDR2 sequence (listed above) is shown in SEQ ID NO: 82; the mutated HCDR2 sequence (below) is shown in SEQ ID NO: 83; the HCDR3 sequence of T0CILIZUMAB is shown in SEQ ID NO: No. 84; the mutated HCDR3 sequence (listed above) is shown in SEQ ID NO: 85; the mutated HCDR3 sequence (below) is shown in SEQ ID NO: 86; the LCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 87; The LCDR1 sequence (listed above) is shown in SEQ ID NO: 88; the mutated LCDR1 sequence (below) is shown in SEQ ID NO: 89; the LCDR3 sequence of TOCIILZUMAB is shown in SEQ ID NO: 90; the mutated LCDR3 sequence (listed above) is shown in Sequence ID 91; the mutated LCDR3 sequence (below) is shown in SEQ ID NO:92. Figure 2 shows the neutralization activity of T0CILIZUMAB and RDC-23 in BaF/gP130. 〇 Figure 3 shows a table of mutation sites that can reduce the isoelectric point of the variable region without significantly reducing T0CILIZUMAB binding to IL-6 receptor. The asterisk in the figure represents a mutation site that has no effect on the isoelectric point but is mutated in order to transform into a human sequence. The HFR1 sequence of T0CILIZUMAB is shown in SEQ ID NO: 93; the HFR1 sequence after canine change is shown in SEQ ID NO: 94; the HCDR1 sequence of tocilizum AB is shown in SEQ ID NO: 95; the hcdri sequence after mutation is shown in SEQ ID NO: 96; T〇CILIZUMAB The HFR2 sequence is shown in the sequence identification number 97' after the HFR2 sequence is shown in the sequence identification number ITOCILIZUMAB. The hidden 2 sequence is shown in SEQ ID NO: 81; the mutation 89 201100100 after the HCDR2 sequence is shown in SEQ ID NO: 99; T0CILIZUMAB HFR4 The sequence is shown in SEQ ID NO: 1 00; the mutated HFR4 sequence is shown in SEQ ID NO: 101; the LFR1 sequence of T0CILIZUMAB is shown in SEQ ID NO: 102; the mutated LFR1 sequence is shown in SEQ ID NO: 103; the LCDR1 sequence of T0CILIZUMAB is shown in SEQ ID NO: 87; the mutated LCDR1 sequence is shown in SEQ ID NO: 1; the LFR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: 105; the mutated LFR2 sequence is shown in SEQ ID NO: 106; the LCDR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: No. 1 07; the LCDR2 sequence after mutation is shown in sequence identification numbers 1 08 and 109; the LFR3 sequence of TOCILIZIMAB is displayed SEQ ID NO 110; LFR3 after the mutant sequence is shown in SEQ ID NO 111; TOCILIZUMAB of LFR4 sequence is shown in SEQ ID NO 112; LFR4 after the mutant sequence is shown in sequence identifier 113. Figure 4 shows the neutralizing activity of TOCILIZUMAB and H53/L28 in BaF/gpl30. Figure 5 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H53/L28 in mice after intravenous administration. Figure 6 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H5 3/L28 in mice after subcutaneous administration. Figure 7 illustrates that an IgG molecule can recombine to another antigen by isolation from a membrane-type antigen in an endosome. Figure 8 shows the mutation site (binding to ρΗ7·4, isolated from ρΗ5·8) which provides pH dependence of TOCILIZUMAB binding to IL-6 receptor. The HFR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 93; the mutated 90 201100100 'HFR1 sequence is shown in SEQ ID NO: 114; the HCDR1 sequence of TOCIILZUMAB is shown in SEQ ID NO: 95; the mutated HCDR1 sequence is shown in SEQ ID NO: 115; T0CILIZUMAB The LCDR1 sequence is shown in sequence identification number 87; the mutated LCDR1 sequence is shown in sequence identification number 116; the LCDR2 sequence in TOCIILZUMBA is shown in sequence identification number 1〇7; and the mutated LCDR2 sequence is shown in sequence identification number 117. Figure 9 shows the neutralizing activity of T0CILIZUMAB and H3pl/L73 in BaF/gpl30. 〇 / Figure 10 shows the temporal changes in plasma concentrations of T0CILIZUMAB and H3pl/L73 in cynomolgus monkeys after intravenous administration. Figure 11 shows the time-varying of plasma concentrations of TOCILIZUMAB and H3pl/L73 in human IL-6 receptor gene-transferred mice after intravenous administration. Figure 12 shows the evaluation of TOCILIZUMAB, T0CILIZUMAB (5 K, using cation exchange chromatography). T0CILIZUMAB (result of heterogeneous Q derived from the C-terminus of 5 GK. Figure 13 shows evaluation using cation exchange chromatography
TOCILIZUMAB-IgG卜TOCILIZUMAB IgG-2、TOCILIZUMAB SKSC 的雙硫鍵衍生的異質性的結果。 圖14顯示利用差示掃描熱量計（])§ c)得到的TOCILIZUMAB-IgG results from the heterogeneity of the disulfide bond of TOCILIZUMAB IgG-2, TOCILIZUMAB SKSC. Figure 14 shows the use of differential scanning calorimeter (]) § c)
TOCILIZUMAB-IgG卜TOCILIZUMAB IgG-2、TOCILIZUMAB SKSC 的變性曲線，及針對各Fab區的Tm值。 圖1 5顯示靜脈内投予後，於人類FcRn轉殖基因小鼠 體内的 TOCILIZUMAB-IgGl 、 T0CILIZUMAB-M44 、 91 201100100 TOCILIZUMAB-M58、TOCILIZUMAB-M73 的血漿濃度的時間變 化0 圖16顯示於BaF/gpl30中之T0CILIZUMAB、對照組及 Fv5-M83的中和活性。 圖 17 顯示於 BaF/gpl30 中之 TOCILIZUMAB、Fv3-M73、 Fv4-M73的中和活性。 圖18顯示靜脈内投予後，於食蟹猴體内的 T0CILIZUMAB、對照組、Fv3-M73、Fv4-M73 及 Fv5-M83 的 血聚·濃度的時間變化。 圖19顯示靜脈内投予後，於食蟹猴體内的 T0CILIZUMAB、對照組、Fv3-M73、Fv4-M73 及 Fv5-M83 的 CRP濃度的時間變化。 圖20顯示靜脈内投予後，於食蟹猴體内的 T0CILIZUMAB、對照組 ' Fv3-M73、Fv4-M73 及 Fv5-M83 的 游離可溶的IL-6受體的百分比的時間變化。 圖21顯示不同配方（A-D)經時形成抗體Fv4-M73之高 分子量物質（HMW)的差異。 圖22顯示於不同的pH值、緩衝液種類、NaC1濃度、 有或無精胺酸，在2個時間點（25〇c )，形成抗體Fv4_M73 之高分子量物質（δ HMW:從起始之增加值）的差異。 圖23顯不於不同的pH值、緩衝液種類、NaC1濃度， 及有或無精胺酸，在2個時間點（託它），形成抗體Fv4_M73 之高分子量物質（HMW)的差異。 圖24顯示於不同的PH值、緩衝液種類、NaC1濃度， 92 201100100 及有或無精胺酸，在3個不同 Fv4-M73之高分子量物質（占 異。 的時間點（4 0 °C )，形成抗體 HMW:從起始之增加值）的差 圖25顯示於不同的 pH值、緩衝液種類、NaCl濃度’ 及有或無精胺酸，在1個眸„飢/ > U %間點（4〇°C )，形成抗體Fv4-M73 之咼分子量物質（HMW)的差異。 圖26顯示於不同的H枯 p值、緩衝液種類、NaCl濃度’ 〇 及有或無精胺酸，在3個+ 1固不冋的時間點（40。(：），形成抗體Denaturation curves of TOCILIZUMAB-IgG, TOCILIZUMAB IgG-2, TOCILIZUMAB SKSC, and Tm values for each Fab region. Figure 15 shows the time change of plasma concentrations of TOCILIZUMAB-IgG1, T0CILIZUMAB-M44, 91 201100100 TOCILIZUMAB-M58, TOCILIZUMAB-M73 in human FcRn transgenic mice after intravenous administration. Figure 16 shows in BaF/ Neutralizing activity of T0CILIZUMAB, control group and Fv5-M83 in gpl30. Figure 17 shows the neutralizing activity of TOCILIZUMAB, Fv3-M73, Fv4-M73 in BaF/gpl30. Figure 18 shows the temporal changes in blood concentration of T0CILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration. Figure 19 shows the temporal changes in CRP concentrations of T0CILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration. Figure 20 shows the time course of the percentage of free soluble IL-6 receptors in T0CILIZUMAB, control groups 'Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration. Figure 21 shows the difference in high molecular weight species (HMW) of the different formulations (A-D) over time to form the antibody Fv4-M73. Figure 22 shows the high molecular weight species (δ HMW: increasing from the initial value) of the antibody Fv4_M73 at different pH values, buffer species, NaC1 concentration, with or without arginine at 2 time points (25 °c). ) the difference. Figure 23 shows the difference in high molecular weight species (HMW) of antibody Fv4_M73 at different pH values, buffer species, NaC1 concentration, and with or without arginine at 2 time points. Figure 24 shows the different pH values, buffer type, NaC1 concentration, 92 201100100 and with or without arginine, at three different Fv4-M73 high molecular weight substances (different time points (40 ° C), The difference between the formation of antibody HMW: the increase from the initial value is shown in Figure 25 at different pH values, buffer type, NaCl concentration' and with or without arginine, at 1 饥 饥 ≥ ≥ ≥ ≥ 4〇°C), the difference in the molecular weight species (HMW) of the antibody Fv4-M73 was formed. Figure 26 shows the different H-values, buffer type, NaCl concentration '〇 and with or without arginine, in 3 + 1 solid time point (40. (:), forming antibodies
Fv4-M73之南分子量物質了…灯 貝I 5 LMW:從起始之增加值）的差 異。 圖27顯示於保存在不同沾„ 啦+网的PH值下的抗體Fv4-M73的 溶液進行陰離子交換層析法的結果。 圖28顯示保存在不同的 +门的pH值、不同的NaC1濃度、精 胺酸及兩種不同的緩衝液 卜抗體Fv4-M73的溶液進行陰 離子交換層析法的結果。 〇 S 29 .㈣針對不同讀的冷"料循環、不同的 紙農度、精胺酸、及2種不同的緩衝液，形成抗體π㈣ 之南分子量物質U HMW:從起始之增加值)的差異。 圖30顯示針對3個不同時間點(於4〇。。及饥)及4 種不同的配方（E-H)，形成抗體Fv4_M73之高分子量物質 (占HMW:從起始之增加值）的差異。 圖31顯示針對2個不同次數的冷康/解凌循環及彳種 不同的配方（E,，形成抗體Fv4_M73之高分子量 HMff:從起始之增加值）的差異。 、 93 201100100 圖32顯示在經過5°C之3個月及6個月後，6種不同 配方形成抗體Fv4-M73之高分子量物質（d HMW:從起始之 增加值）的差異。 圖33顯示在經過-2 0°C之3個月及6個月後，6種不 同配方形成抗體Fv4-M73之高分子量物質（δ HMW:從起始 之增加值）的差異。 【主要元件符號說明】 無 94 201100100 序列表 <110〉 中外製藥股份有限公司 <120〉 含改良抗體分子之醫藥配方 <130> C1-A0905P <150> JP 2009-069095 <151> 2009-03-19 <160〉 117 <170〉 Patentln version 3.4 <210〉 1 <211> 6 <212〉 PRT <213> 含改良抗體分子之醫藥配方 <220〉 <223> 人工合成的多胜肽序列 <400> 1 His Asp His Ala Trp Ser 1 5 <210〉 2 <211> 16 <212> PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400〉 2The south molecular weight of Fv4-M73 is...the difference between the lamp I 5 LMW: the added value from the start). Figure 27 shows the results of anion exchange chromatography performed on a solution of the antibody Fv4-M73 stored at different pH values of the digested net + Fig. 28 shows the pH values, different NaC1 concentrations, which are stored in different + gates, The results of anion exchange chromatography were carried out on a solution of arginine and two different buffer antibodies Fv4-M73. 〇S 29 . (4) Cold "material circulation for different readings, different paper agronomy, arginine And two different buffers, forming a difference in the molecular weight of the antibody π(iv) U HMW: from the initial increase. Figure 30 shows for 3 different time points (in 4 〇 and hunger) and 4 species Different formulations (EH) formed a difference in the high molecular weight material of the antibody Fv4_M73 (accounting for HMW: increasing value from the beginning). Figure 31 shows different formulations for two different times of cold/solvent cycles and E, the difference between the high molecular weight HMff of the antibody Fv4_M73: the increase from the initial value., 93 201100100 Figure 32 shows the formation of the antibody Fv4- in 6 different formulations after 3 months and 6 months at 5 °C. Difference in M73 high molecular weight material (d HMW: increase from the initial value) Figure 33 shows the difference in the formation of high molecular weight substances (δ HMW: increasing value from the initial) of the antibody Fv4-M73 after 6 months and 6 months at -0 °C. Explanation of Symbols None 94 201100100 Sequence Listing <110> Chuwai Pharmaceutical Co., Ltd. <120> Pharmaceutical Formulation Containing Modified Antibody Molecules <130> C1-A0905P <150> JP 2009-069095 <151> 2009-03 -19 <160> 117 <170〉 Patentln version 3.4 <210> 1 <211> 6 <212> PRT <213> Pharmaceutical formula containing modified antibody molecule <220> <223> Synthetic Multi-peptide sequence <400> 1 His Asp His Ala Trp Ser 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220><223> Synthetic Multi-peptide sequence <400〉 2
Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu Gin Gly 15 10 15 1 <210〉 201100100 <211> 10 <212> PRT <213> 人工序列 <220> <223〉 人工合成的多胜狀序列 <400> 3Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu Gin Gly 15 10 15 1 <210> 201100100 <211> 10 <212> PRT <213> Manual sequence <220><223> Synthetic multi-success sequence <400> 3
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210〉 4 <211〉 6 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 4Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 4 <211> 6 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400〉 4
His Asp His Ala Trp Ser 1 5 <210〉 5 <211〉 16 <212> PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400> 5His Asp His Ala Trp Ser 1 5 <210> 5 <211> 16 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu Gin Gly 1 5 10 15Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu Gin Gly 1 5 10 15
<210> 6 <211> 10 <212> PRT 201100100 I <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 6<210> 6 <211> 10 <212> PRT 201100100 I <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 7 <211> 6 <212〉 PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400〉 7Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400〉 7
Asp Asp His Ala Val Ser 1 5 <210> 8 <211> 16 <212> PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 8Asp Asp His Ala Val Ser 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu Gin Asp 15 10 15 <210〉 9 <211> 10 <212> PRT <213> 人工序列 201100100 <220> <223>人工合成的多胜肽序列 <400〉 9Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu Gin Asp 15 10 15 <210> 9 <211> 10 <212> PRT <213> Artificial sequence 201100100 <220><223> Synthetic polypeptide sequence <400〉 9
Leu Leu Ala Arg Ala Thr Ala Met Asp Val 1 5 <210> 10 <211〉 11 <212> PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 10Leu Leu Ala Arg Ala Thr Ala Met Asp Val 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> 10
Gin Ala Ser Arg Asp He Ser Ser His Leu Asn 1 5 10 <210〉 11 <211> 7 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜狀序列 <400> 11Gin Ala Ser Arg Asp He Ser Ser His Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213>Artificial Sequence <220><223> Synthetic Multi-Success Sequence <400> 11
Tyr Gly Ser His Leu Leu Ser 1 5 <210> 12 <211> 9 <212> PRT <213〉 人工序列 <220〉 <223> 人工合成的多胜肽序列 201100100 <400> 12Tyr Gly Ser His Leu Leu Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence<220><223> Synthetic polypeptide sequence 201100100 <400> 12
Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 13 <211〉 11 <212〉 PRT <213> 人工序列 <220> <223〉 人工合成的多胜肽序列 <400〉 13Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400 〉 13
Gin Ala Ser Thr Asp He Ser Ser His Leu Asn 1 5 10 <210> 14 <211> 7 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 14Gin Ala Ser Thr Asp He Ser Ser His Leu Asn 1 5 10 <210> 14 <211> 7 <212> PRT < 213 > Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400〉 14
Tyr Gly Ser His Leu Leu Ser 1 5 <210〉 15 <211〉 9 <212> PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 5 201100100 <400〉 15Tyr Gly Ser His Leu Leu Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence 5 201100100 <400 〉 15
Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 16 <211> 11 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 16GL Leu Pro Tyr Thr 1 5 <210> 〉 16
Gin Ala Ser Gin Asp lie Ser Ser Tyr Leu Asn 1 5 10 <210> 17 <211〉 7 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 17Gin Ala Ser Gin Asp lie Ser Ser Tyr Leu Asn 1 5 10 <210> 17 <211> 7 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400〉 17
Tyr Gly Ser Glu Leu Glu Ser 1 5 <210> 18 <211〉 9 <212> PRT <213〉 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400> 18 * 201100100 ΛTyr Gly Ser Glu Leu Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> * 201100100 Λ
Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210〉 19 <211> 119 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 19Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 19 <211> 119 <212> PRT < 213 > Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400 〉 19
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60Lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60
Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 110Ala 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 7 201100100 115 <210> 20 <211> 119 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 20Thr Leu Val Thr Val Ser Ser 7 201100100 115 <210> 20 <211> 119 <212> PRT < 213 > Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> 20
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser He Ser His Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser He Ser His Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 110Ala 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 8 201100100 <210〉 21 <211> 119 <212〉 PRT <213〉人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 21Thr Leu Val Thr Val Ser Ser 115 8 201100100 <210> 21 <211> 119 <212> PRT < 213 > 213 > artificial sequence<220><223> Synthetic polypeptide sequence <400> twenty one
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30
His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60
Gin Asp Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Q 65 70 75 80Gin Asp Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Q 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 110Ala 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 9 201100100 <211〉 107 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 22Thr Leu Val Thr Val Ser Ser 115 <210> 22 9 201100100 <211> 107 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> twenty two
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr lie Thr Cys Gin Ala Ser Arg Asp He Ser Ser His 20 25 30Asp Ser Val Thr lie Thr Cys Gin Ala Ser Arg Asp He Ser Ser His 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr He Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr He Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu He Glu 100 105 <210> 23 <211> 107 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 10 201100100 <400〉 23Thr Phe Gly Gin Gly Thr Lys Val Glu He Glu 100 105 <210> 23 <211> 107 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence 10 201100100 <400> 23
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr He Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30Asp Ser Val Thr He Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu 100 105 <210> 24 <211> 107 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 24Thr Phe Gly Gin Gly Gly Thr Lys Val Glu lie Glu 100 105 <210> 24 <211> 107 <212> PRT < 213 > Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400> 24
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15 11 201100100Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15 11 201100100
Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu 100 105 <210〉 25 <211> 443 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 25Thr Phe Gly Gin Gly Gly Thr Lys Val Glu lie Glu 100 105 <210> 25 <211> 443 <212> PRT < 213 > Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400> 25
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30 12 201100100Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30 12 201100100
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe He Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Thr Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe He Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Thr Leu 50 55 60
Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 ❹Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 ❹
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Ala 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 125Thr 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 13 201100100Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 13 201100100
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu 210 215 220Ser 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 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro He Glu Lys Thr He Ser Lys 325 330 335Val Ser Asn Lys Gly Leu Pro Ala Pro He Glu Lys Thr He Ser Lys 325 330 335
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380 14 201100100Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380 14 201100100
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400Pro 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 Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 o <210〉 26 <211> 443 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 26His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 o <210> 26 <211> 443 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> 26
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15 ❹Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15 ❹
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
He Gly Phe He Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60He Gly Phe He Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 15 201100100Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 15 201100100
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 110Ala 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 125Thr 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Phe Gly Thr Gin 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 220Ser 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 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255 16 201100100Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255 16 201100100
Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 oGin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 o
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400Pro 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 Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430 17 201100100Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430 17 201100100
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 27 <211> 447 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 27His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 27 <211> 447 <212> PRT <213>Artificial Sequence<220><223> Synthetic Polypeptide Sequence<;400> 27
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30
His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Thr Leu 50 55 60His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Thr Leu 50 55 60
Gin Asp Arg Val Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Gin Asp Arg Val Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 18 201100100Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly 100 105 110 18 201100100
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Thr 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 140Pro 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 160Gly 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 oAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 o
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 19 201100100Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 19 201100100
Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 28 <211> 214 <212〉 PRT <213>人工序列 20 .201100100 <220〉 <223>人工合成的多胜肽序列 <400> 28Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence 20 .201100100 <220> <223> Synthetic polypeptide sequence <400> 28
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr lie Thr Cys Gin Ala Ser Arg Asp lie Ser Ser His 20 25 30Asp Ser Val Thr lie Thr Cys Gin Ala Ser Arg Asp lie Ser Ser His 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu He 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu He 35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95 ❹Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95 ❹
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin 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 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 21 201100100Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 21 201100100
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Ala Cys Glu Val Thr His Gin 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>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 29Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 214 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr He Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30Asp Ser Val Thr He Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu He 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu He 35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 22 201100100Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 22 201100100
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin 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 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 〇Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 〇
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210Phe Asn Arg Gly Glu Cys 210
<210〉 30 <211〉 214 <212> PRT 23 201100100 <213>人工序列 <220〉 <223>人工合成的多胜狀序列 <400> 30<210> 30 <211> 214 <212> PRT 23 201100100 <213> Artificial sequence <220><223> Synthetic multi-success sequence <400> 30
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr He Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30Asp Ser Val Thr He Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin 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 201100100Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 201100100
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 oAla Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 o
Phe Asn Arg Gly Glu Cys 210 <210> 31 <211> 324 <212〉PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 ❹ <400> 31Phe Asn Arg Gly Glu Cys 210 <210> 31 <211>324 <212>PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence ❹ <400>
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Ser 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 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60 25 201100100Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60 25 201100100
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Tyr 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 110Thr 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 125Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140
Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175 〇Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175 〇
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205
Ala Pro He Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220Ala Pro He Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220
Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn 225 230 235 240 26 201100100Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn 225 230 235 240 26 201100100
Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He 245 250 255Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 260 265 270Ala Val Glu Trp Glu Ser Asn Gly Gin 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 285Thr 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 Gin Glu Gly Asn Val Phe Ser Cys 290 295 300Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu 305 310 315 320Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu 305 310 315 320
Ser Leu Ser ProSer Leu Ser Pro
<210> 32 <211> 324 Q <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 32<210> 32 <211> 324 Q <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 32
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 27 201100100Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 27 201100100
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Tyr 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 110Thr 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 125Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140
Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 28 ,201100100Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 28 ,201100100
Ala Pro lie Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220Ala Pro lie Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220
Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn 225 230 235 240Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn 225 230 235 240
Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 245 250 255Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 260 265 270Ala Val Glu Trp Glu Ser Asn Gly Gin 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 285Thr 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 Gin Glu Gly Asn Val Phe Ser Cys 290 295 300Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu 305 310 315 320Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu 305 310 315 320
Ser Leu Ser Pro <210〉 33 <211> 328 <212〉 PRT <213〉人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 33Ser Leu Ser Pro <210> 33 <211> 328 <212> PRT <213> artificial sequence <220><223> Synthetic polypeptide sequence <400>
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 29 201100100 15 10Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 29 201100100 15 10
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Ser 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 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80
Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Tyr lie 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 110Lys 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 125Pro 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 lie Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Lys Pro Lys Asp Thr Leu Met lie 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 160Val 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 175Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 30 201100100 180 185 190Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 30 201100100 180 185 190
His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys Gly 210 215 220Lys Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys Gly 210 215 220
Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240
Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Asn 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 Gin Gin Gly Asn 290 295 300Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr 305 310 315 320Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr 305 310 315 320
Gin Lys Ser Leu Ser Leu Ser Pro 325 <210〉 34 <211> 107 <212> PRT <213>人工序列 31 201100100 <220> <223>人工合成的多胜肽序列 <400〉 34Gin Lys Ser Leu Ser Leu Ser Pro 325 <210> 34 <211> 107 <212> PRT <213> Artificial sequence 31 201100100 <220><223> Synthetic polypeptide sequence <400 〉 34
Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15
Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45
Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin 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 80Thr 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 Gin Gly Leu Ser Ser 85 90 95Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin 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>人工序列1 <220〉 <223>人工合成的多胜肽序列 <400> 35Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 35 <211> 107 <212> PRT <213>Artificial Sequence 1 <220><223> Synthetic Polypeptide Sequence <400> 35
Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu 32 201100100 15 10Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu 32 201100100 15 10
Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45
Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin 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 80Thr 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 Gin Gly Leu Ser Ser 85 90 95Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin 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>人工序列 <220> <223>人工合成的多胜肽序列 <400> 36Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 36 <211> 107 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400> 36
Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15
Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 33 201100100Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 33 201100100
Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45
Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin 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 80Thr 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 Gin Gly Leu Ser Ser 85 90 95Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin 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 60 120 180 240 300 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttgataaPro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 37 <211> 327 <212> DNA <213> Homo sapiens <400> 37 60 120 180 240 300 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttgataa
<210> 38 <211> 107 <212〉 PRT 34 327 .201100100 4 <213> Homo sapiens <400〉 38<210> 38 <211> 107 <212> PRT 34 327 .201100100 4 <213> Homo sapiens <400> 38
Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15
Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45 oTyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45 o
Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin 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 80Thr 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 Gin Gly Leu Ser Ser 85 90 95Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210〉 39 <211> 990Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 39 <211> 990
<212〉 DNA <213> Homo sapiens <400> 39 60 120 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 35 180 201100100 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 960 990 cagaagagcc tctccctgtc tccgggtaaa <210> 40 <211> 330 <212〉 PRT <213> Homo sapiens <400> 40≪ 212> DNA < 213 > Homo sapiens < 400 > 39 60 120 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 35 180 201100100 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 Cggggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa t gggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 990 cagaagagcc tctccctgtc tccgggtaaa < 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 15 10 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 36 201100100Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 36 201100100
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80
Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Tyr lie 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 110Lys 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 125Pro 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 lie Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Lys Pro Lys Asp Thr Leu Met lie 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 160Val 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 175Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190
His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 2⑻ 205 37 201100100His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 2(8) 205 37 201100100
Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220
Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240
Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Asn 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 Gin Gin Gly Asn 290 295 300Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320
Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 41 <211> 984Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 41 <211> 984
<212〉 DNA <213> Homo sapiens <400> 41 60 120 gctagcacca agggcccaic ggtcttcccc ctggcgccct cctccaagag cacctccgag agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 38 180 240 240<212> DNA <213> Homo sapiens <400> 41 60 120 gctagcacca agggcccaic ggtcttcccc ctggcgccct cctccaagag cacctccgag agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 38 180 240 240
201100100 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc aaatcttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt gtggtcagcg tcctcaccgt cgtgcaccag gactggctga acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc tccctgtctc cgggtaaatg ataa <210〉 42 <211> 326 <212> PRT <213> Homo sapiens <400〉 42201100100 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc aaatcttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt gtggtcagcg tcctcaccgt cgtgcaccag gactggctga acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc tccctgtctc cgggtaaatg ataa < 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 15 10 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 300 360 420 480 540 600 660 720 780 840 900 960 984 39 201100100Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 300 360 420 480 540 600 660 720 780 840 900 960 984 39 201100100
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys S5 90 95Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys S5 90 95
Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Thr 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 125Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 yThr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 y
Val Ser His Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160Val Ser His Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 40 201100100Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 40 201100100
Ala Pro lie Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220Ala Pro lie Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220
Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240
Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 245 250 255Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 260 265 270Ala Val Glu Trp Glu Ser Asn Gly Gin 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 285Thr 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 Gin Gin Gly Asn Val Phe Ser Cys 290 295 300Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 305 310 315 320Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 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 60 gctagcacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 41 120 201100100 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〉 44Ser Leu Ser Pro Gly Lys 325 <210> 43 <211> 995 <212> DNA <213> Homo sapiens <400> 43 60 gctagcacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 41 120 201100100 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 gg cttctacc 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 15 10 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 15 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 42 201100100Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 42 201100100
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Phe 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80Leu Ser Ser Val Val Thr Val Pro 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 95Tyr 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 110Arg 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 125Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125
Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 〇Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 〇
Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp 145 150 155 160Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 180 185 190Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 43 201100100 195 2⑻ 205Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 43 201100100 195 2(8) 205
Pro Ser Ser lie Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg 210 215 220Pro Ser Ser lie Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg 210 215 220
Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys 225 230 235 240Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys 225 230 235 240
Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255
He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 260 265 270He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Thr 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 Gin Glu Gly Asn Val Phe Ser 290 295 300Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser 290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 305 310 315 320Cys Ser Val Met His Glu Ala Leu His Ass His Tyr Thr Gin Lys Ser 305 310 315 320
Leu Ser Leu Ser Leu Gly 325 <210〉 45 <211> 4 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 45 44 201100100Leu Ser Leu Ser Leu Gly 325 <210> 45 <211> 4 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400> 45 44 201100100
Gly Gly Gly Ser <210〉 46 <211〉 4 <212〉 PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400〉 46 Ser Gly Gly Gly 1 <210〉 47 <211> 5 <212〉 PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 47Gly Gly Gly Ser <210> 46 <211> 4 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 46 Ser Gly Gly Gly 1 <210> 47 <211> 5 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gly Gly Gly Gly Ser 1 5 <210> 48 <211> 5 <212〉 PRT <213〉人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 48Gly Gly Gly Gly Ser 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Ser Gly Gly Gly Gly 45 201100100 1 5 <210〉 49 <211> 6 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 49Ser Gly Gly Gly Gly 45 201100100 1 5 <210> 49 <211> 6 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gly Gly Gly Gly Gly Ser 1 5 <210> 50 <211> 6 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 50Gly Gly Gly Gly Gly Ser 1 5 <210> 50 <211> 6 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Ser Gly Gly Gly Gly Gly 1 5 <210> 51 <211> 7 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 51Ser Gly Gly Gly Gly Gly 1 5 <210> 51 <211> 7 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400> 51
Gly Gly Gly Gly Gly Gly Ser 1 5 46 201100100 <210> 52 <211> 7 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 52Gly Gly Gly Gly Gly Gly Ser 1 5 46 201100100 <210> 52 <211> 7 <212> PRT <213>Artificial Sequence<220><223> Synthetic Polypeptide Sequence <400>; 52
Ser Gly Gly Gly Gly Gly Gly 1 5 ❹ <210> 53 <211> 449 <212> PRT <21·3>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 53Ser Gly Gly Gly Gly Gly Gly 1 5 ❹ <210> 53 <211> 449 <212> PRT <21·3>Artificial Sequence<220><223> Synthetic Polypeptide Sequence<400> 53
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 47 201100100 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 47 201100100 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 He Ser 48 201100100 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser 48 201100100 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 49 201100100 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 49 201100100 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys <210〉 54 <211> 214 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 54Lys <210> 54 <211> 214 <212> PRT < 213 > artificial sequence <220><223> Synthetic multi-peptide sequence <400> 54
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 50 201100100Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 50 201100100
Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin 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 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 oAla Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 o
Phe Asn Arg Gly Glu Cys 210 <210> 55 <211> 449 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 55Phe Asn Arg Gly Glu Cys 210 <210> 55 <211> 449 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400> 55
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 51 201100100 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 51 201100100 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Val Leu Ala Arg lie Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110Ala Arg Val Leu Ala Arg lie Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 52 201100100 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 52 201100100 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 He Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 53 201100100 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 53 201100100 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys <210> 56 <211> 214 <212〉 PRT <213〉人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 56Lys <210> 56 <211> 214 <212> PRT < 213 > 213 > artificial sequence <220><223> Synthetic polypeptide sequence <400> 56
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30 54 201100100Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30 54 201100100
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp lie Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp lie Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin 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 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 55 201100100Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 55 201100100
Phe Asn Arg Gly Glu Cys 210 <210> 57 <211> 449 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 57Phe Asn Arg Gly Glu Cys 210 <210> 57 <211> 449 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser Asp Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
He Gly Tyr lie Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Ser Leu 50 55 60He Gly Tyr lie Ser Tyr Ser Gly He Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Gly Arg Val Thr lie Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Gly Arg Val Thr lie Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95Leu 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 56 201100100Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 56 201100100
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Thr 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 57 201100100Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 57 201100100
Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335
Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 4⑻Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 4(8)
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys 58 201100100 ’ <210> 58 <211> 214 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 58Lys 58 201100100 '<210> 58 <211> 214 <212> PRT <213> artificial sequence <220><223> Synthetic multi-peptide sequence <400>
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Ser Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Ser Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 59 201100100 130 135 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 59 201100100 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Ala Cys Glu Val Thr His Gin 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>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 59Phe Asn Arg Gly Glu Cys 210 <210> 59 <211> 7 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> 59
Tyr Thr Ser Arg Leu His Ser <210> 60 <211> 7 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 60 201100100 <400> 6060r; 211 >
Tyr Gly Ser Glu Leu His Ser 1 5 <210〉 61 <211> 30 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 61Tyr Gly Ser Glu Leu His Ser 1 5 <210> 61 <211> 30 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400> 61
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr 20 25 30
<210> 62 <211> 30 <212> PRT Q <213〉人工序列 <220> <223>人工合成的多胜肽序列 <400> 62<210> 62 <211> 30 <212> PRT Q < 213 > artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser 20 25 30 <210> 63 61 201100100 <211〉 32 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 63Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser lie Ser 20 25 30 <210> 63 61 201100100 <211> 32 <212> PRT <213> Artificial Sequence <220><223> Multi-peptide sequence <400> 63
Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser Leu Arg 15 10 15Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser Leu Arg 15 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>人工序列 <220> <223>人工合成的多胜肽序列 <400> 64Leu 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 Sequence <220><223> Polypeptide sequence <400> 64
Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 15 10 15Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 15 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>人工序列 <220> <223>人工合成的多胜肽序列 <400> 65 62 .201100100Met 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 Sequence <220><223> Multi-peptide sequence <400> 65 62 .201100100
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15Gin Val Gin Leu Gin 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 lie Ser 20 25 30 <210〉 66 <211> 449 <212〉 PRT <213>人工序列Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser 20 25 30 <210> 66 <211> 449 <212> PRT <213> Artificial Sequence
<220> <223> An artificially synthesized polypeptide sequence <400> 66<220><223> An artificially synthesized polypeptide sequence <400> 66
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30
His Ala His Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp ❹ 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60His Ala His Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp ❹ 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Gly Arg Val Thr lie Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Gly Arg Val Thr lie Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95Leu 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 63 201100100 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 63 201100100 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Thr 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 He Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 64 * 201100100 275 280 285Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 64 * 201100100 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 4⑻Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 4(8)
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys 65 201100100 <210〉 67 <211> 214 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 67Lys 65 201100100 <210> 67 <211> 214 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence <400> 67
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin His lie Ser Ser His 20 25 30Asp Ser Val Thr lie Thr Cys Gin Ala Ser Gin His lie Ser Ser His 20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45
Tyr Tyr Gly Ser His Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Tyr Gly Ser His Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
(J(J
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu He Glu Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Val Glu He Glu Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 66 201100100Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 66 201100100
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Ala Cys Glu Val Thr His Gin 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>人工序列 <220> <223>人工合成的多胜肽序列 <400> 68Phe Asn Arg Gly Glu Cys 210 <210> 68 <211> 448 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30 67 201100100Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30 67 201100100
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205 68 '201100100Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205 68 '201100100
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Ser 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 240Thr 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 lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380 69 201100100Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380 69 201100100
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro V'al Leu 385 390 395 400Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro V'al Leu 385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210〉 69 <211> 447 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 69Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 69 <211> 447 <212> PRT <213> Artificial Sequence <220><223> Multi-peptide sequence <400> 69
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45
He Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60He Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80 70 201100100Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80 70 201100100
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 ❹Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 ❹
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 220Ser 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 240Thr 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 He Ser 245 250 255 71 201100100Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser 245 250 255 71 201100100
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 72 201100100 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 72 201100100 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 70 <211〉 445 <212> PRT <213>人工序列 <220〉Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 70 <211> 445 <212> PRT <213> Artificial Sequence <220>
<223>人工合成的多胜肽序列 <400> 70<223> Synthetic polypeptide sequence <400> 70
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 o lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 o lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110 Ί3 201100100Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110 Ί3 201100100
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Phe Gly Thr Gin 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 220Ser 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 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 201100100Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 201100100
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys 325 330 335 oVal Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys 325 330 335 o
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400Pro 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 Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
<210> 71 <211> 445 <212〉 PRT 75 201100100 <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 71<210> 71 <211> 445 <212> PRT 75 201100100 <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45
He Gly Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60He Gly Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 76 201100100Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 76 201100100
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Phe Gly Thr Gin 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 220Ser 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 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 77 201100100Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 77 201100100
Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400Pro 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 Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 72 <211> 443 <212〉 PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 72His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 72 <211> 443 <212> PRT <213>Artificial Sequence <220><223> Peptide sequence <400〉 72
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15 78 201100100Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15 78 201100100
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 〇Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 〇
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 79 201100100Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 79 201100100
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Phe Gly Thr Gin 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 220Ser 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 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 80 201100100Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365 80 201100100
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400Pro 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 Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210〉 73 <211〉 449 <212> PRT <213>人工序列 〇 <220> <223>人工合成的多胜肽序列 <400〉 73His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 73 <211> 449 <212> PRT < 213 > Artificial Sequence 〇 <220><223> Synthetic Polypeptide Sequence <400〉 73
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 81 201100100 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 81 201100100 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 82 201100100Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 82 201100100
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240Thr 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 lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
OO
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 83 201100100Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 83 201100100
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys f) <210> 74 <211> 447 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 74Lys f) <210> 74 <211> 447 <212> PRT < 213 > artificial sequence <220><223> Synthetic polypeptide sequence <400> 74
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45
He Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60 84 -j 201100100He Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60 84 -j 201100100
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 oSer Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 o
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr He 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 220Ser 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 85 201100100Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 85 201100100
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 86 201100100Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 86 201100100
Ser A.rg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser A.rg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 75 <211> 443 <212> PRT <213>人工序列 <220> 0 <223>人工合成的多胜肽序列 <400〉 75Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 75 <211> 443 <212> PRT <213> Artificial Sequence <220> 0 <223> Multi-peptide sequence <400〉 75
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Asp 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Asp 20 25 30
His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 ❹ lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 ❹ lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu 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 Gin Gly 100 105 110 87 201100100Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110 87 201100100
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Ser 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 140Pro 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 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Phe Gly Thr Gin 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 cSer Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu 210 215 220 c
JJ
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240Cys 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 lie Ser Arg Thr Pro Glu 245 250 255Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 88 201100100Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 88 201100100
Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335
Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350
Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365
Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 〇Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 〇
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430
His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210〉 76 <211> 449 89 201100100 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 76His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 <210> 76 <211> 449 89 201100100 <212> PRT <213>Artificial Sequence<220><223> Synthetic Polypeptide Sequence <400〉 76
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser He Ser Asp Asp 20 25 30Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser He Ser Asp Asp 20 25 30
His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60His Ala Val Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Thr Leu 50 55 60
Gin Asp Arg Val Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Gin Asp Arg Val Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin 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 110Ala 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 125Thr 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 90 201100100Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 90 201100100
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160Gly 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 175Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Val Asn His Lys Pro 195 200 205Ser Ser Leu Gly Thr Gin Thr Tyr He 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 220Ser 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 240Thr 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 lie Ser 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Pro 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 91 201100100Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 91 201100100
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400Ser Asn Gly Gin 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 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
Lys <210〉 77 <211> 446 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 92 201100100 <400〉 77Lys <210> 77 <211> 446 <212> PRT <213>Artificial sequence <220><223> Synthetic polypeptide sequence 92 201100100 <400〉 77
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 10 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr 20 25 30
Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly He Ser Trp Asn Ser Gly Arg lie Gly Tyr Ala Asp Ser Val 50 55 60Ser Gly He Ser Trp Asn Ser Gly Arg lie Gly Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe 65 70 75 80
Leu Gin Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Leu Gin 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 lie Trp Gly Gin Gly Thr Met Val 100 105 110 〇Ala Lys Gly Arg Asp Ser Phe Asp lie Trp Gly Gin Gly Thr Met Val 100 105 110 〇
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Thr 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 140Pro 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 160Val 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 Gin Ser Ser 165 170 175 93 201100100Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser 165 170 175 93 201100100
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190
Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205Gly Thr Gin Thr Tyr lie 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 220Lys 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 240Cys 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 lie Ser Arg Thr Pro 245 250 255Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 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 270Glu 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 285Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285
Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300
Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser 325 330 335Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser 325 330 335
Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 94 201100100 340 345 350Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 94 201100100 340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 355 360 365Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 355 360 365
Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly 370 375 380
Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400Gin 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 415Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415
Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430
Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 78 <211〉 214 <212〉 PRT <213〉人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 78Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 78 <211> 214 <212> PRT <213>Artificial Sequence <220> <223> Peptide Sequence <400> 78
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly 15 10 15
Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Trp 20 25 30 95 201100100Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Trp 20 25 30 95 201100100
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45
Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp Phe Ala Ser Tyr Tyr Cys Gin Gin Ala Asn Ser Phe Pro Tyr 85 90 95Glu Asp Phe Ala Ser Tyr Tyr Cys Gin Gin Ala Asn Ser Phe Pro Tyr 85 90 95
Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala Ala 100 105 110Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin 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 140Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160
Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Glu Ser Val Thr Glu Gin 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 190Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 96 201100100Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 96 201100100
Phe Asn Arg Gly Glu Cys 210 <210> 79 <211> 267 <212> PRT <213> Homo sapiens <400> 79Phe Asn Arg Gly Glu Cys 210 <210> 79 <211> 267 <212> PRT <213> Homo sapiens <400>
Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 15 10 15Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 15 10 15
Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30
Gin Gin Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys 35 40 45Gin Gin Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys 35 40 45
Gly Ala Trp Val Trp Glu Asn Gin Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60Gly Ala Trp Val Trp Glu Asn Gin Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60
Thr Thr Asp Leu Arg lie Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80Thr Thr Asp Leu Arg lie Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80
Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gin Gly Leu Leu Gly Cys 85 90 95Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gin 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 110Glu 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 Gin Gly Thr Trp Gly 115 120 125 97 201100100Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gin Gly Thr Trp Gly 115 120 125 97 201100100
Gly Asp Trp Pro Glu Ala Leu Ala lie Ser Gin Arg Trp Gin Gin Gin 130 135 140Gly Asp Trp Pro Glu Ala Leu Ala lie Ser Gin Arg Trp Gin Gin Gin 130 135 140
Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160Asp 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 175His 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 190Lys 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 205Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu 195 200 205
Gin Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gin Gly 210 215 220Gin Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gin Gly 210 215 220
Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240Asp 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 lie Val Gin His 245 250 255Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys lie Val Gin His 245 250 255
Ala Gly Leu Ala Gin Pro Leu Arg Val Glu Leu 260 265 <210> 80 <211〉 99 <212> PRT <213> Homo sapiens <400> 80 lie Gin Arg Thr Pro Lys lie Gin Val Tyr Ser Arg His Pro Ala Glu 98 201100100 15 10Ala Gly Leu Ala Gin Pro Leu Arg Val Glu Leu 260 265 <210> 80 <211> 99 <212> PRT <213> Homo sapiens <400> 80 lie Gin Arg Thr Pro Lys lie Gin Val Tyr Ser Arg His Pro Ala Glu 98 201100100 15 10
Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30
Ser Asp He Glu Val Asp Leu Leu Lys Asn Gly Glu Arg lie Glu Lys 35 40 45Ser Asp He Glu Val Asp Leu Leu Lys Asn Gly Glu Arg lie Glu Lys 35 40 45
Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 oVal Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 o
Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80Leu 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 Gin Pro Lys He Val Lys Trp Asp 85 90 95Arg Val Asn His Val Thr Leu Ser Gin Pro Lys He Val Lys Trp Asp 85 90 95
Arg Asp Met O <210〉 81 <211> 16 <212> PRT <213〉人工序列 <220> <223>人工合成的多胜肽序列 <400> 81Arg Asp Met O <210> 81 <211> 16 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210〉 82 <211> 16 99 201100100 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 82Tyr He Ser Tyr Ser Gly He Thr Thr Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210> 82 <211> 16 99 201100100 <212> PRT <213> Manual Sequence <220><223> Synthetic polypeptide sequence <400> 82
Phe lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210〉 83 <211> 16 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 83Phe lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210> 83 <211> 16 <212> PRT <213> Artificial Sequence <220><223> Multi-peptide sequence <400> 83
Tyr lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210> 84 <211〉 10 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 84Tyr lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu Lys Ser 15 10 15 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220><223> Polypeptide sequence <400> 84
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 85 <211> 10 <212> PRT <213>人工序列 100 201100100 <220〉 <223>人工合成的多胜肽序列 <400> 85Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence 100 201100100 <220> <223> Synthetic Polypeptide Sequence <400> 85
Leu Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 86 <211> 10 <212> PRT <213>人工序列Leu Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 <210> 86 <211> 10 <212> PRT <213>
<220〉 <223>人工合成的多胜肽序列 <400> 86<220〉 <223> Synthetic polypeptide sequence <400> 86
Ser Leu Ala Arg Ala Thr Ala Met Asp Tyr 1 5 10 <210> 87 <211> 11 <212> PRT <213〉人工序列 ο <220> <223>人工合成的多胜肽序列 <400〉 87Ser Leu Ala Arg Ala Thr Ala Met Asp Tyr 1 5 10 <210> 87 <211> 11 <212> PRT <213>Artificial sequence ο <220><223> Synthetic multi-peptide sequence <400〉 87
Arg Ala Ser Gin Asp He Ser Ser Tyr Leu Asn 1 5 10 <210> 88 <211> 11 <212〉 PRT <213>人工序列 101 <220〉 201100100 <223>人工合成的多胜肽序列 <400> 88Arg Ala Ser Gin Asp He Ser Ser Tyr Leu Asn 1 5 10 <210> 88 <211> 11 <212> PRT <213> Artificial Sequence 101 <220> 201100100 <223> Peptide sequence <400> 88
Arg Ala Ser Thr Asp lie Ser Ser Tyr Leu Asn 1 5 10 <210> 89 <211〉 11 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 89Arg Ala Ser Thr Asp lie Ser Ser Tyr Leu Asn 1 5 10 <210> 89 <211> 11 <212> PRT <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400〉 89
Arg Ala Ser Arg Asp He Ser Ser Tyr Leu Asn 1 5 10 <210〉 90 <211〉 9 <212> PRT <213>人工序列 <220>Arg Ala Ser Arg Asp He Ser Ser Tyr Leu Asn 1 5 10 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220>
<223>人工合成的多胜狀序列 <400> 90<223>Synthetic multi-success sequence <400> 90
Gin Gin Gly Asn Thr Leu Pro Tyr Thr 1 5 <210〉 91 <211〉 9 <212> PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 102 201100100 、 <400> 91Gin Gin Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 91 <211> 9 <212> PRT <213>Artificial Sequence<220><223> Synthetic Polypeptide Sequence 102 201100100 <400> 91
Gly Gin Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 92 <211> 9 <212〉 PRT <213>人工序列 <220>Gly Gin Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220>
<223>人工合成的多胜肽序列 <400> 92<223> Synthetic polypeptide sequence <400> 92
Gin Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 93 <211> 30 <212> PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400> 93Gin Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 <210> 93 <211> 30 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400>; 93
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr 20 <210> 94 <211〉 30 <212〉 PRT <213> 人工序列 103 30 201100100 <220〉 <223>人工合成的多胜肽序列 <400> 94Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr 20 <210> 94 <211> 30 <212> PRT <213> Artificial Sequence 103 30 201100100 <220〉 <223> Synthetic Multi-peptide sequence <400> 94
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser He Ser 20 25 30 <210〉 95 <211〉 6 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 95Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser He Ser 20 25 30 <210> 95 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Peptide sequence <400> 95
Ser Asp His Ala Trp Ser 1 5 <210> 96 <211〉 6 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 96Ser Asp His Ala Trp Ser 1 5 <210> 96 <211> 6 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Asp Asp His Ala Trp Ser 1 5Asp Asp His Ala Trp Ser 1 5
<210> 97 <211> 14 <212〉 PRT ±04 201100100 <213> 人工序列 <220> <223> 人工合成的多胜狀序列 <400〉 97<210> 97 <211> 14 <212> PRT ±04 201100100 <213> Artificial sequence <220><223> Synthetic multi-success sequence <400> 97
Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp lie Gly 1 5 10 <210〉 98 <211> <212〉 〇 <213> 14 PRT 人工序列 <220> <223> 人工合成的多胜肽序列 <400〉 98Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp lie Gly 1 5 10 <210> 98 <211><212>〇<213> 14 PRT Artificial Sequence <220><223> Synthetic Multi-peptide sequence <400> 98
Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp He Gly 1 5 10 <210〉 99 <211〉 <212> 〇 <213> 16 PRT 人工序列 <220> <223> 人工合成的多胜肽序列 <400〉 99Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp He Gly 1 5 10 <210> 99 <211><212>〇<213> 16 PRT Artificial Sequence <220><223> Synthetic Multi-peptide sequence <400> 99
Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu Gin Asp 1 5 10 15 <210〉 <211〉 <212> <213〉 100 11 PRT 人工序列 105 201100100 <220〉 <223>人工合成的多胜肽序列 <400〉 100Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu Gin Asp 1 5 10 15 <210> <211> <212><213> 100 11 PRT Artificial Sequence 105 201100100 <220〉 <223> ; synthetic multi-peptide sequence <400> 100
Trp Gly Gin Gly Ser Leu Val Thr Val Ser Ser 1 5 10 <210〉 101 <211〉 11 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜狀序列 <400> 101Trp Gly Gin Gly Ser Leu Val Thr Val Ser Ser 1 5 10 <210> 101 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Synthetic Multi-Success Sequence <400> 101
Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 102 <211> 23 <212〉 PRT <213>人工序列 <220〉 <223>人工合成的多胜狀序列 <400> 102Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 102 <211> 23 <212> PRT <213>Artificial Sequence <220><223> Synthetic Multi-Success Sequence <400> 102
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Arg Val Thr lie Thr Cys 20Asp Arg Val Thr lie Thr Cys 20
<210〉 103 <211> 23 <212> PRT 106 .201100100 <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400〉 103<210> 103 <211> 23 <212> PRT 106 .201100100 <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 103
Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15
Asp Ser Val Thr lie Thr Cys 20 v <210> 104 <211〉 11 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400> 104Asp Ser Val Thr lie Thr Cys 20 v <210> 104 <211> 11 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gin Ala Ser Gin Asp lie Ser Ser Tyr Leu Asn 1 5 10 ❹ <210〉 105 <211〉 15 <212〉PRT <213>人工序列 <220〉 <223>人工合成的多胜肽序列 <400> 105Gin Ala Ser Gin Asp lie Ser Ser Tyr Leu Asn 1 5 10 ❹ <210> 105 <211> 15 <212>PRT <213>Artificial Sequence<220><223> Synthetic Polypeptide Sequence <400> 105
Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 15 10 15 <210> 106 107 201100100 <211〉 15 <212〉 PRT <213〉 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 106Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 15 10 15 <210> 106 107 201100100 <211> 15 <212> PRT <213> Artificial Sequence <220><223> Synthetic polypeptide sequence <400> 106
Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie Tyr 15 10 15 <210> 107 <211> 7 <212> PRT <213> 人工序列 <220> <223> 人工合成的多胜狀序列 <400> 107Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie Tyr 15 10 15 <210> 107 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Synthetic Multi-success sequence <400> 107
Tyr Thr Ser Arg Leu His Ser 1 5 <210> 108 <211> 7 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 108Tyr Thr Ser Arg Leu His Ser 1 5 <210> 108 <211> 7 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 108
Tyr Thr Ser Glu Leu Glu Ser 1 5 <210> 109 <211〉 7 <212> PRT 108 201100100 <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 109Tyr Thr Ser Glu Leu Glu Ser 1 5 <210> 109 <211> 7 <212> PRT 108 201100100 <213>Artificial Sequence <220><223> Synthetic Polypeptide Sequence <400 〉 109
Tyr Thr Ser Arg Leu Leu Ser 1 5Tyr Thr Ser Arg Leu Leu Ser 1 5
<210> 110 <211> 32 <212> PRT <213>人工序列 <220> <223>人工合成的多胜肽序列 <400〉 110<210> 110 <211> 32 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 110
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15
Phe Thr lie Ser Ser Leu Gin Pro Glu Asp lie Ala Thr Tyr Tyr Cys 20 25 30Phe Thr lie Ser Ser Leu Gin Pro Glu Asp lie Ala Thr Tyr Tyr Cys 20 25 30
O <210> 111 <211〉 32 <212〉 PRT <213〉人工序列 <220> <223>人工合成的多胜肽序列 <400> 111O <210> 111 <211> 32 <212> PRT < 213 > 213 > artificial sequence <220><223> Synthetic polypeptide sequence <400>
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15
Phe Thr lie Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 109 201100100 20 25 30 <210> 112 <211> 10 <212> PRT <213〉 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 112Phe Thr lie Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 109 201100100 20 25 30 <210> 112 <211> 10 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 112
Phe Gly Gin Gly Thr Lys Val Glu lie Lys 1 5 10 <210> 113 <211> 10 <212> PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 113Phe Gly Gin Gly Gly Thr Lys Val Glu lie Lys 1 5 10 <210> 113 <211> 10 <212> PRT <213> Artificial Sequence <220><223> Synthetic Polypeptide Sequence <;400> 113
Phe Gly Gin Gly Thr Lys Val Glu He Glu 1 5 10 <210〉 114 <211〉 30 <212〉 PRT <213> 人工序列 <220> <223> 人工合成的多胜狀序列 <400> 114Phe Gly Gin Gly Gly Thr Lys Val Glu He Glu 1 5 10 <210> 114 <211> 30 <212> PRT <213> Artificial sequence <220><223> Synthetic multi-success sequence <223>;400> 114
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15 110 201100100Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15 110 201100100
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly His Ser lie Thr 20 25 30Thr Leu Ser Leu Thr Cys Thr Val Ser Gly His Ser lie Thr 20 25 30
<210> 115 <211〉 6 <212〉 PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400〉 115 His Asp His Ala Trp Ser 1 5 <210> 116 <211〉 11 <212〉 PRT <213> 人工序列 <220〉 <223> 人工合成的多胜肽序列 <400〉 116<210> 115 <211> 6 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 115 His Asp His Ala Trp Ser 1 5 < ;210> 116 <211> 11 <212> PRT <213> Artificial sequence <220><223> Synthetic polypeptide sequence <400> 116
Arg Ala Ser Gin Asp lie Ser Ser His Leu Asn 1 5 <210〉 117 <211> 7 <212〉 PRT <213> 人工序列 <220> <223> 人工合成的多胜肽序列 <400> 117 111 201100100Arg Ala Ser Gin Asp lie Ser Ser His Leu Asn 1 5 <210> 117 <211> 7 <212> PRT <213> Artificial sequence <220><223> Synthetic multi-peptide sequence <223>;400> 117 111 201100100
Tyr Thr Ser His Leu His Ser 1 5 fTyr Thr Ser His Leu His Ser 1 5 f
112112
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